U.S. patent application number 10/613744 was filed with the patent office on 2005-12-08 for assays for screening compounds which interact with cation channel proteins, mutant prokaryotic cation channel proteins, and uses thereof.
Invention is credited to MacKinnon, Roderick.
Application Number | 20050272093 10/613744 |
Document ID | / |
Family ID | 26722890 |
Filed Date | 2005-12-08 |
United States Patent
Application |
20050272093 |
Kind Code |
A1 |
MacKinnon, Roderick |
December 8, 2005 |
Assays for screening compounds which interact with cation channel
proteins, mutant prokaryotic cation channel proteins, and uses
thereof
Abstract
Assays for screeing potential drugs or agents that can interact
and potentially bind to cation channel proteins, and potentially
have uses in treating conditions related to the function of cation
channel proteins is provided, along with prokaryotic cation channel
proteins mutated to mimic eukaryotic cation channels, which can
then be used in assays of the present invention.
Inventors: |
MacKinnon, Roderick; (New
York, NY) |
Correspondence
Address: |
KLAUBER & JACKSON
411 HACKENSACK AVENUE
HACKENSACK
NJ
07601
|
Family ID: |
26722890 |
Appl. No.: |
10/613744 |
Filed: |
July 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10613744 |
Jul 3, 2003 |
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09275252 |
Mar 24, 1999 |
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6641997 |
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09275252 |
Mar 24, 1999 |
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09054347 |
Apr 2, 1998 |
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09054347 |
Apr 2, 1998 |
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09045529 |
Mar 20, 1998 |
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Current U.S.
Class: |
435/7.1 ;
435/252.3; 435/287.2; 435/320.1; 435/69.1; 506/14; 506/18; 506/9;
530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/705 20130101;
G01N 2500/00 20130101; C07K 14/36 20130101; G01N 33/94 20130101;
G01N 33/6872 20130101; C07K 14/195 20130101; C07K 14/43581
20130101 |
Class at
Publication: |
435/007.1 ;
435/069.1; 435/252.3; 435/320.1; 530/350; 536/023.5 |
International
Class: |
G01N 033/53; C07H
021/04; C07K 014/705; C12P 021/02; C12N 001/21 |
Goverment Interests
[0002] The research leading to the present invention was supported
in part with National Institutes of Health Grant GM 43949. The
government may have rights in the invention.
Claims
What is claimed is:
1. A method of using a functional cation channel protein in an
assay for screening potential drugs or agents which interact with
the cation channel protein, the method comprising the steps of: a)
providing a functional cation channel protein; b) conjugating the
functional cation channel protein to a solid phase resin; c)
contacting the potential drug or agent to the functional cation
channel protein conjugated to the solid phase resin; d) removing
the functional cation channel protein from the solid phase resin;
and e) determining whether the potential drug or agent is bound to
the cation channel protein.
2. The method of claim 1, wherein the providing step comprises: a)
expressing an isolated nucleic acid molecule encoding the cation
channel protein in a unicellular host such that the cation channel
protein is present in the cell membrane of the unicellular host; b)
lysing the unicellular host in a solubilizing solution so that the
cation 4 channel protein is solubilized in the solution; and c)
extracting the cation channel protein from the solublizing solution
with a detergent.
3. The method of claim 2, wherein lysing the unicellular host in a
solubilizing solution comprises sonicating the unicellular host in
a solution comprising 50 mM Tris buffer, 100 mM KCl, 10 mM
MgSO.sub.4, 25 mg DNAse 1, 250 mM sucrose, pepstatin, leupeptin,
and PMSF, pH 7.5.
4. The method of claim 2, wherein the detergent comprises 40 mM
decylmaltoside.
5. The method of claim 1, wherein the conjugating step comprises
binding the cation channel protein to a cobalt resin at protein to
resin ratio that allows for saturation of the resin with the cation
channel protein.
6. The method of claim 1, wherein the removing step comprises
contacting the cation channel protein conjugated to the solid phase
resin to an imidazole solution.
7. The method of claim 1, wherein the isolated nucleic acid
molecule encoding the cation channel protein comprises a DNA
sequence of SEQ ID NO:17, or degenerate variants thereof, or an
isolated nucleic acid molecule hybridizable under standard
hybridization conditions to an isolated nucleic acid molecule
comprising a DNA sequence of SEQ ID NO:17, or degenerate variants
thereof.
8. The method of claim 1, wherein the potential drug or agent is a
member of a libarary of compounds, and the contacting step
comprises contacting the library of compounds to the functional
cation channel protein conjugated to the solid phase resin.
9. The method of claim 8, wherein the library of compounds
comprises a mixture of compounds or a combinatorial library.
10. The method of claim 9, wherein the combinatorial library
comprises a phage display library, or a synthetic peptide
library.
11. A prokaryotic cation channel protein mutated to mimic a
functional eukaryotic cation channel protein.
12. The prokaryotic cation channel protein of claim 11, selected
from the group consisting of a potassium channel protein, a sodium
channel protein, or a calcium channel protein.
13. The prokaryotic cation channel protein of claim 11,
endogenously produced in a prokaryotic organism selected from the
group consisting of E. coli, Streptomyces lividans, Clostridium
acetobutylicum, or Staphylcoccus aureus.
14. The prokaryotic cation channel protein of claim 11, comprising
an amino acid sequence of SEQ ID Nos: 1, 2, 3, or 7.
15. The prokaryotic cation channel protein of claim 11, wherein
said prokaryotic cation channel protein is a potassium channel
protein from Streptomyces lividans.
16. The prokaryotic cation channel of claim 15, encoded by a
nucleic acid comprising a DNA sequence of SEQ ID NO:17, or
degenerate variants thereof.
17. The prokaryotic cation channel protein of claim 15, comprising
an amino acid sequence of SEQ ID NO:1, or conserved variants
thereof.
18. The prokaryotic cation channel protein of claim 11, wherein the
functional eukaryotic cation channel protein comprises a eukaryotic
potassium channel protein, a eukaryotic sodium channel protein, or
a eukaryotic calcium channel protein.
19. The prokaryotic cation channel protein of claim 11, wherein
said functional eukaryotic cation channel protein is endogenously
produced in a eukaryotic organism comprising insects or
mammals.
20. The prokaryotic cation channel protein of claim 19, wherein
said eukaryotic organism comprises Drosophila melanogaster, Homo
sapiens, C. elegans, Mus musculus, Arabidopsis thaliana, paramecium
tetraaurelia or Rattus novegicus.
21. The prokaryotic cation channel protein of claim 11, mutated to
mimic a eukaryotic cation channel protein comprising an amino acid
sequence comprising SEQ ID Nos: 4, 5, 6, 8, 9, 10, 11, 12, 13, or
14.
22. The prokaryotic cation channel protein of claim 21, wherein
said prokaryotic channel protein is a potassium channel protein
from Streptomyces lividans comprising an amino acid sequence of SEQ
ID NO:1, said eukaryotic cation channel is a potassium channel
protein comprising an amino acid sequence of SEQ ID NO:4, and said
mutated prokaryotic channel protein comprises an amino acid
sequence of SEQ ID NO:16, or conserved variants thereof.
23. The prokaryotic cation channel protein of claim 22, wherein
said mutated porkaryotic channel protein is encoded by an isolated
nucleic acid molecule comprising a DNA sequence of SEQ ID NO:17, or
degenerate variants thereof.
24. An isolated nucleic acid molecule which encodes a mutant
K.sup.+ channel protein, comprising a DNA sequence of SEQ ID NO:17,
or degenerate variants thereof.
25. An isolated nucleic acid molecule hybridizable to the isolated
nucleic acid molecule of claim 24 under standard hybridization
conditions.
26. The isolated nucleic acid molecule of claim 24, detectably
labeled.
27. The isolated nucleic acid molecule of claim 25, detectably
labeled.
28. The detectably labeled isolated nucleic acid molecule of either
of claims 26 or 27, wherein said detectable label comprises
radioactive isotopes, compounds which fluoresce, or enzymes.
29. The isolated nucleic acid molecule of either of claims 24 or
25, which encode a polypeptide comprising an amino acid sequence of
SEQ ID NO:16, or conserved variants thereof.
30. An isolated polypeptide comprising an amino acid sequence of
SEQ ID NO:16, or conserved variants thereof.
31. An antibody having a polypeptide of claim 30 as an
immunogen.
32. The antibody of claim 31, wherein said antibody is a monoclonal
antibody.
33. The antibody of claim 32, wherein said antibody is a polyclonal
antibody.
34. The antibody of claim 33, wherein said antibody is a chimeric
antibody.
35. The antibody of any of claims 31-34 detectably labeled.
36. The antibody of claim 35, wherein said detectable label
comprises an enzyme, a chemical which fluoresces, or a radioactive
isotope.
37. A cloning vector comprising an isolated nucleic acid residue of
either of claims 24 or 25, and an origin of replication.
38. The cloning vector of claim 37, wherein said cloning vector is
selected from the group consisting of E. coli, bacteriophages,
plasmids, and pUC plasmid derivatives.
39. The cloning vector of claim 37, wherein bacteriophages further
comprise lambda derivatives, plasmids further comprise pBR322
derivatives, and pUC plasmid derivatives further comprise pGEX
vectors, or pmal-c, pFLAG.
40. An expression vector comprising an isolated nucleic acid
molecule of either of claims 24 or 25, operatively associated with
a promoter.
41. The expression vector of claim 40, wherein said promoter is
selected from the group consisting of the immediate early promoters
of hCMV, early promoters of SV40, early promoters of adenovirus,
early promoters of vaccinia, early promoters of polyoma, late
promoters of SV40, late promoters of adenovirus, late promoters of
vaccinia, late promoters of polyoma, the lac the trp system, the
TAC system, the TRC system, the major operator and promoter regions
of phage lambda, control regions of fd coat protein,
3-phosphoglycerate kinase promoter, acid phosphatase promoter, and
promoters of yeast .alpha. mating factor.
42. A unicellular host transformed with an expression vector of
claim 40.
43. The unicellular host of claim 42, wherein said host is selected
from the group consisting of E. coli, Pseudonomas, Bacillus,
Strepomyces, yeast, CHO. R1.1, B-W, L-M, COS1, COS7, BSC1,BSC40,
BMT10 and St9 cells.
44. A method of producing a mutant cation channel protein
comprising an amino acid sequence of SEQ ID NO:16, or conserved
variants thereof, comprising the steps of: a) culturing a
unicellular host of claim 42 under conditions that provide for
expression of said mutant cation channel protein; and b) recovering
said mutant cation channel protein from said unicellular host.
45. A method of screening for compounds which selectively bind to a
potassium ion channel protein comprising: (a) complexing a
functional two-transmembrane-domain-type potassium ion channel
protein to a solid, support; (b) ontacting the complexed
protein/solid support with an aqueous solution said solution
containing a compound that is being screened for the ability to
selectively bind to the ion channel protein; (c) determining
whether the compound selectively binds to the ion channel protein
with the provisoes that the potassium ion channel protein is in the
form of a tetrameric protein; and, when the protein is mutated to
correspond to the agitoxin2 docking site of a Shaker K.sup.+
channel protein by substituting amino acid residues permitting the
mutated protein to bind agitoxin2, the protein will bind agitoxin 2
while bound to the solid support, said substituting of residues
being within the 36 amino acid domain defined by -25 to +5 of the
selectivity filter where the 0 residue is either the phenylalanine
or the tyrosine of the filter's signature sequence selected from
the group consisting of glycine-phenylalanine-glycine or
glycine-tyrosine-glycine.
46. A method of claim 45 wherein the solid supports are selected
from the group comprising: cobalt, insoluble polystyrene beads,
PVDF, and polyethylene, glycol.
47. A method of claim 45 wherein the two-transmembrane-domain-type
ion channel protein is a prokaryote.
48. A method of claim 45, wherein the two-transmembrane-domain-type
ion channel protein is from Steptomyces lividans.
49. A method of claim 45 wherein the two-transmembrane-domain-type
ion channel protein is KcsA.
50. A method of claim 45 wherein the two-transmembrane-domain-type
ion channel protein is mutated from a wild-type protein.
51. A method of claim 50 where the mutation is within the 36 amino
acid domain defined by -25 to +5 of the selectivity filter where
the 0 residue is either the phenylalanine or the tyrosine of the
filter's signature sequence selected from the group consisting of
glycine-phenylalanine-glyc- ine or glycine-tyrosine-glycine.
52. A method of claim 50 wherein the mutation deletes a subsequence
of the native amino acid sequence and replaces that the native with
a subseqeunce from the corresponding domain of a second and
different ion channel protein.
53. A method of claim 52 wherein the second ion channel protein is
from a eukaryote.
54. A method claim 45 wherein the aqueous solution comprises a
non-ionic detergent.
55. A non-natural and functional two-transmembrane-domain-type
potassium ion channel protein wherein the non-natural protein is
mutated in its amino acid sequence from a corresponding natural
protein whereby the mutation does not prevent the non-natural
protein from binding agitoxin2 when the non-natural protein is
further mutated to correspond to the agitoxin2 docking site of a
Shaker K.sup.+ channel protein said docking site created by
substituting amino acid residues selected from within the 36 amino
acid domain defined by -25 to +5 of the Shaker K.sup.- selectivity
filter where the 0 residue is either the phenylalanine or the
tyrosine of the filter's signature sequence selected from the group
consisting of glycine-phenylalanine-glycine or
glycine-tyrosine-glycine.
56. A non-natural protein of claim 55 wherein the protein binds to
a channel blocking protein toxin with at least a 10 fold increase
in affinity over the native ion channel.
57. A non-natural protein of claim 55 wherein the natural protein
is the KcsA from Streptomyces lividans.
58. A method of assessing the adequacy of the structural
conformation of a two-transmembrane-domain-type potassium ion
channel protein for high through put assays comprising the steps
of: (a) complexing a two-transmembrane-domain-type potassium ion
channel protein having a tetrameric form to a non-lipid solid
support under aqueous conditions; (b) contacting the complexed
two-transmembrane-domain-type potassium ion channel protein with a
substance known to bind to the two-transmembrane-domain-type
potassium ion channel protein when bound to lipid membrane wherein
the substance also modulates potassium ion flow in that channel
protein; and, (c) detecting the binding of the substance to the
complexed two-transmembrane-domain-type potassium ion channel
protein.
59. A method of claim 58 wherein the two-transmembrane-domain-type
potassium ion channel protein is mutated from a wild type
two-transmembrane-domain-type potassium ion channel protein by
substitution of amino acids.
60. A method of claim 58 wherein the contacting is done in the
presence of a non-ionic detergent.
60. A method of claim 58 where in the substance is a channel
blocker.
62. A method of claim 58 wherein the substance is a toxin.
63. A prescreening method for identifying potential modulators of
potassium ion channel function comprising: (a) binding a soluble
potassium ion channel protein to a solid support where the ion
channel has the scaffold of a two-transmembrane-domain-type
potassium ion channel and has a tetrameric confirmation; (b)
contacting the soluble potassium ion channel protein of step i with
a compound in an aqueous solution; and, (c) determining the binding
of the compound to the soluble potassium ion channel protein.
64. A method of claim 63 wherein the contacting takes place in the
presence of a detergent.
65. A method of claim 63 wherein the ion channel can pass potassium
ions when expressed in a cell.
66. A method of claim 63 which further comprises the contacting of
the compound to cell expressing a two-transmembrane-domain-type
potassium ion channel protein said cell cultured in an aqueous
media containing potassium and determining modulation of potassium
flow between the inside of the cell and the media.
67. A column comprising a solid support having bound thereto an ion
channel having the scaffold of a two-transmembrane-domain-type
potassium ion channel and having a tetrameric confirmation.
68. A column of claim 25 wherein the ion channel is a non-natural
and functional two-transmembrane-domain-type potassium ion channel
protein wherein the non-natural protein is mutated in its amino
acid sequence from a corresponding natural protein whereby the
mutation does not prevent the non-natural protein from binding
agiroxin2 when the non-natural protein is further mutated to
correspond to the agitoxin2 docking site of a Shaker K.sup.+
channel protein said docking site created by substituting amino
acid residues selected from within the 36 amino acid domain defined
by -25 to +5 of the Shaker K.sup.+ selectivity filter where the 0
residue is either the phenylalanine or the tyrosine of the filter's
signature sequence selected from the group consisting of
glycine-phenylalanine-glycine or glycine-tyrosine-glycine.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation in part of copending U.S.
application Ser. No. 09/054,347 filed on Apr. 2, 1998, which is a
continuation in part of copending U.S. application Ser. No.
09/045,529 filed on Mar. 20, 1998, wherein both U.S. Ser. Nos.
09/054,347 and 09/045,529 are hereby incorporated by reference in
their entireties.
FIELD OF INVENTION
[0003] The present invention relates to a crystal of a cation
channel protein, and methods of using such a crystal in screening
potential drugs and therapeutic agents for use in treating
conditions related to the function of such channels in vivo.
BACKGROUND OF INVENTION
[0004] Although numerous types of channel proteins are known, the
main types of ion channel proteins are characterized by the method
employed to open or close the channel protein to either permit or
prevent specific ions from permeating the channel protein and
crossing a lipid bilayer cellular membrane. One important type of
channel protein is the voltage-gated channel protein, which is
opened or closed (gated) in response to changes in electrical
potential across the cell membrane. Another type of ion channel
protein are celled mechanically gated channel proteins, for which a
mechanical stress on the protein opens or closes the channel. Still
another type is called a ligand-gated channel, which opens or
closes depending on whether a particular ligand is bound the
protein. The ligand can be either an extracellular moiety, such as
a neurotransmitter, or an intracellular moiety, such as an ion or
nucleotide.
[0005] Presently, over 100 types of ion channel proteins have been
described, with additional ones being discovered. Basically, all
ion channels have the same basic structure regarding the permeation
of their specific ion, although different gating mechanisms (as
described above) can be used. One of the most common types of
channel proteins, found in the membrane of almost all animal cells,
permits the specific permeation of potassium ions (K.sup.+) across
a cell membrane. In particular, potassium ions permeate rapidly
across cell membranes through K.sup.+ channel proteins (up to
10.sup.8 ions per second). Moreover, potassium channel proteins
have the ability to distinguish among potassium ions, and other
small alkali metal ions, such as Li.sup.+ or Na.sup.+ with great
fidelity. In particular, potassium ions are at least ten thousand
times more permeant than sodium ions. In light of the fact that
both potassium and sodium ions are generally spherical in shape,
with radii of about 1.33 .ANG. and 0.95 .ANG. respectively, such
selectivity is remarkable.
[0006] Broadly, potassium channel proteins comprise four (usually
identical) subunits. Presently two major types of subunits are
known. One type of subunit contains six long hydrophobic segments
(presumably membrane-spanning), while the other type contains two
hydrophobic segments. Regardless of what type of subunits are used,
potassium channel proteins are highly selective for potassium ions,
as explained above.
[0007] Among their many functions, potassium channel proteins
control the pace of the heart, regulate the secretion of hormones
such as insulin into the blood stream, generate electrical impulses
underlying information transfer in the nervous system, and control
airway and vascular smooth muscle tone. Thus, potassium channels
participate in cellular control processes that are abnormal, such
as cardiac arrhythmia, diabetes mellitus, seizure disorder, asthma
and hypertension, to name only a few.
[0008] Although potassium channel proteins are involved in such a
wide variety of homeostatic functions, few drugs or therapeutic
agents are available that act on potassium channel proteins to
treat abnormal processes. A reason for a lack of presently
available drugs that act on potassium channel proteins is that
isolated potassium channel proteins are not available in great
abundance, mainly because an animal cell requires only a very
limited number of such channel proteins in order to function.
Consequently, it has been very difficult to isolate and purify
potassium channel proteins, reducing the amount of drug screening
efforts in search of potassium channel protein acting drugs.
[0009] Hence, what is needed is accurate information regarding the
structure of cation channel proteins so that drugs or therapeutic
agents having an appropriate structure to potentially interact with
a cation channel protein can be selected.
[0010] What is also needed is an ability to overcome the physical
limitations regarding the isolation and purification of cation
channel proteins, particularly potassium ion channel proteins.
[0011] What is also needed is a reliable method of utilizing cation
channel proteins in screening potential drugs or agents for their
possible use in treating conditions related to the function of
cation channel proteins in vivo.
[0012] What is also needed are novel methods of using accurate
information regarding the structure of cation channel proteins so
that drugs or therapeutic agents can be screened for potential
activity in treating abnormal control processes of the body.
[0013] The citation of any reference herein should not be construed
as an admission that such reference is available as "Prior Art" to
the instant application.
SUMMARY OF THE INVENTION
[0014] There is provided, in accordance with the present invention,
a method of preparing a functional cation channel protein for use
in an assay for screening potential drugs or other agents which
interact with a cation channel protein, which permits the screening
of potential drugs or agents that may be used as potential
therapeutic agents in treating conditions related to the function
of cation channel proteins in vivo.
[0015] More specifically, the method comprising the steps of
providing a functional cation channel protein, conjugating the
functional cation channel protein to a solid phase resin,
contacting the potential drug or agent to the functional cation
channel protein conjugated to the solid phase resin, removing the
functional cation channel protein from the solid phase resin, and
determining whether the potential drug or agent is bound to the
cation channel protein.
[0016] In particular, the present invention extends to a method of
preparing a functional cation channel protein for use in an assay
as described above, wherein the providing step of the method
comprises expressing an isolated nucleic acid molecule encoding the
cation channel protein in a unicellular host, such that the cation
channel protein is present in the cell membrane of the unicellular
host, lysing the unicellular host in a solubilizing solution so
that the cation channel protein is solubilized in the solution, and
extracting the cation channel protein from the solubilizing
solution with a detergent. In a preferred embodiment, the isolated
nucleic acid molecule comprises a DNA sequence of SEQ ID NO:17, or
degenerate variants thereof, or an isolated nucleic acid molecule
hybridizable under standard hybridization conditions to an isolated
nucleic acid molecule having a DNA sequence of SEQ ID NO:17, or
degenerate variants thereof.
[0017] Numerous methods of lysing a unicellular host are known to
the skilled artisan, and have applications in the present
invention. In a preferred embodiment, lysing the unicellular host
in a solubilizing solution comprises sonicating the unicellular
host in a protein solubilizing solution comprising 50 mM Tris
buffer, 100 mM KCl, 10 mM MgSO.sub.4, 25 mg DNAse 1, 250 mM
sucrose, pepstatin, leupeptin, and PMSF, pH 7.5.
[0018] Furthermore, a skilled artisan is aware of numerous
detergents that can be used to extract an integral membrane bound
protein, such as a cation channel protein, from a solubilizing
solution described above. Examples of such detergents include SDS,
Triton-100, Tween 20, Tween 80, glycerol, or decylmaltoside, to
name only a few. Preferably, 40 mM decylmaltoside is used to
extract the cation channel protein from the solubilizing
solution.
[0019] Moreover, numerous solid phase resins to which a functional
cation channel protein can be conjugated have applications in a
method of preparing a functional cation channel protein for use in
an assay, as described above. For example, a solid phase resin
comprising insoluble polystyrene beads, PVDF, polyethylene glycol,
or a cobalt resin, to name only a few have application in the
present invention. Preferably, a cation channel protein is
conjugated to a cobalt resin at a protein to resin ratio that
allows for saturation of the resin with the cation channel protein.
Moreover, after conjugation, the cobalt resin is preferably used to
line a column having a volume of about 1 ml.
[0020] After the cation channel protein is conjugated to a solid
phase resin, it is contacted with a potential drug or agent, which
is given an opportunity to bind to the cation channel protein.
[0021] Subsequently, the cation channel protein is removed from the
solid phase resin, and analyzed to determine whether the potential
drug or agent is bound thereto. Numerous methods of removing the
cation channel protein from the solid phase resin are known to
those of ordinary skill in the art. In a preferred embodiment,
wherein the solid phase resin is a cobalt resin, the removing step
comprises contacting the cation channel protein conjugated to the
solid phase resin with an imidazole solution. This solution readily
cleaves any bonds conjugating the cation channel protein to the
resin, so that the protein can removed from the resin, and
collected for further analysis to determine whether the potential
drug or agent is bound to the protein.
[0022] After the cation channel protein has been removed from the
resin, it must be examined to determine whether the potential drug
or agent is bound thereto. If bound, the drug or agent may have
uses involved in modulation of the function of a cation channel
protein in vivo, including uses as a therapeutic agent in treating
conditions related to the function of cation channel proteins.
Numerous analytical methods are presently available to the skilled
artisan for determining whether the potential ligand is bound to
the cation channel protein. Examples of such methods include
molecular weight analysis with SDS-PAGE, immunoassays using an
antibody to the drug or agent. HPLC, or mass spectrometry.
[0023] Furthermore, the present invention extends to a method of
using a functional cation channel protein in an assay for screening
potential drugs or agents which interact with the cation channel
protein, wherein the potential drug or agent is a member of a
library of compounds, which is contacted to the cation channel
protein. Examples of libraries having applications in the present
invention include, but are not limited to, a mixture of compounds,
or a combinatorial library of compounds. Furthermore, examples of
combinatorial compounds having applications in the present
invention include, but are not limited to, a phage display library,
or a synthetic peptide library, to name only a few.
[0024] In another embodiment, the present invention extends to a
prokaryotic cation channel protein mutated to mimic a functional
eukaryotic cation channel protein. More specifically. Applicant has
discovered that all cation channel proteins from all organisms have
a conserved structure. Hence, placing mutations in a potassium
channel from a prokaryotic organism, for example, can permit the
use of the prokaryotic cation channel protein in screening assays
for drugs that may interact with specific eukaryotic cation channel
proteins. For example, a prokaryotic potassium channel protein can
be mutated to mimic a cardiac potassium channel protein, a venous
potassium channel protein, or a neuro potassium channel of a human,
to name only a few.
[0025] Hence, pursuant to the present invention, a prokaryotic
potassium channel protein, a prokaryotic sodium channel protein, or
a prokaryotic calcium channel protein can be mutated to mimic a
eukaryotic cation channel protein.
[0026] Examples of prokaryotic organisms from which a prokaryotic
cation channel protein can be taken and mutated to mimic a
eukaryotic cation channel protein include E. coli, Streptomyces
lividans, Clostridium acetobutylicum, or Staphylcoccus aureus, to
name only a few. Furthermore, such prokaryotic cation channel
proteins can comprise an amino acid sequence of SEQ ID Nos: 1, 2,
3, or 7, or conserved variants thereof. In a preferred embodiment,
the prokaryotic cation channel protein mutated to mimic a
eukaryotic cation channel protein, wherein the prokaryotic cation
channel protein is a potassium channel protein from Streptomyces
lividans.
[0027] Furthermore, pursuant to the present invention, a
prokaryotic cation channel protein can be mutated to mimic
eukaryotic potassium channel protein, a eukaryotic sodium channel
protein, or a eukaryotic calcium channel protein. Preferably, the
eukaryotic cation channel protein is produced endogenously in a
eukaryotic organism, such as an insect or a mammal, for example.
More specifically, pursuant to the present invention, a prokaryotic
cation channel protein is mutated to mimic a eukaryotic cation
channel protein endogenously produced in a eukaryotic organism
selected from the group consisting of Drosophila melanogaster, Homo
sapiens, C. elegans, Mus musculus, Arabidopsis thaliana, paramecium
tetraaurelia or Rattus novegicus, or having an amino acid sequence
comprising SEQ ID Nos: 4, 5, 6, 8, 9, 10, 11, 12, 13, or 14, or
conserved variants thereof.
[0028] In a preferred embodiment, the present invention extends to
a prokaryotic cation channel protein mutated to mimic a functional
eukaryotic channel protein, wherein the prokaryotic cation channel
protein is a potassium channel protein from Streptomyces lividans
comprising an amino acid sequence of SEQ ID NO:1 or degenerate
variants thereof, and the eukaryotic cation channel is a potassium
channel protein comprising an amino acid sequence of SEQ ID NO:4 or
conserved variants thereof. As a result, the mutated prokaryotic
channel protein comprises an amino acid sequence of SEQ ID NO:16,
or conserved variants thereof, which is encoded by an isolated
nucleic acid molecule comprising a DNA sequence of SEQ ID NO:17, or
degenerate variants thereof.
[0029] In another embodiment, the present invention extends to a
method of using a crystal of a cation channel protein, as described
herein, in an assay system for screening drugs and other agents for
their ability to modulate the function of a cation channel protein,
comprising the steps of initially selecting a potential drug or
agent by performing rational drug design with the three-dimensional
structure determined for a crystal of the present invention,
wherein the selecting is performed in conjunction with computer
modeling. After potential drugs or agents have been selected, a
cation channel protein is contacted with the potential drug or
agent. If the drug or therapeutic agent has potential use for
modulating the function of a cation channel protein, a change in
the function of the cation channel after contact with the agent,
relative to the function of a similar cation channel protein not
contacted with the agent, or the function of the same cation
channel protein prior to contact with the agent. Hence, the change
in function is indicative of the ability of the drug or agent to
modulate the function of a cation channel protein.
[0030] Furthermore, the present invention extends to extends to a
method of using a crystal of a cation channel protein as described
herein, in an assay system for screening drugs and other agents for
their ability to modulate the function of a cation channel protein,
wherein the crystal comprises a Na.sup.+ channel protein, a K.sup.+
channel protein, or a Ca.sup.2+ channel protein.
[0031] The present invention further extends to a method of using a
crystal of a cation channel protein in an assay for screening drugs
or other agents for their ability to modulate the function of a
cation channel protein, wherein the crystal of the cation channel
protein comprises an amino acid sequence of:
[0032] residues 23 to 119 of SEQ ID NO:1 (Streptomyces
lividans);
[0033] residues 61 to 119 of SEQ ID NO:2 (E. coli);
[0034] residues 61 to 119 of SEQ ID NO:3 (Clostridium
acetobutylicum);
[0035] residues 61 to 119 of SEQ ID NO:4 (Drosophila
melanogaster);
[0036] residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
[0037] residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
[0038] residues 61 to 119 of SEQ ID NO:7 (Paramecium
tetraaurelia);
[0039] residues 61 to 119 of SEQ ID NO:8 (C. elegans);
[0040] residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
[0041] residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
[0042] residues 61 to 119 of SEQ ID NO:11 (Arabidopsis
thaliana);
[0043] residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
[0044] residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus);
or
[0045] residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
[0046] or conserved variants thereof.
[0047] In a preferred embodiment of a method of using a crystal of
a cation channel protein in an assay for screening drugs or other
agents for their ability to modulate the function of a cation
channel protein, the crystal comprises a potassium channel protein,
comprising amino acid residues 23 to 119 of SEQ ID NO:1, a space
grouping of C2, and a unit cell of dimensions of a=128.8 .ANG.,
b=68.9 .ANG., c=112.0 .ANG., and .beta.=124.6.degree..
[0048] Moreover, it is important to note that a drug's or agent's
ability to modulate the function of a cation channel protein
includes, but is not limited to, increasing or decreasing the
cation channel protein's permeability to the specific cation
relative the permeability of the same or a similar not contacted
with the drug or agent, or the same cation channel protein prior to
contact with the drug or agent.
[0049] In a further embodiment, the present invention extends to a
method of using a crystal of a cation channel protein, as set forth
herein, in an assay system for screening drugs and other agents for
their ability to treat conditions related to the function of cation
channel proteins in vivo, and particularly in abnormal cellular
control processes related to the functioning of cation channel
protein. Such a method comprises the initial step of selecting a
potential drug or other agent by performing rational drug design
with the three-dimensional structure determined for a crystal of
the invention, wherein the selecting is performed in conjunction
with computer modeling. After potential drugs or therapeutic agents
are selected, a cation channel protein is contacted with the
potential drug or agent. If an interaction of the potential drug or
other agent with the cation channel is detected, it is indicative
of the potential use of the drug or agent to treat conditions
related the function of cation channel proteins in vivo. Examples
of such conditions include, but are not limited to, cardiac
arrhythmia, diabetes mellitus, seizure disorder, asthma or
hypertension, to name only a few.
[0050] Furthermore, a crystal of a cation channel protein used in
the method for screening drugs or agents for their ability to
interact with a cation channel comprises an Na.sup.+ channel
protein. K.sup.+ channel protein, or Ca.sup.2+ channel protein.
Hence, the method of the present invention can be used to screen
drugs or agents capable of treating conditions related to the
function of such channels.
[0051] Moreover, the present invention extends to a crystal used in
the method for screening drugs or agents for their ability to
interact with a cation channel protein comprising an amino acid
sequence of:
[0052] residues 23 to 119 of SEQ ID NO:1 (Streptomyces
lividans);
[0053] residues 61 to 119 of SEQ ID NO:2 (E. coli);
[0054] residues 61 to 119 of SEQ ID NO:3 (Clostridium
acetobutylicum);
[0055] residues 61 to 119 of SEQ ID NO:4 (Drosophila
melanogaster);
[0056] residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
[0057] residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
[0058] residues 61 to 119 of SEQ ID NO:7 (Paramecium
tetraaurelia);
[0059] residues 61 to 119 of SEQ ID NO:8 (C. elegans);
[0060] residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
[0061] residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
[0062] residues 61 to 119 of SEQ ID NO:11 (Arabidopsis
thaliana);
[0063] residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
[0064] residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus);
or
[0065] residues 61 to 119 of SEQ ID NO:14 (Homo sapiens),
[0066] or conserved variants thereof.
[0067] In a preferred embodiment, a crystal used in a method for
screening drugs or agents for their ability to interact with a
cation channel, comprises amino acid residues 23 to 119 of SEQ ID
NO:1, has a space grouping of C2, and a unit cell of dimensions of
a=128.8 .ANG., b=68.9 .ANG., c=112.0 .ANG., and
.beta.=124.6.degree..
[0068] In yet another embodiment, the present invention extends to
a method of using a crystal of a cation channel protein described
herein, in an assay system for screening drugs and other agents for
their ability to permeate through a cation channel protein,
comprising an initial step of selecting a potential drug or other
agent by performing rational drug design with the three-dimensional
structure determined for the crystal, wherein the selecting of the
potential drug or agent is performed in conjunction with computer
modeling. After a potential drug or agent has been selected, a
cation channel protein can be prepared for use in the assay. For
example, preparing the cation channel protein can include isolating
the cation channel protein from the membrane of a cell, and then
inserting the cation channel protein into a membrane having a first
and second side which is impermeable to the potential drug or
agent. As a result, the cation channel protein traverses the
membrane, such that the extracellular portion of the cation channel
protein is located on the first side of the membrane, and the
intracellular portion of the cation channel protein is located on
the second side of the membrane. The extracellular portion of the
cation channel membrane can then be contacted with the potential
drug or agent. The presence of the drug or agent in the second side
of the membrane is indicative of the drug's or agent's potential to
permeate the cation channel protein, and the drug or agent is
selected based on its ability to permeate the cation channel
protein.
[0069] In addition, a crystal used in a method for screening drugs
or agents for their ability to permeate a cation channel can
comprise a Na.sup.+ channel protein, a K.sup.+ protein channel, or
a Ca.sup.2+ protein channel.
[0070] Furthermore, the present invention extends to the use of a
crystal in an assay system for screening drugs and other agents for
their ability to permeate through a cation channel protein, wherein
the crystal comprises an amino acid sequence of:
[0071] residues 23 to 119 of SEQ ID NO:1 (Streptomyces
lividans);
[0072] residues 61 to 119 of SEQ ID NO:2 (E. coli);
[0073] residues 61 to 119 of SEQ ID NO:3 (Clostridium
acetobutylicum);
[0074] residues 61 to 119 of SEQ ID NO:4 (Drosophila
melanogaster);
[0075] residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
[0076] residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
[0077] residues 61 to 119 of SEQ ID NO:7 (Paramecium
tetraaurelia);
[0078] residues 61 to 119 of SEQ ID NO:8 (C. elegans);
[0079] residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
[0080] residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
[0081] residues 61 to 119 of SEQ ID NO:11 (Arabidopsis
thaliana);
[0082] residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
[0083] residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus);
or
[0084] residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
[0085] or conserved variants thereof.
[0086] In a preferred embodiment, the crystal used in an assay
system of the present invention for screening drugs and other
agents for their ability to permeate through a cation channel
protein comprises amino acid residues 23 to 119 of SEQ ID NO:1, has
a space grouping of C2, and a unit cell of dimensions of a=128.8
.ANG., b=68.9 .ANG., c=112.0 .ANG., and .beta.=124.6.degree..
[0087] Naturally, the present invention extends to an isolated
nucleic acid molecule encoding a mutant K.sup.+ channel protein,
comprising a DNA sequence of SEQ ID NO:17, or degenerate variants
thereof.
[0088] Furthermore, the present invention extends to an isolated
nucleic acid molecule hybridizable to an isolated nucleic acid
molecule encoding a mutant K.sup.+ channel protein under standard
hybridization conditions.
[0089] Moreover, isolated nucleic acid molecules of the present
invention, and described above, can be detectably labeled. Examples
of detectable labels having applications in the present invention
include, but are not limited to, radioactive isotopes, compounds
which fluoresce, or enzymes.
[0090] The present invention further extends to an isolated nucleic
acid molecule encoding a mutant K.sup.+ channel protein, or
degenerate variants thereof, comprising an amino acid sequence of
SEQ ID NO:16, or conserved variants thereof.
[0091] In addition, the present invention extends to an isolated
nucleic acid molecule encoding a polypeptide comprising an amino
acid sequence of SEQ ID NO:16, or conserved variants thereof,
wherein the isolated nucleic acid molecule is hybridizable under
standard hybridization conditions to an isolated nucleic acid
molecule encoding a K.sup.+ channel protein, or degenerate variants
thereof.
[0092] Furthermore, the present invention extends to a mutant
cation channel protein comprising an amino acid sequence of SEQ ID
NO:16, or conserved variants thereof.
[0093] In addition, the present invention extends to a cloning
vector comprising an isolated nucleic acid molecule, or degenerate
variants thereof, which encodes a mutant cation channel protein of
the present invention, or conserved variants thereof, and an origin
of replication. The present invention also extends to a cloning
vector comprising an origin of replication and an isolated nucleic
acid molecule hybridizable under standard hybridization conditions
to an isolated nucleic acid molecule, or degenerate variants
thereof, which encodes a mutant cation channel protein of the
present invention.
[0094] Examples of cloning vectors having applications in the
present invention include, but are not limited to, E. coli,
bacteriophages, plasmids, and pUC plasmid derivatives. More
specifically, examples of bacteriophages, plasmids, and pUC plasmid
derivatives having applications herein comprise lambda derivatives,
pBR322 derivatives, and pGEX vectors, or pmal-c, pFLAG,
respectively.
[0095] Naturally, the present invention extends to an expression
vector comprising an isolated nucleic acid molecule comprising a
DNA sequence of SEQ ID NO:17, or degenerate variants thereof,
operatively associated with a promoter. In another embodiment, an
expression vector comprises an isolated nucleic acid molecule
hybridizable under standard hybridization conditions to an isolated
nucleic acid comprising a DNA sequence of SEQ ID NO:17, or
degenerate variants thereof, operatively associated with a
promoter.
[0096] Examples of promoters having applications in expression
vectors of the present invention comprise immediate early promoters
of hCMV, early promoters of SV40, early promoters of adenovirus,
early promoters of vaccinia, early promoters of polyoma, late
promoters of SV40, late promoters of adenovirus, late promoters of
vaccinia, late promoters of polyoma, the lac the trp system, the
TAC system, the TRC system, the major operator and promoter regions
of phage lambda, control regions of fd coat protein,
3-phosphoglycerate kinase promoter, acid phosphatase promoter, or
promoters of yeast .alpha. mating factor.
[0097] Furthermore, the present invention extends to a unicellular
host transformed or transfected with an expression vector of the
present invention. Such a unicellular host can be selected from the
group consisting of E. coli, Pseudonomas, Bacillus, Strepomyces,
yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 and Sf9
cells.
[0098] Naturally, the present invention extends to a method of
producing a mutant cation channel protein, comprising the steps of
culturing a unicellular host transformed or transfected with an
expression vector of the present invention under conditions that
provide for expression of the isolated nucleic acid molecule of the
expression vector and recovering the mutant cation channel protein
from the unicellular host. Moreover, such a method can also be used
wherein the expression vector comprises a an isolated nucleic acid
molecule hybridizable under standard hybridization conditions to an
isolated nucleic acid molecule comprising a DNA sequence of SEQ ID
NO:17, or degenerate variants thereof, operatively associated with
a promoter.
[0099] The present invention further extends to an antibody having
a mutant cation channel protein of the present invention as an
immunogen. More specifically, an antibody of the present invention
can be a monoclonal antibody, a polyclonal antibody, or a chimeric
antibody. Furthermore, an antibody of the present invention can be
detectably labeled. Examples of detectable labels having
applications in the present invention include, but are not limited
to, an enzyme, a chemical which fluoresces, or a radioactive
isotope.
[0100] Broadly, the present invention extends to a crystal of a
cation channel protein having a central pore, which is found
natively in a lipid bilayer membrane of an animal cell, such that
the central pore communicates with extracellular matrix and
cellular cytosol, wherein the crystal effectively diffracts x-rays
to a resolution of greater than 3.2 angstroms.
[0101] Moreover, the present invention extends to a crystal of a
cation channel protein as described above, wherein the cation
channel protein comprises a first layer of aromatic amino acid
residues positioned to extend into the lipid bilayer membrane
proximate to the interface an extracellular matrix and lipid
bilayer membrane, a second layer of aromatic amino acid residues
positioned to extend into the lipid bilayer membrane proximate to
the interface of cellular cytosol and said lipid bilayer membrane,
a tetramer of four identical transmembrane subunits, and a central
pore formed by the four identical transmembrane subunits.
[0102] Moreover, the present invention extends to a crystal of a
cation channel protein described above, wherein each transmembrane,
subunit comprises an inner transmembrane alpha-helix which has a
kink therein, an outer transmembrane alpha-helix, and a pore
alpha-helix, wherein each subunit is inserted into the tetramer of
the cation channel protein so that the outer transmembrane helix of
each subunit contacts the first and second layers of aromatic amino
acid residues described above, and abuts the lipid bilayer
membrane. Moreover, the inner transmembrane helix of each subunit
abuts the central pore of the cation channel protein, contacts the
first and second layers of aromatic amino acid residues, is tilted
by about 25.degree. with respect to the normal of the lipid bilayer
membrane, and is packed against inner transmembrane alpha helices
of other transmembrane subunits at the second layer of aromatic
amino acid residues forming a bundle of helices at the second
layer. The pore alpha-helix of each subunit is located at the first
layer of said aromatic amino acid residues, and positioned between
inner transmembrane alpha-helices of adjacent subunits, and are
directed, in an amino to carboxyl sense, towards the center of the
central pore.
[0103] Furthermore, the present invention extends to a crystal
described above, comprising a cation channel protein having a
central pore, which comprises a pore region located at the first
layer of aromatic amino acid residues, and connected to the inner
and outer transmembrane alpha-helices of said subunits. More
particularly, the pore region comprises about 25-45 amino acid
residues, a turret connected to the pore alpha-helix and the outer
alpha-helix, wherein turret is located at the interface of said
extracellular matrix and the lipid bilayer membrane. The pore
region further comprises an ion selectivity filter connected to the
pore alpha-helix and the inner transmembrane alpha-helix of each
subunit. The ion selectivity filter extends into the central pore
of the cation channel protein, and comprises a signature amino acid
residue sequence having main chain atoms which create a stack of
sequential oxygen atoms along the selectivity filter that extend
into the central pore, and amino acid residues having side chains
that interact with the pore helix. It is the signature sequence
which enables a cation channel protein to discriminate among the
cation intended to permeate the protein, and other cations, so that
only the cation intended to permeate the channel protein is
permitted to permeate.
[0104] The central pore further comprises a tunnel into the lipid
bilayer membrane which communicates with the cellular cytosol, and
a cavity located within the lipid bilayer membrane between the pore
region and the tunnel, and connected to the them, such that the
central pore crosses the membrane.
[0105] Furthermore, the structure of all ion channel proteins share
common features, which are set forth in the crystal of a cation
channel protein described above. Consequently, the present
invention extends to a crystal of a cation channel protein having a
central pore and structure, as described above, wherein the cation
is selected from the group consisting of: Na.sup.31, K.sup.+, and
Ca.sup.2-. Hence, the present invention extends to crystals of
potassium channel proteins, sodium channel proteins, and calcium
ion channels, to name only a few. In a preferred embodiment, the
crystal of a cation channel protein comprises a crystal of a
potassium ion channel protein.
[0106] In addition, a crystal of a cation channel protein of a
present invention comprises the amino acid sequence of any
presently known, or subsequently discovered cation protein channel.
Consequently, the present invention extends to a crystal of a
cation channel protein having a central pore, which is found
natively in a lipid bilayer membrane of an animal cell, such that
the central pore communicates with extracellular matrix and
cellular cytosol, wherein the crystal comprises an amino acid
sequence of:
[0107] residues 23 to 119 of SEQ ID NO:1 (Streptomyces
lividans):
[0108] residues 61 to 119 of SEQ ID NO:2 (E. coli);
[0109] residues 61 to 119 of SEQ ID NO:3 (Clostridium
acetobutylicum):
[0110] residues 61 to 119 of SEQ ID NO:4 (Drosophila
melanogaster):
[0111] residues 61 to 119 of SEQ ID NO:5 (Homo sapiens):
[0112] residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
[0113] residues 61 to 119 of SEQ ID NO:7 (Paramecium
tetraaurelia);
[0114] residues 61 to 119 of SEQ ID NO:8 (C. elegans);
[0115] residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
[0116] residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
[0117] residues 61 to 119 of SEQ ID NO:11 (Arabidopsis
thaliana);
[0118] residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
[0119] residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or
residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
[0120] or conserved variants thereof.
[0121] In a preferred embodiment, a crystal of the present
invention having a central pore, which is found natively in a lipid
bilayer membrane of an animal cell, such that the central pore
communicates with extracellular matrix and cellular cytosol,
comprises an amino sequence of amino acid residues 23 to 119 of SEQ
ID NO:1, has a space grouping of C2, a unit cell of dimensions of
a=128.8 .ANG., b=68.9 .ANG., c=112.0 .ANG., and
.beta.=124.6.degree..
[0122] Furthermore, the present invention extends to a crystal of a
cation channel protein having a central pore, which is found
natively in a lipid bilayer membrane of an animal cell, such that
the central pore communicates with extracellular matrix and
cellular cytosol, wherein the channel protein comprises a signature
sequence comprising:
[0123] Thr-Val-Gly-Tyr-Gly-Asp (SEQ ID NO:15).
[0124] In another embodiment, the present invention extends to a
method for growing a crystal of a cation channel protein having a
central pore, which is found natively in a lipid bilayer membrane
of an animal cell, such that the central pore communicates with
extracellular matrix and cellular cytosol, by sitting-drop vapor
diffusion. Such a method of the present invention comprises the
steps of providing the cation channel protein, removing a
predetermined number of carboxy terminal amino acid residues from
the cation channel protein to form a truncated cation channel
protein, dissolving the truncated cation channel protein in a
protein solubilizing solution, such that the concentration of
dissolved truncated channel protein is about 5 to about 10 mg/ml,
and mixing equal volumes of protein solubilizing solution with
reservoir mixture at 20.degree. C. Preferably, the reservoir
mixture comprises 200 mM CaCl.sub.2, 100 mM Hepes, 48% PEG 400, pH
7.5, and the protein solution comprises (150 mM KCl, 50 mM Tris, 2
mM DTT, pH 7.5).
[0125] Moreover, the present invention extends to a method of
growing a crystal of a cation channel protein as described above,
wherein a crystal can be grown comprising any kind of cation
channel protein. In particular, the present invention can be used
to grow crystals of potassium channel proteins, sodium channel
proteins, or calcium channel proteins, to name only a few.
[0126] Furthermore, the present invention extends to a method of
growing a crystal of a cation channel protein, as described herein,
wherein the crystal comprises an amino acid sequence of:
[0127] residues 23 to 119 of SEQ ID NO:1 (Streptomyces
lividans);
[0128] residues 61 to 119 of SEQ ID NO:2 (E. coli);
[0129] residues 61 to 119 of SEQ ID NO:3 (Clostridium
acetobutylicum);
[0130] residues 61 to 119 of SEQ ID NO:4 (Drosophila
melanogaster);
[0131] residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
[0132] residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
[0133] residues 61 to 119 of SEQ ID NO:7 (Paramecium
tetraaurelia);
[0134] residues 61 to 119 of SEQ ID NO:8 (C. elegans);
[0135] residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
[0136] residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
[0137] residues 61 to 119 of SEQ ID NO:11 (Arabidopsis
thaliana);
[0138] residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
[0139] residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus);
or
[0140] residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
[0141] or conserved variants thereof.
[0142] Numerous methods can be used to provide a cation channel
protein, for use in growing a crystal. For example, traditional
purification techniques such as gel filtration, HPLC, or
immunoprecipitation can be used to purify cation channel proteins
from the membranes of numerous cells. In another method,
recombinant DNA technology can be used, wherein a nucleic acid
molecule encoding the particular cation channel protein can be
inserted into an expression vector, which is then used to transfect
a unicellular host. After transfection, the host can be induced to
express the nucleic acid molecule, and the particular cation
channel protein can be harvested from the membrane of the
unicellular host.
[0143] Moreover, numerous methods are available for removing a
predetermined number of carboxy terminal amino acid residues from
the cation channel protein to form a truncated cation channel
protein. For example, chemical techniques can be used to cleave a
peptide bond between two particular amino acid residues in the
carboxy terminus of the cation channel protein. In another
embodiment, the cation channel protein can be contacted with a
proteolytic enzyme, so that the predetermined number of residues
from the carboxy terminus are enzymatically removed from the
carboxy terminus of the cation channel protein, forming a truncated
cation channel protein. In a preferred embodiment, the cation
channel protein comprises a potassium channel protein having an
amino acid sequence of SEQ ID NO:1, which is contacted with
chymotripsin so that residues 1-22 are removed, forming a truncated
potassium channel protein comprising an amino acid sequence of
residues 23-119 of SEQ ID NO:1.
[0144] This invention further provides for a prescreening method
for identifying potential modulators of potassium ion channel
function comprising the steps of: (i) binding a soluble potassium
ion channel protein to a solid support where the ion channel has
the scaffold of a two-transmembrane-domain-type potassium ion
channel and has a tetrameric confirmation; (ii) contacting the
soluble potassium ion channel protein of step i with a compound in
an aqueous solution; and, (iii) determining the binding of the
compound to the soluble potassium ion channel protein.
[0145] In addition, this invention provides for a method of
screening for compounds which selectively bind to a potassium ion
channel protein comprising: (i) complexing a functional
two-transmembrane-domain-type potassium ion channel protein to a
solid support; (ii) contacting the complexed protein/solid support
with an aqueous solution said solution containing a compound that
is being screened for the ability to selectively bind to the ion
channel protein; and, (iii) determining whether the compound
selectively binds to the ion channel protein with the provisoes
that the potassium ion channel protein is in the form of a
tetrameric protein; and, when the protein is mutated to correspond
to the agitoxin2 docking site of a Shaker K.sup.+ channel protein
by substituting amino acid residues permitting the mutated protein
to bind agitoxin2, the protein will bind agitoxin2 while bound to
the solid support, said substituting of residues being within the
36 amino acid domain defined by -25 to +5 of the selectivity filter
where the 0 residue is either the phenylalanine or the tyrosine of
the filter's signature sequence selected from the group consisting
of glycine-phenylalanine-glyc- ine or glycine-tyrosine-glycine.
[0146] In a particular embodiment of the method for screening for
compounds as described, above, a prokaryote
two-transmembrane-domain-type ion channel protein is used, such as
from Steptomyces lividans especially, the KcsA channel. The
channels can be either wild-type or mutated from a wild-type
protein. One mutation is confined to the 36 amino acid domain
defined by -25 to +5 of the selectivity filter where the 0 residue
is either the phenylalanine or the tyrosine of the filter's
signature sequence selected from the group consisting of
glycine-phenylalanine-glycine or glycine-tyrosine-glycine. The
method of this invention includes the use of channel mutations
where the protein alteration involves the deletion of a subsequence
of the native amino acid sequence and replacement of that native
sequence with a subseqeunce from the corresponding domain of a
second and different ion channel protein. The second ion channel
protein can be from either a prokaryote or an eukaryote cell.
[0147] The methods described above may be conducted using an
aqueous solution comprises a nonionic detergent.
[0148] In addition to the methods of this invention, the invention
further comprises a column having the channel proteins of this
invention bound thereto. The proteins are as described herein.
[0149] The invention also provides for a non-natural and functional
two-transmembrane-domain-type potassium ion channel protein wherein
the non-natural protein is mutated in its amino acid sequence from
a corresponding natural protein whereby the mutation does not
prevent the non-natural protein from binding agitoxin2 when the
non-natural protein is further mutated to correspond to the
agitoxin2 docking site of a Shaker K.sup.+ channel protein said
docking site created by substituting amino acid residues selected
from within the 36 amino acid domain defined by -25 to +5 of the
Shaker K.sup.+ selectivity filter where the 0 residue is either the
phenylalanine or the tyrosine of the filter's signature sequence
selected from the group consisting of glycine-phenylalanine-glyc-
ine or glycine-tyrosine-glycine. It is preferred that the
non-natural protein so modified will binds to a channel blocking
protein toxin with at least a 10 fold increase in affinity over the
native ion channel. The non-natural proteins include those
mutations described above for use on a solid support to identify
modulators of potassium ion function.
[0150] The invention further provides for a means to assess the
adequacy of the structural conformation of a
two-transmembrane-domain-type potassium ion channel protein for
high through put assays comprising the steps of: (i) complexing a
two-transmembrane-domain-type potassium ion channel protein having
a tetrameric form to a non-lipid solid support under aqueous
conditions; (ii) contacting the complexed
two-transmembrane-domain-type potassium ion channel protein with a
substance known to bind to the two-transmembrane-domain-type
potassium ion channel protein when bound to lipid membrane wherein
the substance also modulates potassium ion flow in that channel
protein; and, (iii) detecting the binding of the substance to the
complexed two-transmembrane-domain-type potassium ion channel
protein. The channel proteins can be wildtype proteins or modified
as described above. Optionally the contacting is done in the
presence of a non-ionic detergent and the substance for binding is
either a channel blocker or other modulator including a toxin.
[0151] What's more, the present invention extends to columns having
applications in the methods of the invention. In particular, the
present invention extends to a column comprising a solid support
having bound thereto an ion channel having the scaffold of a
two-transmembrane-domain-- type potassium ion channel and having a
tetrameric confirmation.
[0152] Furthermore, the present invention extends to a column as
described above, wherein the ion channel is a non-natural and
functional two-transmembrane-domain-type potassium ion channel
protein wherein the non-natural protein is mutated in its amino
acid sequence from a corresponding natural protein. Such a mutation
does not prevent the non-natural protein from binding a toxin, such
as agitoxin2 when the non-natural protein is further mutated to
correspond to the agitoxin2 docking site of a Shaker K.sup.+
channel protein. Numerous means are available to the skilled
artisan to create the docking. A particular means to create the
docking site comprises substituting amino acid residues selected
from within the 36 amino acid domain defined by -25 to +5 of the
Shaker K.sup.+ selectivity filter where the 0 residue is either the
phenylalanine or the tyrosine of the filter's signature sequence
selected from the group consisting of glycine-phenylalanine-glycine
or glycine-tyrosine-glycine.
[0153] Accordingly, it is a principal object of the present
invention to provide a crystal comprising a cation channel
protein.
[0154] It is another object of the present invention to provide a
method for growing a crystal comprising a cation channel
protein.
[0155] It is yet another object of the present invention to utilize
information on the structure of a cation channel protein obtained
from a crystal of the present invention, in an assay system for
screening potential drugs or agents that may interact with a cation
channel protein. Interaction of the potential drug or agent with a
cation channel protein includes binding to a cation channel
protein, or modulating the function of a cation channel protein,
wherein modulation involves increasing the function of a cation
channel protein to allow more specific cations to cross a cell
membrane, or decrease the function of a cation channel protein to
limit or prevent specific cations from permeating through the
protein and crossing the cell membrane. Such drugs or therapeutic
agents may have broad applications in treating a variety of
abnormal conditions, such as cardiac arrhythmia, diabetes mellitus,
seizure disorder, asthma or hypertension, to name only a few.
[0156] It is vet another object of the present invention to provide
mutant form of a cation channel protein, preferably a potassium
channel protein from Streptomyces lividans, which binds to
Agitoxin2, a toxin found in scorpion venom, in a manner very
similar to that in which eukaryotic potassium channel proteins bind
to Agitoxin2. Consequently, a mutant cation channel protein of the
present invention mimics a functional eukaryotic potassium channel
protein, and can serve as a model therefor in screening potential
drugs or agents that may interact with a eukaryotic potassium
channel protein.
[0157] It is still yet another object of the present invention to
provide a method of preparing functional cation channel proteins
for use in screen systems for assaying potential drugs or
therapeutic agents which may have applications in treating
conditions related to the function of cation channel proteins in
vivo.
[0158] It is yet another object of the present invention to provide
mutated prokaryotic cation channel proteins which mimic eukaryotic
cation channel proteins. With these mutated prokaryotic cation
channel proteins, drugs or other can be screened for potential
interaction with cation channel proteins in vivo, and hence,
potential use as therapeutic agents in treating conditions related
to the function of cation channel proteins in vivo, such as cardiac
arrhythmia, diabetes mellitus, seizure disorder, asthma or
hypertension, to name only a few.
[0159] These and other aspects of the present invention will be
better appreciated by reference to the following drawings and
Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0160] FIG. 1. (A) Sequence alignment of selected K.sup.+ channels
and cyclic nucleotide-gated channels. The numbering and secondary
structural elements for the Streptomyces lividans K.sup.+; channel
(kcsa) is given above the sequences. Selectivity filter, red;
lining of the cavity and inner pore, blue: residues in which the
nature of the side chain is preserved (>50% similarity), grey.
The sequences are: kcsa, Streptomyces lividans accession number
(acc) 2127577 (SEQ ID NO:1): kch, Escherichia coli acc 902457 (SEQ
ID NO:2); clost. Clostridium acetoburylicum (Genome Therapeutics
Corp.) (SEQ ID NO:3); Shaker, Drosophila melanogaster acc 85110
(SEQ ID NO:4); hKv1.1, Homo sapiens acc 1168947 (SEQ ID NO:5);
hDRK, Homo sapiens acc 345875 (SEQ ID NO:6); Parame, Paramecium
tetraaurelia acc 643475 (SEQ ID NO:7); Caenorhabiditis elegans acc
2218158 (SEQ ID NO:8); mSlo, Mus musculus acc 539800 (SEQ ID NO:9);
cal_act, Homo sapiens acc 2832249 (SEQ ID NO:10); AKT1, Arabidopsis
thaliana acc 2129673 (SEQ ID NO:11); herg. Homo sapiens acc 2135973
(SEQ ID NO:12): romk, Rattus norvegicus acc 547736 (SEQ ID NO:13):
hgirk. Homo sapiens acc 1042217 (SEQ ID NO:14): olCNG, Homo sapiens
acc 2493743 (SEQ ID NO:18): rodCNG, Homo sapiens acc 539557 (SEQ ID
NO:19). The last two sequences, separate from the rest, are from
cyclic nucleotide-gated channels, which are not K.sup.+
selective.
[0161] FIG. 2. Experimental electron density map. Stereo views of
the experimental electron-density map contoured at 1.sigma.
covering nearly an entire subunit (removed from the tetramer) of
the final model. The map was calculated at 3.2 .ANG. resolution
with the following Fourier coefficients: native-sharpened
amplitudes and MIR solvent flattened averaged phases. (A)
Foreground: map showing inner helix, loop structures and
selectivity filter; background: the pore helix and outer helix. CPK
spheres show positions of mercury atoms used as residue markers
(from the top, marked residues are Leu86. Leu90 and Val93). (B)
Alternative view, Foreground: pore helix and part of outer helix;
background: selectivity filter and turret. CPK sphere marks
position of Ala42. (C) Close up view of electron density.
[0162] FIG. 3. Views of the tetramer. (A) Stereo view of ribbon
representation illustrating the three-dimensional fold of the kcsa
tetramer viewed from the extracellular side. The four subunits are
distinguished by color. (B) Stereo view from another perspective,
perpendicular to that in (A). (C) Ribbon representation of the
tetramer as an integral-membrane protein. Aromatic amino acids
present on the membrane-facing surface are displayed in black. (D)
Inverted tepee architecture of the tetramer. These diagrams were
prepared with MOLSCRIPT and RASTER-3D (33 of Example I).
[0163] FIG. 4. Mutagenesis studies on Shaker: Mapping onto the kcsa
structure. Mutations in the voltage-gated Shaker K.sup.+ channel
that affect function are mapped to the equivalent positions in kcsa
based on the sequence alignment. Two subunits of kcsa are shown.
Mutation of any of the white side chains significantly alters the
affinity of agitoxin2 or charybdotoxin for the Shaker K.sup.+
channel (12 of Example I). Changing the yellow side chain affects
both agitoxin2 and tetraethylammonium ion (TEA) binding from the
extracellular solution (14 of Example I). This residue is the
external TEA site. The mustard-colored side chain at the base of
the selectivity filter affects TEA binding from the intracellular
solution (the internal TEA site (15 of Example I)). The side chains
colored green, when mutated to cysteine, are modified by
cysteine-reactive agents whether or not the channel gate is open,
whereas those colored pink react only when the channel is open (16
of Example I). Finally, the residues colored red (GYG, main chain
only) are absolutely required for K.sup.+ selectivity (4 of Example
I). This figure was prepared with MOLSCRIPT and RASTER-3D.
[0164] FIG. 5. Molecular surface of kcsa and contour of the pore.
(A) A cutaway Stereo view displaying the solvent-accessible surface
of the K.sup.+ channel colored according to physical properties.
Electrostatic potential was calculated with the program GRASP,
assuming an ionic strength equivalent to 150 mM KCl and dielectric
constants of 2 and 80 for protein and solvent, respectively. Side
chains of lysine, arginine, glutamate and aspartate residues were
assigned single positive or negative charges as appropriate, and
the surface coloration varies smoothly from blue in areas of high
positive charge through white to red in negatively charged regions.
The yellow areas of the surface are colored according to carbon
atoms of the hydrophobic (or partly so) side chains of several
semi-conserved residues in the inner vestibule (Thr75. Ile100,
Phe103, Thr107, Ala108, Ala111, Val115). The green CPK spheres
represent potassium ion positions in the conduction pathway. (B)
Stereo view of the internal pore running the length of the ion
channel. Within a stick model of the channel structure is a three
dimensional representation of the minimum radial distance from the
center of the channel pore to the nearest van der Waals protein
contact.
[0165] The display was created with the program HOLE (34 of Example
I).
[0166] FIG. 6. Identification of permeant ion positions in the
pore. (A) A Rb.sup.- difference Fourier map calculated to 4.0 .ANG.
and contoured at 6.sigma. identify two strong peaks corresponding
to ions in the selectivity filter (inner and outer ions) and a
weaker peak corresponding to ions in the cavity (cavity ion). The
inner ion density has two closely-spaced peaks. (B) A Cs.sup.+
difference Fourier map calculated to 5.0 .ANG. and contoured at
6.sigma. shows the inner and outer ion peaks in the selectivity
filter. Both difference Fourier maps were calculated with Fourier
coefficients: F(soak)-F(native-unsharpened) and MIR phases. (C)
Electron density map contoured at 1 (showing diffuse density at the
cavity ion position. This map was calculated with the following
Fourier coefficients: unsharpened native amplitudes and MIR solvent
flattened phases (no averaging information was included).
[0167] FIG. 7. Two mechanisms by which the K.sup.+ channel
stabilizes a cation in the middle of the membrane. First, a large
aqueous cavity stabilizes an ion (green) in the otherwise
hydrophobic membrane interior. Second, oriented helices point their
partial negative charge (carboxyl end, red) towards the cavity
where a cation is located.
[0168] FIG. 8. Detailed views of the K.sup.+ channel selectivity
filter. (A) Stereo view of the experimental electron-density
(green) in the selectivity filter. The map was calculated with
native-sharpened amplitudes and MIR-solvent-flattened-averaged
phases. The selectivity filter of three subunits is shown as a
stick representation with several signature sequence residues
labeled. The Rb.sup.+ difference map (yellow) is also shown. (B)
Stereo view of the selectivity filter in a similar orientation to
(A) with the chain closest to the viewer removed. The three chains
represented are comprised of the signature sequence amino acids
Thr, Val, Gly, Tyr, Gly (SEQ ID NO:15) running from bottom to top,
as labeled in single letter code. The Val and Tyr side chains are
directed away from the ion conduction pathway, which is lined by
the main chain carbonyl oxygen atoms. Two K.sup.+ ions (green) are
located at opposite ends of the selectivity filter, roughly 7.5
.ANG. apart, with a single water molecule (red) in between. The
inner ion is depicted as in rapid equilibrium between adjacent
coordination sites. The filter is surrounded by inner and pore
helices (white). Although not shown, the model accounts for
hydrogen bonding of all amide nitrogen atoms in the selectivity
filter except for that of Gly77. (C) A section of the model
perpendicular to the pore at the level of the selectivity filter
and viewed from the cytoplasm. The view highlights the network of
aromatic amino acids surrounding the selectivity filter. Tyrosine
78 from the selectivity filter (Y78) interacts through hydrogen
bonding and van der Waals contacts with two Trp residues (W67. W68)
from the pore helix.
[0169] FIG. 9. Sequence alignment of residues 51 to 86 of kcsa
K.sup.+ (SEQ. ID NO:1) and Shaker K.sup.- (SEQ. ID NO:4) channel
pore regions. The numbering for kcsa is given above the sequences.
Structural elements are indicated (5 of Example 11). Asterisks mark
several Shaker K.sup.+ channel amino acid locations where mutations
influence Agitoxin2 binding (4, 8, 9 of Example II). Arrows mark
the three kcsa K.sup.+ channel amino acids mutated in this study.
The sequences are: kcsa, Streptomyces lividans accession number
(acc) 2127577 and Shaker, Drosophila melanogaster acc 85110.
[0170] FIG. 10. Mass Spectra of scorpion toxins before and after
channel column purification. MALDI-TOF mass spectra of venom before
purification (A) and after elution from a cobalt column in the
absence (B) and presence (C) of attached mutant kcsa K.sup.+
channel. The accuracy of the mass measurements (.+-.0.3 Da)
permitted identification of most of the major peaks in the mass
spectra searched from databases of known toxins of the Leiurus
quinquestriatus hebraeus scorpion (D). The kcsa-binding component
labeled * could not be assigned to a known scorpion toxin. The
component labeled X (4193.0 Da) binds nonspecifically to the column
and was not identified. MALDI_MS was performed with the MALDI
matrix 4-hydroxy-.alpha.-cyano-cinnamic acid (16 of Example
II).
[0171] FIG. 11. Binding affinity of wild type and mutant Agitoxin2
to the mutant kcsa K.sup.+ channel. (A) Quantity of radiolabeled
Agitoxin2 bound to 0.3 .mu.l of cobalt resin saturated with the
mutant kcsa K.sup.+ channel is shown as a function of the
radiolabeled Agitoxin2 concentration (17 of Example II). Each point
is the mean.+-.SEM of 4 measurements, except for the 0.03 .mu.M and
1.5 .mu.M concentrations which are the mean.+-.range of mean of two
measurements. The curve corresponds to equation Bound
Agitoxin2=A*{1+K.sub.d/[Agitoxin2]}.sup.-1, with equilibrium
dissociation constant K.sub.d=0.62 .mu.M and resin capacity A=16
pMoles. (B) Remaining bound fraction of radiolabeled wild type
toxin is graphed as a function of the concentration of unlabeled
wild type toxin or mutant toxins K27A or N30A (17). Each point is
mean.+-.SEM of 4 measurements for wild type Agitoxin2 (squares) or
mean.+-.range of mean of 2 measurements for K27A (circles) and N30A
(triangles) Agitoxin2 mutants. The curves correspond to equation
Remaining Bound
Fraction={1+Kdhot/[Thot]}*{1+(Kdhot/[Thot])*(1+[Tcold]/Kd-
cold)}.sup.-1 with labeled toxin concentration Thot=0.06 .mu.M,
wild type toxin Kdhot=0.62 .mu.M, and competing toxin dissociation
constant Kdcold=0.62 .mu.M (wild type), 81 .mu.M(K27A), and 27
.mu.M (N30A). (C) CPK model of Agitoxin2 viewing the interaction
surface. Side chains of functionally important amino acids are
shown in red (4 of Example II). This figure was prepared using the
program GRASP (19 of Example II).
[0172] FIG. 12. Docking of Agitoxin2 onto the kcsa K.sup.+ channel.
(A) Molecular surface of the pore entryway of the kcsa K.sup.+
channel (left) and Agitoxin2 (right). The colors indicate locations
of interacting residues on the toxin and channel surfaces as
determined by thermodynamic mutant cycle analysis of the Shaker
K.sup.+ channel-Agitoxin2 interaction (4,8 of Example II). The
three pore mutations of the kcsa K.sup.+ channel used in this study
(Q58A. T61S, R64D) were introduced into the channel model using the
program O (19 of Example II). Indicated residues on the channel
surface correspond to the positions of the Shaker K channel
equivalent residues (See FIG. 9) which couple to the indicated
Agitoxin2 residues. (B) The pattern of colors in (A) suggests the
docking orientation shown by the main worm representation of
Agitoxin2 placed manually onto the pore entryway. The side chain
colors match the colored patches in (A). Gly10 is shown as a green
band on the worm. The mutant cycle coupling between residues at
Shaker 425 (mutant kcsa 58) and residue 10 of Agitoxin2 comes about
through substitution of a bulky side chain residue at either
position (4, 7 of Example II). Pictures were made using the program
GRASP (19 of Example II).
DETAILED DESCRIPTION OF THE INVENTION
[0173] The present invention is based on the discovery of a crystal
of a cation channel protein, in particular a potassium channel
protein from Streptomyces lividans, and a method of forming such
crystals. Moreover, the present invention is based on the
recognition that, based on the structure of the crystalline cation
channel protein, potential drugs and therapeutic agents which can
bind to cation channel protein can be screened for their use in
treating conditions related to the function of cation channel
proteins, particularly potassium channel proteins, in vivo.
[0174] Furthermore, the present invention is based upon the
discovery that cation channel proteins from prokaryotic organisms,
such as a potassium channel protein from Streptomyces lividans,
have much similarity and conservation with eukaryotic potassium
channel proteins. In particular, a mutated prokaryotic potassium
channel protein binds to a particular scorpion toxin in much the
same way a eukaryotic potassium channel protein binds to the same
toxin.
[0175] For purposes of this Application, the term "positioned to
extend into the lipid bilayer membrane proximate to the interface .
. . " indicates that aromatic side groups of amino acid residues
interject into the lipid bilayer membrane from about 0 .ANG. to
about 5 .ANG. from the interface of the lipid bilayer with either
the extracellular matrix of the cellular cytosol, i.e., the point
at which the lipid bilayer membrane meets either the extracellular
matrix or the cellular cytosol.
[0176] Moreover, for purposes of this Application, the term "kink"
indicates the inner transmembrane alpha-helix comprises a slight
bend in its structure. Moreover, the angle of the tilt of the inner
transmembrane helix "normal of the lipid bilayer" indicates the
amount of tilt in the inner membrane helix relative to a line
perpendicular to the lipid bilayer membrane at a point at which the
inner transmembrane alpha-helix would have intersected the lipid
bilayer membrane, had the inner transmembrane alpha-helix extended
thereto.
[0177] Moreover, for purposes of this Application the "specific
ion" refers the the ion species intended to permeate a particular
cation channel protein. For example, if the K.sup.+ is the specific
ion for a potassium channel protein, Na.sup.+ is the specific ion
for a sodium channel protein, and Ca.sup.2+ is the specific ion for
calcium channel protein.
[0178] Furthermore, an .alpha.-helix in a protein is found when a
stretch of consecutive residues all have a phi,psi angle pair of
approximately -60.degree. and -50.degree., corresponding to the
allowed region of a Ramachandran plot (Branden, C. And Tooze, J.
Introduction to Protein Structure, Garland Publishing. Inc. New
York and London, 1991 p. 12 (this reference is incorporated by
reference herein in its entirety).
[0179] Moreover, the term "bundle" of .alpha.-helices, as used
herein, refers to the packing at least two .alpha.-helices closely
together by intercalating side chains of residues of the helices in
the physically interact with Agitoxin2 and are primarily
responsible for conferring the ability of a channel protein to bind
to Agitoxin2.
[0180] As used herein, the term "functional" refers to a channel
protein which is in a tetrameric form and having a confirmation
that is sufficiently reflective of the native protein in its
natural environment so that when a compound binds to the functional
channel protein that same compound would also bind to that protein
in its natural environment. The test for determining if a channel
protein is functional is provided below and relies upon the ability
of the protein to bind Agitoxin2 when deliberately mutated to bind
the toxin.
[0181] "Non-natural" refers to a potassium ion channel protein that
has been modified or altered from a corresponding wild type
protein. Typically the protein is altered in its primary amino acid
sequence but fusions and chimera to the N and C terminus are
included as well as addition of non-protein components to available
reactive sites.
[0182] As used herein, "natural" refers to a potassium ion channel
protein which is found in nature. This is referred to as a
wildtype.
[0183] The term "mutated" as used herein refers to a potassium ion
channel protein that has been altered by deletion, substitution of
addition of amino acids.
[0184] As used herein, the phrase "selectivity filter" refers to
the domain of channel ion protein that is responsible for the
ability of the protein to exclude one or a group of ions and to
allow other ions to pass.
[0185] As used herein, the phrase "signature sequence" refers to a
sequence of amino acids which define the protein as that protein or
as belonging to a group or family of proteins. For specific
proteins the signature sequence may be very conserved and be a
unique identifier. For signature sequences that define a family,
the sequence would be relatively hypervariable but conserved across
the family.
[0186] Also, as used herein, "solid supports" refer to any
non-soluble matrix upon which the potassium ion channel proteins of
this invention may be attached.
[0187] As used herein, the phrase "structural confomation" refers
to a physical relationship between amino acids within a protein. It
is a relative state which alters with salt concentration,
temperature and hydrophobic nature of the solvent being used.
Structural confirmation is best defined by function.
[0188] The phrase "tetrameric protein" used herein refers to a
protein having quaternary structure comprising 4 subunits which may
be the same or different.
[0189] As used herein, the phrase "two-transmembrane-domain type
potassium ion channel protein" refers to potassium channel monomer
having two regions of hydrophobicity with sufficient length to form
transmembrane segments. Between these two segments must be found
the potassium channel signature sequence. When using the tyrosine
or phenylalanine residue of the signature sequence as a zero
reference point, the first transmembrane segment would begin within
approximately -61 residues of the reference point and the second
transmembrane would end within approximately +42 amino acids of the
reference point. To identify the two transmembrane domains one can
construct a a Kyte-Dolittle hydropathy plot of the amino acids.
[0190] As used herein, the phrase "wild-type" protein refers to a
protein such as a potassium ion channel protein which is presented
with a primary amino acid sequence that is found in nature.
Isolation of a Functional Cation Channel Protein for Use in Assays
to Screen Potential Drugs and Therapeutic Agents
[0191] This method of the present invention overcomes limitations
of using cation channel proteins in the development of drugs or
therapeutic agents to treat conditions related to the function of
cation channel proteins, and particularly potassium cation channel
proteins in vivo, such as cardiac arrhythmia, diabetes mellitus,
seizure disorder, asthma or hypertension, to name only a few.
[0192] In particular, since cells need very few potassium channels
in order to function, it is difficult to isolate functional
potassium channels in great quantities. Moreover, recombinant
techniques to have a cell produce excess potassium channel proteins
has met with only limited success. As a result, very few drugs or
agents are currently available which act on potassium channel
proteins.
[0193] However, Applicant has discovered a method to isolate cation
channel proteins, particularly potassium cation channel proteins,
which can then be used in efficient assays to screen potential
drugs and agents for interaction with such proteins. In particular,
disclosed herein is a method of using a functional cation channel
protein in an assay for screening for potential drugs or agents
that may bind to a cation channel protein comprising, wherein the
assay comprises the steps of providing a functional cation channel
protein, conjugating the functional cation channel protein to a
solid phase resin, contacting the potential drug or agent to the
functional cation channel protein conjugated to the solid phase
resin, removing the functional cation channel protein from the
solid phase resin, and determining whether the potential drug or
agent is bound to the cation channel protein.
[0194] Since cation channel proteins are trans membrane bound
proteins, care should be taken in their isolation. In particular,
to prevent denaturation and a loss of functional activity, they
require a hydrophobic environment. In a preferred embodiment, a
functional cation channel protein is provided by expressing an
isolated nucleic acid molecule encoding the cation channel protein
in a unicellular host such that the cation channel protein is
present in the cell membrane of the unicellular host, lysing the
unicellular host in a solubilizing solution so that the cation
channel protein is solubilized in the solution, and extracting the
cation channel protein from the solubilizing solution with a
detergent.
[0195] Many solubilizing solutions are presently known to one of
ordinary skill in art, which can solubilize a cation channel
protein, and prevent its denaturation or proteolytic digestion. All
such solutions are encompassed by the present invention. In a
preferred embodiment, the solubilizing solution comprises Tris
buffer, 100 mM KCl, 10 mM MgSO.sub.4, 25 mg DNAse 1, 250 mM
sucrose, pepstatin, leupeptin, and PMSF at pH 7.5.
[0196] Moreover, many detergents are available to the skilled
artisan for extracting solubilized cation channel protein from a
solubilizing solution of the present invention. Examples of
detergents having applications herein include SDS, Triton 100,
glycerol, decylmaltoside, Tween-20, or Tween-80, to name only a
few. In a preferred embodiment, a 40 mM decylmaltoside is used to
extract the cation channel protein from a solubilizing solution of
the present invention.
[0197] Furthermore, Applicant has discovered that cation channel
proteins, particularly potassium cation channel proteins, can be
conjugated chemically to a solid phase resin. As a result, the
channel proteins are immobilized and readily available in assays
for screening drugs or agents that may bind to a cation channel
protein. In a preferred embodiment, a cation channel protein is
conjugated to a cobalt resin through a carboxyl terminal
hexahistidine tag.
[0198] In preferred embodiment, cation channel proteins are
conjugated to a cobalt resin at a protein to resin ratio that
allows for saturation of the resin with the cation channel protein.
As a result, numerous cation channel proteins are immobilized and
available for contact with a potential drug or therapeutic agent to
be screened pursuant to the present invention.
[0199] Moreover, numerous screening methods are available and
encompassed by the present invention. For example, the resin with
the cation channel conjugated thereto can be incubated in a
solution comprising the potential drug or therapeutic agent. In
another embodiment, the resin can be used to line a column, to
which the potential drug or agent is added. Preferably, a potassium
ion channel protein from Streptomyces lividans comprising an amino
acid sequence of SEQ ID NO:1, or conserved variants thereof, is
mutated to mimic a eukaryotic potassium channel, such as a
potassium channel protein of Drosophila melanogaster comprising an
amino acid sequence of SEQ ID NO:4, or conserved variants.
Consequently, the mutated potassium channel protein of Streptomyces
lividans comprising an amino acid sequence of SEQ ID NO:16 is
conjugated to a cobalt resin, which is then used to line a 1 ml
column. A composition comprising the potential drug or agent to be
screened for interaction with a eukaryotic cation channel protein
is then poured into the column, so that the potential drug or agent
can contact the mutated prokaryotic cation channel protein
conjugated to the cobalt membrane.
[0200] After contact, the cation channel proteins are removed from
the resin, and examined for interaction binding with the potential
drug or agent. Numerous methods of cleaving a protein from a solid
phase resin are available to the skilled artisan, and included in
the present invention. In a preferred embodiment, the removing step
comprises contacting the cation channel protein conjugated to the
resin to an imidazole solution. The cation channel proteins can
then be collected, and examined for interaction, i.e. binding, with
the potential drug or therapeutic agent.
[0201] Furthermore, determining whether the drug or therapeutic
agent is bound to the cation channel protein can be done with
numerous methods. For example, molecular weight determinations can
be made with SDS-PAGE comparing the molecular weight of the cation
channel protein not contacted with the drug, to the molecular
weight of the cation channel protein contacted with the drug.
Furthermore, other analytical methods, such as HPLC, mass
spectrometry, or spectrophotometry, to name only a few, can be used
to determine whether the drug or agent is bound to a cation channel
protein previously conjugated to a solid phase resin.
[0202] Moreover, screening potential drugs or agents which may bind
a cation channel protein may be performed on an individual basis,
i.e. one potential drug or agent at a time, or the present
invention can be used to screen whole libraries of compounds at one
time such as a mixture of compounds or a combinatorial library, for
potential drugs or agents which potentially bind to a cation
channel protein. For example, combinatorial libraries which can be
screened with the present invention include, but are not limited
to, a phage display library, in which numerous proteins and
polypeptides are being express simultaneously, libraries comprising
synthetic peptides.
Two-Transmembrane-Domain Type Potassium Ion Channel Proteins
[0203] As set forth above, two-transmembrane type potassium ion
channel proteins are well known and structurally constitute one of
the classes of potassium channels. They are found in a wide variety
of organisms, both prokaryotic and eukaryotic where they serve the
purpose of controlling the influx or efflux of potassium ions
across cell membranes. Potassium channels as a class are tetrameric
membrane proteins characterized by multiple transmembrane segments
and a pore region through which potassium ions flow. These channels
may be homotetrameric, that is, consisting of four identical
monomers, or heterotetrameric, consisting of four monomers which
are not necessarily identical. The individual monomers of the
heterotetrameric forms are usually structurally related, and may or
may not form a functional potassium channel when reconstituted as
homotetramers of themselves. The pore region contains a signature
sequence consisting of glycine-tyrosine-glycine or
glycine-phenylalanine-glycine. Each monomer in the tetrameric
structure contributes to the formation of the pore region, and each
subunit contains a signature sequence.
[0204] To identify a putative protein as a two-transmembrane
potassium channel monomer, a Kyte-Dolittle hydropathy plot of the
amino acid may be constructed, and it should demonstrate two
regions of hydrophobicity with sufficient length to form
transmembrane segments. Between these segments must be found the
potassium channel signature sequence. When using the tyrosine or
phenylalanine residue of the signature sequence as a zero reference
point, the first transmembrane segment would begin within
approximately -61 residues of the reference point and the second
transmembrane would end within approximately +42 amino acids of the
reference point.
[0205] Potassium channel monomer subunits may be obtained by a
variety of methods, including cloning by nucleic acid
hybridization, cloning by antibody selection of expressed proteins,
and using the polymerase chain reaction (PCR) with homologous or
degenerate primer sets. One of skill in the art would be able to
readily obtain DNA sequence encoding such potassium channels given
a known DNA sequence or an antibody against the channel itself.
[0206] Examples of proteins which have been cloned and identified
as two-transmembrane potassium ion channels include IRK3 as
described in Koyama H, et al. Molecular cloning, functional
expression and localization of a novel inward rectifier potassium
channel in the rat brain. FEBS Lett 341:303-7 1994; IRK3 as
described in Morishige et al., Molecular cloning and functional
expression of a novel brain-specific inward rectifier potassium
channel. FEBS Lett 346: 251-6, 1994; UKATP reported in Inagaki et
al. Cloning and functional characterization of a novel
ATP-sensitive potassium channel ubiquitously expressed in rat
tissues, including pancreatic islets, pituitary, skeletal muscle,
and heart. J Biol Chem 270:5691-4; and GIRK2 reported in Ferrer et
al., Pancreatic islet cells express a family of inwardly rectifying
K.sup.+ channel subunits which interact to form G-protein-activated
channels. J Biol Chem 270:26086-91 1995.
Mutations of Two-Transmembrane-Domain Type Potassium Ion Channel
Proteins
[0207] The present invention further extends to introducing
Agitoxin2 docking sites into two-transmembrane-domain type
potassium ion channel protein. Any two transmembrane cation channel
protein presently known, or subsequently discovered, can routinely
be modified to bind agitoxin2 using the protocols described infra.
As explained herein, scorpion toxins, such as agitoxin2, bind to an
ion channel by making contact with all four subunits where they
come together to form the pore. Hence, such toxins will only bind
to the channel if the subunits have been properly assembled. As a
result, the binding of a toxin, such as agitoxin2, to a non-natural
two transmembrane cation channel protein can be used to confirm the
template channel integrity or function, i.e., to confirm the
two-transmembrane cation channel protein has been properly modified
to mimic a functional eukaryotic two-transmembrane cation channel
protein.
[0208] The general method for creating an agitoxin (or related
scorpion toxin) binding site on the template channel is now
described. Particular examples of pore region sequences (toxin
binding sequences) of four two-transmembrane cation channel
proteins having applications in the present invention are described
below:
1 Shaker aeagsensffksipdafwwavvtmttvgygdmtpvgfwgk Romk1
anhtpcveningltsaflfsletqvtigygfrcvteqcat Mjan
esvilmtvegwdfftafytavvtistvgygdytpqtflgkls KcsA
vlaerpgaqlitypralwwsvetattvgygdlypvtlwgr
[0209] Shaker is a six-membrane spanning K channel from Drosophila
melanogaster, ROMK1 is a two membrane-spanning K channel from rat
renal outer medulla (kidney). Mjan is a two membrane-spanning K
channel from Methanococcus janschii, and KcsA is a two
membrane-spanning K channel from the bacterium Streptomyces
lividans.
[0210] As explained herein, cation channle proteins have a high
degree of sequence conservation, particularly in the region of the
selectivity filter. Hence, gyg sequence should be used as a
reference to align the sequences. The underlined amino acids on the
Shaker channel sequence are known to be important for binding of
agitoxin, as described infra. In particular, described herein is
the mutating of several of the underlined amino acids, using
standard techniques. As a result of these mutations, the KcsA K
channel became sensitive to agitoxin binding. Similarly, other
channels can be subjected to the same analysis. Therefore, using
the teachings set forth infra. Mjan or Romk1 channels can readily
be modified by those of ordinary skill. Numerous techniques are
readily available to the skilled artisan to convert the appropriate
(underlined) amino acids of the pore relations of the
two-transmembrane cation channel proteins described above to the
amino acid residues found in the corresponding position of the
Shaker K channel. A particular technique which can be in this
modification process is directed mutagenesis.
[0211] Also, the present invention involves introducing mutations
into the two-transmembrane-domain type potassium ion channel
protein which allow it to mimic other potassium ion channel
proteins. In particular, the present invention contemplates the use
of two-transmembrane proteins as a scaffold for studying or
identifying modulators of potassium ion channel function. The
proteins can be modified in a variety of different ways to mimic or
simulate properties of related potassium ion channels including
conferring properties found in six membrane domain type ion
channels. Accordingly, one can create channel proteins that have
been minimally altered from their corresponding wild type for
convenience of purification, i.e. removing protease cleavage sites
in noncritical domains, or attaching binding domains to facilitate
chromatographic purifications such as FLAG or polyHis. Because the
overall structure of potassium ion proteins is conserved,
modifications can be introduced that can transfer properties of one
channel protein to the two-transmembrane proteins that is being
used as a scaffold. Among these modifications are venom docking
sites as exemplified herein as well as binding sites for modulators
such as to the transmembrane domains and alterations to the ion
filter region.
[0212] Recombinant genetics has a variety of techniques for
introducing and for determining the domains and in many cases the
specific amino acids which are responsible for the physical
properties of channel proteins. In brief, these methods consists of
manipulating the amino acid sequence of a protein in order to
identify which part of the protein is involved in the structure or
function of the molecule and then transferring that domain and its
properties to proteins that do not naturally have that property.
These methods have already been widely applied in the study of ion
channels. The study of ion channels lends itself very well to such
methods, because these proteins exist in a number of functional
families within which are numerous structurally related yet
biophysically and pharmacologically distinct subfamily members. For
example, the superfamily of potassium channels all share the pore
signature sequence gly-tyr-gly or gly-phe-gly, and are tetrameric;
subfamily monomers may have two transmembrane segments or 6
transmembrane segments, and may be gated by membrane potential,
intracellular calcium concentration, intracellular cyclic
nucleotides, membrane deformation, and pH: they may be inwardly
rectifying, outwardly rectifying, or nonrectifying: and their
activation and inactivation kinetics, and conductances may vary
tremendously.
[0213] As exemplified in this application, a number of scorpion and
bee venom, toxins can bind with high affinity to one subfamily
member while being inactive on a closely related subfamily members.
It is therefore not surprising that amino acid sequence mutations
which confer the properties of one ion channel upon another are a
tool which has been commonly employed by ion channel researchers
and this invention takes advantage of this plieomorphic property in
the super family of potassium channels.
[0214] Mutations may be introduced using a number of approaches,
each with its own particular strengths. Often a combination of
these may be used to generate a channel with altered properties.
Examples of these approaches are deletions of amino acids, domain
replacement of one channel with that of a different channel
(chimeras), replacement of amino acids with different amino acid in
a nontargeted or semi-targeted way (e.g. alanine-scanning
mutagenesis) and replacement of targeted amino acids with different
amino acids (site-directed mutagenesis). Although each method may
be applied independently, oftentimes several or all of these may be
employed to arrive at a mutant channel with the desired
characteristics. Examples of changed characteristics include
channel gating, voltage response, rectification, ion preference,
and the binding of small organic molecules and peptides to the
channel.
[0215] Mutagenesis is especially powerful when an ion channel with
novel toxin or small organic molecule-binding characteristics is
required. Using this approach, channels which do not show
significant binding of a particular toxin or small organic molecule
may be engineered to bind strongly to these molecules. Conversely,
channels which strongly bind a particular toxin or small organic
molecule may be engineered to lose that property.
[0216] Examples of the use of the chimeric and site-directed
approach are many. In Ishii, T. M., Maylie, J. and Adelman, J. P.
(1997) J. Biol. Chem 272: 23195-200, the authors were able to
confer apamin sensitivity on a channel which did not possess this
property. Similar studies have been performed on the Kv1.3 and
Kv2.1 potassium channels by Gross et al. (1994), Neuron 13: 961-6.
In their study, they transferred scorpion toxin sensitivity from
the highly sensitive Kv1.3 potassium channel to the insensitive
Kv2.1 potassium channel by transferring the stretch of amino acids
between transmembrane domains 5 and 6. Conversely, alanine-scanning
mutagenesis was used by Hanner et al. (1998). J Biol Chem 273:
16289-96, to impair charybdotoxin binding to the maxi-K channel,
and direct point mutations were employed by Wang and Wang (1998),
Proc Natl Acad Sci USA 95:2653-8, to remove batrachotoxin
sensitivity from sodium channels.
[0217] Mutagenesis may also be employed to alter the biophysical
properties of ion channels, in effect causing one channel to have
characteristics similar to those of another. For example,
voltage-gated potassium channels of the Shaker subfamily open in
response to changes in membrane potential. Members of this
subfamily of potassium channels have the intrinsic property of
opening at different membrane potentials depending on the
particular family member, and have the characteristic of delayed
rectification. Liman et al., (1991). Nature, 353:752-6, were able
to demonstrate that mutations in the S4 voltage sensor domain of
Shaker changed the opening potential; by mutating several amino
acid residues in the S4 voltage sensor domain of Shaker, Miller and
Aldrich (1996), Neuron. 16:853-8, were able to convert this channel
from a delayed rectifier into a voltage-gated inward rectifier.
Chimeric constructs may use related domains from different channel
types. The rat CNG olfactory channel is a member of the
voltage-gated subfamily of potassium channels, but is itself
voltage-independent and is not entirely selective for potassium
ions as compared with the eag channel. Tang and Papazian (1997), J
Gen Physiol, 109:301-11, were able to convert the human eag
potassium channel from a voltage sensitive to a voltage-independent
channel by substituting the S3-S4 domain of the rat
cyclic-nucleotide gated (CNG) olfactory channel.
[0218] It is therefore clear that mutagenesis may be readily used
to confer the pharmacological and biophysical properties of one
channel upon another, and that this methodology applies to not only
potassium, but sodium and calcium channels.
[0219] Determining if the two-transmembrane-domain type potassium
ion channel protein has maintained function using Agitoxin2
binding. Beyond the ability of the channel proteins of this
invention to pass ions under ex vivo conditions or using liposomes,
their functionality can measured by the ability to be modified to
accept or recognize agitoxin2. To accomplish this one follows the
mutagenesis methods described above both generically for mutation
of any channel protein and for the introduction of an agitoxin2
docking site into any two transmembrane-type domain potassium ion
channel protein.
[0220] Once mutated, the proteins are tested by any number of
binding assay formats including homogenous assays where both
agitoxin2 and the channel protein are free in solution and
heterogeneous assay formats where one of the binding members is
bound to a solid support. Either member can be labelled using the
labels described herein. The preferred method for assaying for
agitoxin2-binding uses the cobalt resin and procedures described in
Example II.
Binding the Two-Transmembrane-Domain Type Potassium Ion Channel
Protein to Solid Supports
[0221] The potassium channels of the invention can be bound to a
variety of solid supports. Solid supports of this invention include
membranes (e.g., nitrocellulose or nylon), a microtiter dish (e.g.
PVC, polypropylene, or polystyrene), a test tube (glass or
plastic), a dip stick (e.g., glass, PVC, polypropylene,
polystyrene, latex and the like), a microfuge tube, or a glass,
silica, plastic, metallic or polymer bead or other substrate such
as paper. A preferred solid support uses a cobalt or nickel column
which binds with specificity to a histadine tag engineered onto the
channel proteins.
[0222] Adhesion of the channel proteins to the solid support can be
direct (i.e. the protein contacts the solid support) or indirect (a
particular compound or compounds are bound to the support and the
target protein binds to this compound rather than the solid
support). One can immobilize channel proteins either covalently
(e.g., utilizing single reactive thiol groups of cysteine residues
(see, e.g., Colliuod et al. Bioconjugate Chem. 4:528-536 (1993)) or
non-covalently but specifically (e.g., via immobilized antibodies
(Schuhmann et al. Adv. Mater. 3:388-391 (1991); Lu et al. Anal.
Chem. 67:83-87 (1995), the biotin/strepavidin system (Iwane et al.
Biophys. Biochem. Res. Comm. 230:76-80 (1997) or metal chelating
Langmuir-Blodgett films (Ng et al. Langmuir 11:4048-55 (1995);
Schmitt et al. Angew. Chem. Int. Ed. Engl. 35:317-20 (1996); Frey
et al. Proc. Natl. Acad. Sci. USA 93:493741 (1996); Kubalek et al.
J. Struct. Biol. 113:117-123 (1994)) and metal-chelating
self-assembled monolayers (Sigal et al. Anal. Chem. 68:490497
(1996)) for binding of polyhistidine fusions.
[0223] Indirect binding can be achieved using a variety of linkers
which are commercially available. The reactive ends can be any of a
variety of functionalities including, but not limited to: amino
reacting ends such as N-hydroxysuccinimide (NHS) active esters,
imidoesters, aldehydes, epoxides, sulfonyl halides, isocyanate,
isothiocyanate, and nitroaryl halides; and thiol reacting ends such
as pyridyl disulfides, maleimides, thiophthalimides, and active
halogens. The heterobifunctional crosslinking reagents have two
different reactive ends, e.g., an amino-reactive end and a
thiol-reactive end, while homobifunctional reagents have two
similar reactive ends, e.g., bismaleimidohexane (BMH) which permits
the cross-linking of sulfhydryl-containing compounds. The spacer
can be of varying length and be aliphatic or aromatic. Examples of
commercially available homobifunctional cross-linking reagents
include, but are not limited to, the imidoesters such as dimethyl
adipimidate dihydrochloride (DMA); dimethyl pimelimidate
dihydrochloride (DMP); and dimethyl suberimidate dihydrochloride
(DMS).
[0224] Heterobifunctional reagents include commercially available
active halogen-NHS active esters coupling agents such as
N-succinimidyl bromoacetate and
[0225] N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) and the
sulfosuccinimidyl derivatives such as
sulfosuccinimidyl(4-iodoacetyl)amin- obenzoate (sulfo-SIAB)
(Pierce). Another group of coupling agents is the
heterobifunctional and thiol cleavable agents such as
N-succinimidyl 3-(2-pyridyidithio)propionate (SPDP) (Pierce).
[0226] Antibodies are also available for binding channel proteins
to a solid support. This can be done directly by binding channel
protein specific antibodies to the column and allowing channel
proteins to bind or it can be done by creating chimeras constructed
from the channel protein linked to an appropriate immunoglobulin
constant domain sequence, they are termed immunoadhesins and they
are known in the art. Immunoadhesins reported in the literature
include Gascoigne et al., Proc. Natl. Acad. Sci. USA 84,. 2936-2940
(1987), Capon et al. Nature 377, 525-531 (1989); and Traunecker et
al., Nature 33, 68-70 (1989).
[0227] By manipulating the solid support and the mode of attachment
of the target molecule to the support, it is possible to control
the orientation of the target molecule. Thus, for example, where it
is desirable to attach a target molecule to a surface in a manner
that leaves the molecule tail free to interact with other
molecules, a tag (e.g., FLAG, myc. GST, polyhis, etc.) may be added
t) the target molecule at a particular position in the target
sequence.
[0228] It is also possible to reconstitute of channels in lipid,
membranes or liposomes. For example the following references teach
how to reconstitute the channel proteins of this invention in
membranes. The very channels of this invention, SliK, the K+
channel encoded by the Streptomyces KcsA gene, was expressed,
purified, and reconstituted in liposomes. See, Heginbotham L et al.
J Gen Physiol 1998 June; 111(6):741-9 and in Cuello L G, et al.,
Biochemistry 1998 Mar. 10; 37(10):3229-36. In Shin, J H et al. FEBS
Lett 1997 Oct. 6; 415(3):299-302 where the authors demonstrated
that nitric oxide could activate a calcium-activated potassium
channel from rat using the planar lipid bilayer technique.
Santacruz-Toloza L et al. Biochemistry 1994 Feb. 15; 33(6):
1295-9.
Assays
[0229] Once bound there are a variety of assay formats that can be
used to screen for modulators of the channel proteins. Various
molecules that interact with a potassium channel can be identified
by 1) attaching the potassium channel ("the target") to a solid
support, 2) contacting a second molecule with the support coated
with the potassium channel, and 3) detecting the binding of the
second molecule to the potassium channel. Molecules that interact
or bind with the target are then eluted, with or without the
target, thereby isolating molecules that interact with the
target.
[0230] For a general description of different formats for binding
assays, see BASIC AND CLINICAL IMMUNOLOGY, 7.sup.th Ed. (D. Stiles
and A. Terr, ed.)(1991): ENZYME IMMUNOASSAY. E. T. Maggio, ed., CRC
Press, Boca Raton, Fla. (1980); and "Practice and Theory of Enzyme
Immunoassays" in P. Tijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY
AND MOLECULAR BIOLOGY, Elsevier Science Publishres. B. V. Amsterdam
(1985), each of which is incorporated by reference.
[0231] In competitive binding assays, the test compound competes
with a second compound for specific binding sites on a target
molecule attached to the solid support. Binding is determined by
assessing the amount of second compound associated with the target
molecule. The amount of second compound associated with the target
molecule is inversely proportional to the ability of a test
compound to compete in the binding assay.
[0232] The amount of inhibition or stimulation of binding of a
labeled target by the test compound depends on the binding assay
conditions and on the concentrations of binding agent, labeled
analyte and test compounds used. Under specified assay conditions,
a compound is said to be capable of inhibiting the binding of a
second compound to a target compound to the amount of bound second
compound is decreased by 50% or preferably 90% or more compared to
a control sample.
[0233] Alternatively, various known or unknown compounds, including
proteins, carbohydrates, and the like, can be assayed for their
ability to bind to the channels of this invention. In one
embodiment, samples from various tissues are contacted with the
target to isolate molecules that interact with the target. In
another embodiment, small molecule libraries and high throughput
screening methods are used to identify compounds that bind to the
target.
Labels for Use in Assays
[0234] The amount of binding of the second compound to a target
channel protein can be assessed by directly labeling the second
compound with a detectable moiety, or by detecting the binding of a
labeled ligand that specifically binds to the second compound. A
wide variety of labels can be used. The detectable labels of the
invention can be primary labels (where the label comprises an
element that is detected or that produces a directly detectable
signal) or secondary labels (where the detected label binds to a
primary label, e.g., as is common in immunological labeling). An
introduction to labels, labeling procedures and detection of labels
is found in Polak and Van Noorden (1997) Introduction to
Immunochemistry, 2.sup.nd ed., Springer Verlag, N.Y. and in
Haugland (1996) Handbook of Fluorescent Probes and Research
Chemicals, a combined catalog and handbook published by Molecular
Probes. Inc., Eugene, Oreg. Useful primary and secondary labels of
the present invention can include spectral labels such as
fluorescein isothiocyanate (FITC) and Oregon Green.TM., rhodamine
and derivatives (e.g. Texas red, tetrarhodimine isothiocyanate
(TRITC), etc.), digoxigenin, biotin, phycoerythrin, AMCA,
CyDyes.TM., and the like), radiolabels (e.g., .sup.3H, .sup.125I,
.sup.35S, .sup.14C or .sup.32P), enzymes (e.g. horseradish
peroxidase, alkaline phosphotase, etc.), spectral calorimetric
labels such as colloidal gold and colored glass or plastic (e.g.
polysytrene, polypropylene, latex, etc.) beads. The choice of label
depends on sensitivity required, ease of conjugation with the
compound, stability requirements, and available
instrumentation.
[0235] In general, a detector that monitors a particular probe or
probe combination is used to detect the recognition reagent label.
Typical detectors include spectrophotometers, phototubes and
photodiodes, microscopes, scintillation counters, cameras, film and
the like, as well as combinations thereof. Examples of suitable
detectors are widely available from a variety of commercial sources
known to persons of skill.
High-Throughput Screening of Candidate Agents that Modulate
Potassium Channel Proteins
[0236] Conventionally, new chemical entities with useful properties
are generated by identifying a chemical compound (called a "lead
compound") with some desirable property or activity, creating
variants of the lead compound, and evaluating the property and
activity of those variant compounds. However, the current trend is
to shorten the time scale for all aspects of drug discovery.
Because of the ability to test large numbers quickly and
efficiently, high throughput screening (HTS) methods are replacing
conventional lead compound identification methods.
[0237] In one preferred embodiment, high throughput screening
methods involve providing a library containing a large number of
potential therapeutic compounds (candidate compounds). Such
"combinatorial chemical libraries" are then screened in one or more
assays, as described herein, to identify those library members
(particular chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
Combinatorial Chemical Libraries
[0238] Combinatorial chemical libraries are a preferred means to
assist in the generation of new chemical compound leads. A
combinatorial chemical library is a collection of diverse chemical
compounds generated by either chemical synthesis or biological
synthesis by combining, a number of chemical "building blocks" such
as reagents. For example, a linear combinatorial chemical library
such as a polypeptide library is formed by combining a set of
chemical building blocks called amino acids in every possible way
for a given compound length (i.e., the number of amino acids in a
polypeptide compound). Millions of chemical compounds can be
synthesized through such combinatorial mixing of chemical building
blocks. For example, one commentator has observed that the
systematic, combinatorial mixing of 100 interchangeable chemical
building blocks results in the theoretical synthesis of 100 million
tetrameric compounds or 10 billion pentameric compounds (Gallop et
al. (1994) 37(9): 12331250).
[0239] Preparation and screening of combinatorial chemical
libraries are well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka (1991)
Int. J. Pept. Prot. Res., 37: 487-493. Houghton et al. (1991)
Nature, 354: 84-88). Peptide synthesis is by no means the only
approach envisioned and intended for use with the present
invention. Other chemistries for generating chemical diversity
libraries can also be used. Such chemistries include, but are not
limited to: peptoids (PCT Publication No. WO 91/19735, 26 Dec.
1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct.
1993), random biooligomers (PCT Publication WO 92/00091, 9 Jan.
1992), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993)
Proc. Nat. Acad. Sci. USA 90: 69096913), vinylogous polypeptides
(Hagihara et al. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal
peptidomimetics with a Beta D Glucose scaffolding (Hirschmann et
al. (1992) J. Amer. Chem. Soc. 114: 92179218), analogous organic
syntheses of small compound libraries (Chen et al. (1994) J. Amer.
Chem. Soc. 116: 2661), oligocarbamates (Cho. et al., (1993) Science
261:1303), and/or peptidyl phosphonates (Campbell et al., (1994) J.
Org. Chem. 59: 658). See, generally, Gordon et al., (1994) J. Med.
Chem. 37:1385, nucleic acid libraries, peptide nucleic acid
libraries (see, e.g. U.S. Pat. No. 5,539,083) antibody libraries
(see, e.g., Vaughn et al. (1996) Nature Biotechnology, 14(3):
309-314), and PCT/US96/10287), carbohydrate libraries (see, e.g.
Liang et al. (1996) Science, 274: 1520-1522, and U.S. Pat. No.
5,593,853), and small organic molecule libraries (see, e.g.,
benzodiazepines, Baum (1993) C&EN, January 18, page 33,
isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones and
metathiazanones U.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat.
Nos. 5,525,735 and 5,519,134, morpholino compounds U.S. Pat. No.
5,506,337, benzodiazepines U.S. Pat. No. 5,288,514, and the
like).
[0240] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS. Advanced Chem
Tech, Louisville Ky., Symphony. Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore. Bedford,
Mass.).
[0241] A number of well known robotic systems have also been
developed for solution phase chemistries. These systems include
automated workstations like the automated synthesis apparatus
developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and
many robotic systems utilizing robotic arms (Zymate II, Zymark
Corporation, Hopkinton, Mass.; Orca, HewlettPackard, Palo Alto,
Calif.) which mimic the manual synthetic operations performed by a
chemist. Any of the above devices are suitable for use with the
present invention. The nature and implementation of modifications
to these devices (if any) so that they can operate as discussed
herein will be apparent to persons skilled in the relevant art. In
addition, numerous combinatorial libraries are themselves
commercially available (see, e.g., ComGenex, Princeton. N.J.,
Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd.
Moscow, RU, 3D Pharmaceuticals, Exton, Pa. Martek Biosciences,
Columbia, Md., etc.).
High Throughput Assays of Chemical Libraries
[0242] Any of the assays for compounds capable of modulating
potassium ion channel proteins described herein are amenable to
high throughput screening. High throughput screening systems are
commercially available (see, e.g., Zymark Corp., Hopkinton, Mass.;
Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc.
Fullerton. CA; Precision Systems, Inc., Natick, Mass., etc.). These
systems typically automate entire procedures including all sample
and reagent pipetting, liquid dispensing, timed incubations, and
final readings of the microplate in detector(s) appropriate for the
assay. These configurable systems provide high thruput and rapid
start up as well as a high degree of flexibility and customization.
The manufacturers of such systems provide detailed protocols the
various high throughput. Thus, for example, Zymark Corp. provides
technical bulletins describing screening systems for detecting the
modulation of gene transcription, ligand binding, and the like.
Assays for Modulation of Potassium Flow
[0243] The activity of functional potassium channels of this
invention can be assessed using a variety of in vitro and in vivo
assays, e.g., measuring voltage, current, measuring membrane
potential, measuring ion flux, e.g., potassium or rubidium,
measuring potassium concentration, measuring second messengers and
transcription levels, and using e.g., voltage-sensitive dyes,
radioactive tracers, and patch-clamp electrophysiology. In
particular such assays can be used to test for modulators both
inhibitors and activators of channels.
[0244] Modulators of the potassium channels are tested using
biologically active, functional two-transmembrane domain type
potassium ion channels, either recombinant or naturally occurring.
In recombinantly based assays, the subunits are typically expressed
and modulation is tested using one of the in vitro or in vivo
assays described below.
[0245] In brief, samples or assays that are treated with a
potential channel inhibitors or activators are compared to control
samples without the test compound, to examine the extent of
modulation. Control samples e.g., those untreated with activators
or inhibitors are assigned a relative potassium channel activity
value of 100. Inhibition is present when potassium channel activity
value relative to the control is about 90%, preferably 50%, more
preferably 25%. Activation of channels is achieved when the select
potassium channel activity value relative to the control is 110%,
more preferably 150%, more preferable 200% higher.
[0246] Changes in ion flux may be assessed by determining changes
in polarization (i.e., electrical potential) of the cell or
membrane expressing the potassium channels of this invention A
preferred means to determine changes in cellular polarization is by
measuring changes in current (thereby measuring changes in
polarization) with voltage-clamp and patch-clamp techniques, e.g.,
the "cell-attached" mode, the "inside-out" mode, and the "whole
cell" mode (see, e.g., Ackerman et al., New Engl. J. Med.
336:1575-1595 (1997)). Whole cell currents are conveniently
determined using the standard methodology (see, e.g., Hamil et al.,
PFlugers. Archiv. 391:85 (1981). Other known assays include:
radiolabeled rubidium flux assays and fluorescence assays using
voltage-sensitive dyes (see, e.g. Vestergarrd-Bogind et al., J
Membrane Biol. 88:67-75 (1988); Daniel et al., J. Pharmacol. Meth.
25: 185-193 (1991): Holevinsky et al. J. Membrane Biology 137:59-70
(1994)). Assays for compounds capable of inhibiting or increasing
potassium flux through the channel proteins can be performed by
application of the compounds to a bath solution in contact with and
comprising cells having an channel of the present invention (see,
e.g., Blatz et al., Nature 323:718-720 (1986); Park, J. Physiol.
481:555-570 (1994)). Generally, the compounds to be tested are
present in the range from 1 pM to 100 mM.
[0247] The effects of the test compounds upon the function of the
channels can be measured by changes in the electrical currents or
ionic flux or by the consequences of changes in currents and flux.
Changes in electrical current or ionic flux are measured by either
increases or decreases in flux of cations such as potassium or
rubidium ions. The cations can be measured in a variety of standard
ways. They can be measured directly by concentration changes of the
ions or indirectly by membrane potential or by radiolabeling of the
ions. Consequences of the test compound on ion flux can be quite
varied. Accordingly, any suitable physiological change can be used
to assess the influence of a test compound on the channels of this
invention. The effects of a test compound can be measured by a
toxin binding assay. When the functional consequences are
determined using intact cells or animals, one can also measure a
variety of effects such as transmitter release (e.g., dopamine),
hormone release (e.g., insulin), transcriptional changes to both
known and uncharacterized genetic markers (e.g., northern blots),
cell volume changes (e.g., in red blood cells), immunoresponses
(e.g., T cell activation), changes in cell metabolism such as cell
growth or pH changes, and changes in intracellular second
messengers such as [Ca.sup.2+].
Prokaryotic Cation Channel Protein Mutated to Mimic a Functional
Eukaryotic Cation Channel Protein
[0248] Furthermore, as explained above, the present invention
extends to prokaryotic cation channel proteins mutated to mimic a
functional eukaryotic cation channel protein. These mutated cation
channel proteins have broad applications in assays for screening
potential drugs or therapeutic agents which potentially can
interact with eukaryotic cation channel proteins, and be used to
treat numerous conditions related to the function of cation channel
proteins in vivo, such as cardiac arrhythmia, diabetes mellitus,
seizure disorder, asthma or hypertension, to name only a few.
[0249] Presently available recombinant DNA techniques, such as site
directed mutagenesis for example, can be used to readily mutate one
or a number of codons of an isolated nucleic acid molecule
encoding. A prokaryotic cation channel protein which can then be
expressed to produce a mutated prokaryotic cation channel protein
which mimics a eukaryotic cation channel protein.
[0250] Furthermore, prokaryotic cation channel proteins having
applications in this aspect of the present invention comprise
prokaryotic potassium channel proteins, prokaryotic sodium channel
proteins, or prokaryotic calcium channel proteins. Such prokaryotic
cation channel proteins can be obtained from varying prokaryotic
organisms, such as E. coli, Streptomyces lividans, Clostridium
acetobutylicum, or Staphylcoccus aureus, to name only a few. More
specifically, a prokaryotic potassium channel protein comprising an
amino acid sequence of SEQ ID NOs:1, 2, 3, or 7, or conserved
variants thereof, can be mutated to mimic the physiological
functions and chemical properties of numerous eukaryotic cation
channel proteins. In a preferred embodiment, a potassium channel
protein from Streptomyces lividans is mutated to mimic the
physiological functions and chemical properties of a eukaryotic
cation channel protein, such as a eukaryotic potassium channel
protein, a eukaryotic sodium channel protein, or a eukaryotic
calcium channel protein. Consequently, a potential drug or agent
which interacts with a mutated prokaryotic channel protein of the
present invention, such as binding thereto for example, should
undergo the same or similar interactions with a eukaryotic cation
channel protein the prokaryotic cation channel protein was mutated
to mimic. Hence, a mutated prokaryotic cation channel protein of
the present invention can serve as a model for a specific
eukaryotic cation channel protein in screening potential drugs or
therapeutic agents for interaction therewith.
[0251] Moreover, pursuant to the present invention, and using
recombinant DNA techniques, a prokaryotic cation channel protein
can be mutated to mimic eukaryotic cation channel proteins from
numerous eukaryotic organisms, such as, for example, insects or
mammals. More specifically, a prokaryotic cation channel protein
can be mutated to mimic eukaryotic cation channel proteins from a
wide variety of eukaryotic organisms, such as Drosophila
melanogaster, Homo sapiens, C. elegans, Mus musculus, Arabidopsis
thaliana, or Rattus novegicus, to name only a few. Such eukaryotic
cation channel proteins comprise an amino acid sequence comprising
SEQ ID Nos: 4, 5, 6, 8, 9, 10, 11, 12, 13, or 14, or conserved
variants thereof.
[0252] In a preferred embodiment of the present invention, the
prokaryotic cation channel protein comprises a potassium channel
protein from Streptomyces lividans comprising an amino acid
sequence of SEQ ID NO:1, or conserved variants thereof, which is
mutated to comprise an amino acid sequence of SEQ ID NO:16, or
conserved variants thereof, in order to mimic the physiological
functions and chemical properties of a eukaryotic cation channel
protein comprising an amino acid sequence of SEQ ID NO:4. Moreover,
such a mutated prokaryotic cation channel protein of the present
invention is encoded by an isolated nucleic acid molecule
comprising a DNA sequence of SEQ ID NO:17, or degenerate variants
thereof.
[0253] Mutant Cation Channel Protein
[0254] Moreover, the present invention is directed to a mutant
cation channel protein. More specifically, the present invention
comprises a mutant potassium channel protein comprising an amino
acid sequence of SEQ ID NO:16, or conserved variants thereof.
[0255] The nomenclature used to define the polypeptides is that
specified by Schroder & Lubke, "The Peptides", Academic Press
(1965), wherein in accordance with conventional representation the
amino group at the N-terminal appears to the left and the carboxyl
group at the C-terminal to the right. NH.sub.2, refers to the amide
group present at the carboxy terminus when written at the right of
a polypeptide sequence.
[0256] Accordingly, conserved variants of an isolated mutant cation
channel protein of the present invention displaying substantially
equivalent activity to an isolated cation channel protein of the
present invention, are likewise contemplated for use in the present
invention. These modifications can be obtained through peptide
synthesis utilizing the appropriate starting material.
[0257] In keeping with standard polypeptide nomenclature, J. Biol.
Chem., 243:3552-59 (1969), abbreviations for amino acid residues
are shown in the following Table of Correspondence:
2 TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyr
tyrosine G Gly glycine F Phe phenylalanine M Met methionine A Ala
alanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine
V Val valine P Pro proline K Lys lysine H His histidine Q Gln
glutamine E Glu glutamic acid W Trp tryptophan R Arg arginine D Asp
aspartic acid N Asn asparagine C Cys cysteine
[0258] It should be noted that all amino-acid residue sequences are
represented herein by formulae whose left and right orientation is
in the conventional direction of amino-terminus to
carboxy-terminus. Furthermore, it should be noted that a dash at
the beginning or end of an amino acid residue sequence indicates a
peptide bond to a further sequence of one or more amino-acid
residues. The above Table is presented to correlate the
three-letter and one-letter notations which may appear alternately
herein.
[0259] Hence, an amino acid in the mutant cation channel protein of
the present invention can be changed in a non-conservative manner
(i.e., by changing an amino acid belonging to a grouping of amino
acids having a particular size or characteristic to an amino acid
belonging to another grouping) or in a conservative manner (i.e.,
by changing an amino acid belonging to a grouping of amino acids
having a particular size or characteristic to an amino acid
belonging to the same grouping). Such a conservative change
generally leads to less change in the structure and function of the
resulting polypeptide. The present invention should be considered
to include analogs whose sequences contain conservative changes
which do not significantly alter the activity or binding
characteristics of the resulting polypeptide.
[0260] The following is one example of various groupings of amino
acids:
[0261] Amino Acids with Nonpolar R Groups
[0262] Alanine
[0263] Valine
[0264] Leucine
[0265] Isoleucine
[0266] Proline
[0267] Phenylalanine
[0268] Tryptophan
[0269] Methionine
[0270] Amino Acids with Uncharged Polar R Groups
[0271] Glycine
[0272] Serine
[0273] Threonine
[0274] Cysteine
[0275] Tyrosine
[0276] Asparagine
[0277] Glutamine
[0278] Amino Acids with Charged Polar R Groups (Negatively Charged
at pH 6.0)
[0279] Aspartic acid
[0280] Glutamic acid
[0281] Basic Amino Acids (Positively Charged at pH 6.0)
[0282] Lysine
[0283] Arginine
[0284] Histidine (at pH 6.0)
[0285] Another grouping may be those amino acids with aromatic
groups:
[0286] Phenylalanine
[0287] Tryptophan
[0288] Tyrosine
[0289] Another grouping may be according to molecular weight (i.e.,
size of R groups):
[0290] Glycine 75
[0291] Alanine 89
[0292] Serine 105
[0293] Proline 115
[0294] Valine 117
[0295] Threonine 119
[0296] Cysteine 121
[0297] Leucine 131
[0298] Isoleucine 131
[0299] Asparagine 132
[0300] Aspartic acid 133
[0301] Glutamine 146
[0302] Lysine 146
[0303] Glutamic acid 147
[0304] Methionine 149
[0305] Histidine (at pH 6.0) 155
[0306] Phenylalanine 165
[0307] Arginine 174
[0308] Tyrosine 181
[0309] Tryptophan 204
[0310] Particularly preferred substitutions are:
[0311] Gin for Arg or Lys; and
[0312] His for Lys or Arg.
[0313] Amino acid substitutions may also be introduced to
substitute an amino acid with a particularly preferable property.
For example, a Cys may be introduced a potential site for disulfide
bridges with another Cys, or with a carrier of the present
invention. A His may be introduced as a particularly "catalytic"
site (i.e., His can act as an acid or base and is the most common
amino acid in biochemical catalysis). Pro may be introduced because
of its particularly planar structure, which induces .beta.-turns in
the polypeptide's structure. Alternately, D-amino acids can be
substituted for the L-amino acids at one or more positions.
Antibodies to an Isolated Mutant Cation Channel Protein of the
Invention
[0314] As explained above, the present invention further extends to
antibodies of a cation channel protein of the present invention, or
conserved variants thereof. Such antibodies include but are not
limited to polyclonal, monoclonal, chimeric, single chain, Fab
fragments, and an Fab expression library. The anti-mutant channel
cation protein antibodies of the invention may be cross reactive,
e.g., they may recognize cation channel proteins from different
species, and even different types of cation channel proteins, i.e.
potassium, sodium, calcium channel proteins, or their numerous
variants which are gated with different mechanisms (i.e.
voltage-gated, mechanical gated, ligand binding gated, etc.).
Polyclonal antibodies have greater likelihood of cross
reactivity.
[0315] Various procedures known in the art may be used for the
production of polyclonal antibodies to an isolated mutant cation
channel protein, or conserved variants thereof, of the present
invention. For the production of antibody, various host animals can
be immunized by injection with a mutant cation channel protein, or
conserved variants thereof, including but not limited to rabbits,
mice, rats, sheep, goats, etc. Furthermore, a mutant cation channel
protein, or conserved variants thereof, of the present invention,
may be conjugated to an immunogenic carrier, e.g., bovine serum
albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants
may be used to increase the immunological response, depending on
the host species, including but not limited to Freund's (complete
and incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and Corynebacterium parvum.
[0316] For preparation of monoclonal antibodies directed toward a
mutant cation channel protein of the present invention, or
conserved variants thereof, any technique that provides for the
production of antibody molecules by continuous cell lines in
culture may be used. These include but are not limited to the
hybridoma technique originally developed by Kohler and Milstein
[Nature 256:495-497 (1975)], as well as the trioma technique, the
human B-cell hybridoma technique [Kozbor et al., Immunology Today
4:72 1983): Cote et al., Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030
(1983)], and the EBV-hybridoma technique to produce human
monoclonal antibodies [Cole et al., in Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss. Inc., pp. 77-96 (1985)]. In an
additional embodiment of the invention, monoclonal antibodies can
be produced in germ-free animals utilizing recent technology
[PCT/US90/02545]. In fact, according to the invention, techniques
developed for the production of "chimeric antibodies" [Morrison et
al., J. Bacteriol. 159:870 (1984); Neuberger et al., Nature
312:604-608 (1984); Takeda et al., Nature 314:452454 (1985)] by
splicing the genes from a mouse antibody molecule specific for an
isolated mutant cation channel protein of the present invention, or
conserved variants thereof, together with a fragment of a human
antibody molecule of appropriate biological activity can be used;
such antibodies are within the scope of this invention.
[0317] According to the invention, techniques described for the
production of single chain antibodies [U.S. Pat. Nos. 5,476,786 and
5,132,405 to Huston; U.S. Pat. No. 4,946,778] can be adapted to
produce single chain antibodies specific for an isolated mutant
cation channel protein of the invention or conserved variants
thereof. An additional embodiment of the invention utilizes the
techniques described for the construction of Fab expression
libraries [Huse et al. Science 246:1275-1281 (1989)] to allow rapid
and easy identification of monoclonal Fab fragments with the
desired specificity for an isolated mutant cation channel protein
of the present invention, or conserved variants thereof.
[0318] Antibody fragments which contain the idiotype of the
antibody molecule can be generated by known techniques. For
example, such fragments include but are not limited to: the
F(ab').sub.2 fragment which can be produced by pepsin digestion of
the antibody molecule; the Fab' fragments which can be generated by
reducing the disulfide bridges of the F(ab').sub.2, fragment, and
the Fab fragments which can be generated by treating the antibody
molecule with papain and a reducing agent.
[0319] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.,
radioimmunoassay, ELISA (enzyme-linked immunosorbant assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitin reactions, immunodiffusion assays, in situ immunoassays
(using colloidal gold, enzyme or radioisotope labels, for example),
western blots, precipitation reactions, agglutination assays (e.g.,
gel agglutination assays, hemagglutination assays), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labeled. Many means are known in the art for detecting binding in
an immunoassay and are within the scope of the present invention.
For example, to select antibodies which recognize a specific
epitope of an isolated mutant cation channel protein of the present
invention, or conserved variants thereof, one may assay generated
hybridomas for a product which binds to a fragment of an isolated
mutant cation channel protein, or conserved variants thereof,
containing such epitope. For selection of an
[0320] The foregoing antibodies can be used in methods known in the
art relating to the localization and activity of an isolated mutant
cation channel protein, or conserved variants thereof, e.g., for
Western blotting, imaging such a cation channel protein in situ,
measuring levels thereof in appropriate physiological samples, etc.
using any of the detection techniques mentioned above or known in
the art.
[0321] In a specific embodiment, antibodies that agonize or
antagonize the activity of an isolated mutant cation channel
protein of the present invention, or conserved variants thereof,
can be generated. Such antibodies can be tested using the assays
described infra for identifying ligands.
Detectably Labeled Antibodies of an Isolated Mutant Cation Channel
Protein of the Present Invention, or Conserved Variants Thereof
[0322] Moreover, the present invention extends to antibodies
described above, detectably labeled. Suitable detectable labels
include enzymes, radioactive isotopes, fluorophores (e.g.,
fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red
(TR), rhodamine, free or chelated lanthanide series salts,
especially Eu.sup.3+, to name a few fluorophores), chromophores,
radioisotopes, chelating agents, dyes, colloidal gold, latex
particles, ligands (e.g., biotin), and chemiluminescent agents.
When a control marker is employed, the same or different labels may
be used for the receptor and control marker.
[0323] In the instance where a radioactive label, such as the
isotopes .sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.36Cl,
.sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe, .sup.90Y, .sup.125I,
.sup.131I, and .sup.186Re are used, known currently available
counting procedures may be utilized. In the instance where the
label is an enzyme, detection may be accomplished by any of the
presently utilized colorimetric, spectrophotometric,
fluorospectrophotometric, amperometric or gasometric techniques
known in the art.
[0324] Direct labels are one example of labels which can be used
according to the present invention. A direct label has been defined
as an entity, which in its natural state, is readily visible,
either to the naked eye, or with the aid of an optical filter
and/or applied stimulation, e.g. U.V. light to promote
fluorescence. Among examples of colored labels, which can be used
according to the present invention, include metallic sol particles,
for example, gold sol particles such as those described by
Leuvering (U.S. Pat. No. 4,313,734): dye sole particles such as
described by Gribnau et al. (U.S. Pat. No. 4,373,932) and May et
al. (WO 88/08534): dyed latex such as described by May, supra,
Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated in
liposomes as described by Campbell et al. (U.S. Pat. No.
4,703,017). Other direct labels include a radionucleotide, a
fluorescent moiety or a luminescent moiety. In addition to these
direct labeling devices, indirect labels comprising enzymes can
also be used according to the present invention. Various types of
enzyme linked immunoassays are well known in the art, for example,
alkaline phosphatase and horseradish peroxidase, lysozyme,
glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease,
these and others have been discussed in detail by Eva Engvall in
Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70,
419-439, 1980 and in U.S. Pat. No. 4,857,453.
[0325] Suitable enzymes include, but are not limited to, alkaline
phosphatase and horseradish peroxidase.
[0326] Other labels for use in the invention include magnetic beads
or magnetic resonance imaging labels.
[0327] As explained above, the present invention contemplates an
isolated nucleic molecule, or degenerate variants thereof, which
encode a mutant cation channel protein, or conserved variants
thereof. Accordingly, with the present invention, there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"): DNA
Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed.
1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization[B. D. Hames & S. J. Higgins eds. (1985)];
Transcription And Translation[B. D. Hames & S. J. Higgins, eds.
(1984)]; Animal Cell Culture[R. I. Freshney, ed. (1986)];
Immobilized Cells And Enzymes[IRL Press, (1986)]; B. Perbal, A
Practical Guide To Molecular Cloning (1984): F. M. Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons. Inc. (1994).
[0328] Therefore, if appearing herein, the following terms shall
have the definitions set out below.
[0329] A "vector" is a replicon, such as plasmid, phage or cosmid,
to which another DNA segment may be attached so as to bring about
the replication of the attached segment. A "replicon" is any
genetic element (e.g., plasmid, chromosome, virus) that functions
as an autonomous unit of DNA replication in vivo, i.e., capable of
replication under its own control.
[0330] A "cassette" refers to a segment of DNA that can be inserted
into a vector at specific restriction sites. The segment of DNA
encodes a polypeptide of interest, and the cassette and restriction
sites are designed to ensure insertion of the cassette in the
proper reading frame for transcription and translation.
[0331] A cell has been "transfected" by exogenous or heterologous
DNA when such DNA has been introduced inside the cell. A cell has
been "transformed" by exogenous or heterologous DNA when the
transfected DNA effects a phenotypic change. Preferably, the
transforming DNA should be integrated (covalently linked) into
chromosomal DNA making up the genome of the cell.
[0332] "Heterologous" DNA refers to DNA not naturally located in
the cell, or in a chromosomal site of the cell. Preferably, the
heterologous DNA includes a gene foreign to the cell.
[0333] A "nucleic acid molecule" refers to the phosphate ester
polymeric form of ribonucleosides (adenosine, guanosine, uridine or
cytidine: "RNA molecules") or deoxyribonucleos ides
(deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine:
"DNA molecules"), or any phosphoester analogs thereof, such as
phosphorothioates and thioesters, in either single stranded form,
or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and
RNA-RNA helices are possible. The term nucleic acid molecule, and
in particular DNA or RNA molecule, refers only to the primary and
secondary structure of the molecule, and does not limit it to any
particular tertiary forms. Thus, this term includes double-stranded
DNA found, inter alia, in linear or circular DNA molecules (e.g.,
restriction fragments), plasmids, and chromosomes. In discussing
the structure of particular double-stranded DNA molecules,
sequences may be described herein according to the normal
convention of giving only the sequence in the 5' to 3' direction
along the nontranscribed strand of DNA (i.e., the strand having a
sequence homologous to the mRNA). A "recombinant DNA molecule" is a
DNA molecule that has undergone a molecular biological
manipulation.
[0334] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength (see Sambrook et al.,
supra). The conditions of temperature and ionic strength determine
the "stringency" of the hybridization. For preliminary screening
for homologous nucleic acids, low stringency hybridization
conditions, corresponding to a T.sub.m of 55.degree., can be used,
e.g., 5.times.SCC, 0.1% SDS, 0.25% milk, and no formamide; or 30%
formamide, 5.times.SCC, 0.5% SDS). Moderate stringency
hybridization conditions correspond to a higher T.sub.m, e.g., 40%
formamide, with 5.times. or 6.times.SCC. High stringency
hybridization conditions correspond to the highest T.sub.m, e.g.,
50% formamide, 5.times. or 6.times. SCC. Hybridization requires
that the two nucleic acids contain complementary sequences,
although depending on the stringency of the hybridization,
mismatches between bases are possible. The appropriate stringency
for hybridizing nucleic acids depends on the length of the nucleic
acids and the degree of complementation, variables well known in
the art. The greater the degree of similarity or homology between
two nucleotide sequences, the greater the value of T.sub.m for
hybrids of nucleic acids having those sequences. The relative
stability (corresponding to higher T.sub.m) of nucleic acid
hybridizations decreases in the following order: RNA:RNA, DNA:RNA,
DNA:DNA. For hybrids of greater than 100 nucleotides in length,
equations for calculating T.sub.m have been derived (see Sambrook
et al., supra, 9.50-0.51). For hybridization with shorter nucleic
acids, i.e., oligonucleotides, the position of mismatches becomes
more important, and the length of the oligonucleotide determines
its specificity (see Sambrook et al., supra, 11.7-11.8). Preferably
a minimum length for a hybridizable nucleic acid is at least about
12 nucleotides; preferably at least about 18 nucleotides; and more
preferably the length is at least about 27 nucleotides: and most
preferably 36 nucleotides.
[0335] In a specific embodiment, the term "standard hybridization
conditions" refers to a T.sub.m of 55.degree. C., and utilizes
conditions as set forth above. In a preferred embodiment, the
T.sub.m is 60.degree. C.; in a more preferred embodiment, the
T.sub.m is 65.degree. C.
[0336] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in a cell in
vitro or in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxyl) terminus. A coding
sequence can include, but is not limited to, prokaryotic sequences,
cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic
(e.g., mammalian) DNA, and even synthetic DNA sequences. If the
coding sequence is intended for expression in a eukaryotic cell, a
polyadenylation signal and transcription termination sequence will
usually be located 3' to the coding sequence.
[0337] Transcriptional and translational control sequences are DNA
regulatory sequences, such as promoters, enhancers, terminators,
and the like, that provide for the expression of a coding sequence
in a host cell. In eukaryotic cells, polyadenylation signals are
control sequences.
[0338] A "promoter sequence" or "promoter" is a DNA regulatory
region capable of binding RNA polymerase in a cell and initiating
transcription of a downstream (3' direction) coding sequence. For
purposes of defining the present invention, the promoter sequence
is bounded at its 3' terminus by the transcription initiation site
and extends upstream (5' direction) to include the minimum number
of bases or elements necessary to initiate transcription at levels
detectable above background. Within the promoter sequence will be
found a transcription initiation site (conveniently defined for
example, by mapping with nuclease S1), as well as protein binding
domains (consensus sequences) responsible for the binding of RNA
polymerase.
[0339] A coding sequence is "under the control" of transcriptional
and translational control sequences in a cell when RNA polymerase
transcribes the coding sequence into mRNA, which is then trans-RNA
spliced and translated into the protein encoded by the coding
sequence.
[0340] As used herein, the term "sequence homology" in all its
grammatical forms refers to the relationship between proteins that
possess a "common evolutionary origin," including proteins from
superfamilies (e.g., the immunoglobulin superfamily) and homologous
proteins from different species (e.g., myosin light chain, etc.)
[Reeck et al., Cell, 50:667 (1987)].
[0341] Accordingly, the term "sequence similarity" in all its
grammatical forms refers to the degree of identity or
correspondence between nucleic acid or amino acid sequences of
proteins that do not share a common evolutionary origin [see Reeck
et al., 1987, supra]. However, in common usage and in the instant
application, the term "homologous" when modified with an adverb
such as "highly," may refer to sequence similarity and not a common
evolutionary origin.
[0342] In a specific embodiment, two DNA sequences are
"substantially homologous" or "substantially similar" when at least
about 50% (preferably at least about 75%, and most preferably at
least about 90 or 95%) of the nucleotides match over the defined
length of the DNA sequences. Sequences that are substantially
homologous can be identified by comparing the sequences using
standard software available in sequence data banks, or in a
Southern hybridization experiment under, for example, stringent
conditions as defined for that particular system. Defining
appropriate hybridization conditions is within the skill of the
art. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I &
II, supra, Nucleic Acid Hybridization, supra.
[0343] Similarly, in a particular embodiment, two amino acid
sequences are "substantially homologous" or "substantially similar"
when greater than 30% of the amino acids are identical, or greater
than about 60% are similar (functionally identical). Preferably,
the similar or homologous sequences are identified by alignment
using, for example, the GCG (Genetics Computer Group, Program
Manual for the GCG Package, Version 7, Madison, Wis.) pileup
program.
[0344] The term "corresponding to" is used herein to refer similar
or homologous sequences, whether the exact position is identical or
different from the molecule to which the similarity or homology is
measured. Thus, the term "corresponding to" refers to the sequence
similarity, and not the numbering of the amino acid residues or
nucleotide bases.
[0345] Moreover, due to degenerate nature of codons in the genetic
code, a mutant cation channel protein of the present invention can
be encoded by numerous isolated nucleic acid molecules. "Degenerate
nature" refers to the use of different three-letter codons to
specify a particular amino acid pursuant to the genetic code. It is
well known in the art that the following codons can be used
interchangeably to code for each specific amino acid:
[0346] Phenylalanine (Phe or F) UUU or UUC
[0347] Leucine (Leu or L) UUA or UUG or CUU or CUC or CUA or
CUG
[0348] Isoleucine (Ile or I) AUU or AUC or AUA
[0349] Methionine (Met or M) AUG
[0350] Valine (Val or V) GUU or GUC of GUA or GUG
[0351] Serine (Ser or S) UCU or UCC or UCA or UCG or AGU or AGC
[0352] Proline (Pro or P) CCU or CCC or CCA or CCG
[0353] Threonine (Thr or T) ACU or ACC or ACA or ACG
[0354] Alanine (Ala or A) GCU or GCG or GCA or GCG
[0355] Tyrosine (Tyr or Y) UAU or UAC
[0356] Histidine (His or H) CAU or CAC
[0357] Glutamine (Gln or Q) CAA or CAG
[0358] Asparagine (Asn or N) AAU or AAC
[0359] Lysine (Lys or K) AAA or AAG
[0360] Aspartic Acid (Asp or D) GAU or GAC
[0361] Glutamic Acid (Glu or E) GAA or GAG
[0362] Cysteine (Cys or C) UGU or UGC
[0363] Arginine (Arg or R) CGU or CGC or CGA or CGG or AGA or
AGG
[0364] Glycine (Gly or G) GGU or GGC or GGA or GGG
[0365] Tryptophan (Trp or W) UGG
[0366] Termination codon UAA (ochre) or UAG (amber) or UGA
(opal)
[0367] It should be understood that the codons specified above are
for RNA sequences. The corresponding codons for DNA have a T
substituted for U.
[0368] Furthermore, the present invention extends to an isolated
nucleic acid molecule, or degenerate variants thereof encoding a
mutant cation channel protein, detectably labeled, and a detectably
labeled isolated nucleic acid molecule hybridizable under standard
hybridization conditions to an isolated nucleic acid molecule, or
degenerate variants thereof, encoding a cation channel protein of
the present invention. Suitable detectable labels include enzymes,
radioactive isotopes, fluorophores (e.g., fluorescene
isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR),
rhodamine, free or chelated lanthanide series salts, especially
Eu.sup.3+, to name a few fluorophores), chromophores,
radioisotopes, chelating agents, dyes, colloidal gold, latex
particles, ligands (e.g., biotin), and chemiluminescent agents.
When a control marker is employed, the same or different labels may
be used for the receptor and control marker.
[0369] In the instance where a radioactive label, such as the
isotopes .sup.3H, .sup.14C, .sup.32P, .sup.35S, .sup.36CL,
.sup.51CR, .sup.57Co, .sup.58Co, .sup.59Fe, .sup.90Y, .sup.125I,
.sup.131I, and .sup.156Re, are used, known currently available
counting procedures may be utilized. In the instance where the
label is an enzyme, detection may be accomplished by any of the
presently utilized calorimetric, spectrophotometric,
fluorospectrophotometric, amperometric or gasometric techniques
known in the art.
[0370] Direct labels are one example of labels which can be used
according to the present invention. A direct label has been defined
as an entity, which in its natural state, is readily visible,
either to the naked eye, or with the aid of an optical filter
and/or applied stimulation, e.g. U.V. light to promote
fluorescence. Among examples of colored labels, which can be used
according to the present invention, include metallic sot particles,
for example, gold sot particles such as those described by
Leuvering (U.S. Pat. No. 4,313,734); dye sole particles such as
described by Gribnau et al. (U.S. Pat. No. 4,373,932) and May et
al. (WO 88/08534): dyed latex such as described by May, supra,
Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated in
liposomes as described by Campbell et al. (U.S. Pat. No.
4,703,017). Other direct labels include a radionucleotide, a
fluorescent moiety or a luminescent moiety. In addition to these
direct labeling devices, indirect labels comprising enzymes can
also be used according to the present invention. Various types of
enzyme linked immunoassays are well known in the art, for example,
alkaline phosphatase and horseradish peroxidase, lysozyme,
glucose-6-phosphate dehydrogenase, lactate dehydrogenase, urease,
these and others have been discussed in detail by Eva Engvall in
Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology,
70.419-439, 1980 and in U.S. Pat. No. 4,857,453.
[0371] Suitable enzymes include, but are not limited to, alkaline
phosphatase and horseradish peroxidase.
[0372] Other labels for use in the invention include magnetic beads
or magnetic resonance imaging labels.
Cloning Vectors
[0373] The present invention also extends to cloning vectors
comprising an isolated nucleic acid molecule of the present
invention, or degenerate variants thereof, and an origin of
replication. For purposes of this Application, an "origin of
replication refers to those DNA sequences that participate in DNA
synthesis.
[0374] As explained above, in an embodiment of the present
invention, an isolated nucleic acid molecule, or degenerate
variants thereof, encoding a mutant cation channel protein of the
present invention, along with isolated nucleic acid molecules
hybridizable under standard hybridization conditions to an isolated
nucleic acid, or degenerate variants thereof, which encodes a
mutant cation channel protein of the present invention, can be
inserted into an appropriate cloning vector in order to produce
multiple copies of the isolated nucleic acid. A large number of
vector-host systems known in the art may be used. Possible vectors
include, but are not limited to, plasmids or modified viruses, but
the vector system must be compatible with the host cell used.
Examples of vectors include, but are not limited to, E. coli,
bacteriophages such as lambda derivatives, or plasmids such as
pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors,
pmal-c, pFLAG, etc. The insertion into a cloning vector can, for
example, be accomplished by ligating an isolated nucleic acid
molecule of the present invention or degenerate variants thereof,
or an isolated nucleic acid hybridizable thereto under standard
hybridization conditions, into a cloning vector which has
complementary cohesive termini. However, if the complementary
restriction sites used to fragment the isolated nucleic acid or
degenerate variants thereof, or an isolated nucleic acid
hybridizable thereto under standard hybridization conditions, are
not present in the cloning vector, the ends of the isolated nucleic
acid molecule or degenerate variants thereof, or an isolated
nucleic acid molecule hybridizable under standard hybridization
conditions thereto may be enzymatically modified. Alternatively,
any site desired may be produced by ligating nucleotide sequences
(linkers) onto the DNA termini; these ligated linkers may comprise
specific chemically synthesized oligonucleotides encoding
restriction endonuclease recognition sequences. Such recombinant
molecules can then be introduced into host cells via
transformation, transfection, infection, electroporation, etc., so
that many copies of an isolated nucleic acid molecule of the
present invention, or degenerate variants thereof, or an an
isolated nucleic acid molecule hybridizable thereto under standard
hybridization conditions, can be generated. Preferably, the cloned
isolated nucleic acid molecule is contained on a shuttle vector
plasmid, which provides for expansion in a cloning cell, e.g., E.
coli, and facile purification for subsequent insertion into an
appropriate expression cell line, if such is desired. For example,
a shuttle vector, which is a vector that can replicate in more than
one type of organism, can be prepared for replication in both E.
coli and Saccharomyces cerevisiae by linking sequences from an E.
coli plasmid with sequences from the yeast 2.mu. plasmid.
[0375] In an alternative method, an isolated nucleic acid molecule
of the present invention, or degenerate variants thereof, or an
isolated nucleic acid molecule hybridizable thereto under standard
hybridization conditions may be identified and isolated after
insertion into a suitable cloning vector in a "shot gun" approach.
Enrichment for an isolated nucleic acid molecule, for example; by
size fractionation, can be done before insertion into the cloning
vector.
Expression Vectors
[0376] As stated above, the present invention extends to an
isolated nucleic acid molecule encoding a mutant cation channel
protein of the present invention, degenerate variants thereof, or
an isolated nucleic acid hybridizable thereto under standard
hybridization conditions.
[0377] Isolated nucleic acid molecules of the present invention can
be inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted protein-coding sequence. Such elements
are termed herein a "promoter." Thus, an isolated nucleic acid
molecule, or degenerate variants thereof, which encodes a mutant
cation channel protein of the present, along with isolated nucleic
acid molecules hybridizable thereto under standard hybridization
conditions is operatively associated with a promoter in an
expression vector of the invention. A DNA sequence is "operatively
associated" to an expression control sequence, such as a promoter,
when the expression control sequence controls and regulates the
transcription and translation of that DNA sequence. The term
"operatively associated" includes having an appropriate start
signal (e.g., ATG) in front of the DNA sequence to be expressed and
maintaining the correct reading frame to permit expression of the
DNA sequence under the control of the expression control sequence
and production of the desired product encoded by the DNA sequence.
If an isolated nucleic acid molecule of the present invention does
not contain an appropriate start signal, such a start signal can be
inserted into the expression vector in front of (5' of) the
isolated nucleic acid molecule.
[0378] Both cDNA and genomic sequences can be cloned and expressed
under control of such regulatory sequences. An expression vector
also preferably includes a replication origin.
[0379] The necessary transcriptional and translational signals can
be provided on a recombinant expression vector, or they may be
supplied by the native gene encoding the wild type variant of a
mutant cation channel protein of the present invention, and/or its
flanking regions.
[0380] Potential host-vector systems include but are not limited to
mammalian cell systems infected with virus (e.g., vaccinia virus,
adenovirus, etc.); insect cell systems infected with virus (e.g.,
baculovirus); microorganisms such as yeast containing yeast
vectors; or bacteria transformed with bacteriophage, DNA, plasmid
DNA, or cosmid DNA. The expression elements of vectors vary in
their strengths and specificities. Depending on the host-vector
system utilized, any one of a number of suitable transcription and
translation elements may be used.
[0381] Moreover, an isolated nucleic acid molecule of the present
invention may be expressed chromosomally, after integration of the
coding sequence by recombination. In this regard, any of a number
of amplification systems may be used to achieve high levels of
stable gene expression (See Sambrook et al. 1989, supra).
[0382] A unicellular host containing a recombinant vector
comprising an isolated nucleic acid molecule, or degenerate
variants thereof, which encodes a mutant cation channel protein of
the present invention, or an isolated nucleic acid molecule
hybridizable under standard hybridization conditions to an isolated
nucleic acid molecule, or degenerate variants thereof, which
encodes a mutant cation channel protein of the present invention,
is cultured in an appropriate cell culture medium under conditions
that provide for expression of the isolated nucleic acid molecule
by the cell.
[0383] Any of the methods previously described for the insertion of
DNA fragments into a cloning vector may be used to construct
expression vectors comprising an isolated nucleic acid molecule of
the present invention, and appropriate
transcriptional/translational control signals and the protein
coding sequences. These methods may include in vitro recombinant
DNA and synthetic techniques and in vivo recombination (genetic
recombination).
[0384] Expression of an isolated nucleic acid molecule of the
present invention, degenerate variants thereof, or an isolated
nucleic acid molecule hybridizable thereto under standard
hybridization conditions, along with a an isolated mutant cation
channel protein encoded by isolated nucleic acid molecules of the
present invention, degenerate variants thereof, or an isolated
nucleic acid molecule hybridizable thereto under standard
hybridization conditions, may be controlled by any
promoter/enhancer element known in the art, but these regulatory
elements must be functional in the host selected for expression.
Promoters which may be used to control expression include, but are
not limited to, the SV40 early promoter region (Benoist and
Chambon, 1981, Nature 290:304-310), the promoter contained in the
3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al.,
1980, Cell 22:787-797), the herpes thymidine kinase promoter
(Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445),
the regulatory sequences of the metallothionein gene (Brinster et
al., 1982. Nature 296:3942): prokaryotic expression vectors such as
the .beta.-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc.
Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer,
et al., 1983. Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also
"Useful proteins from recombinant bacteria" in Scientific American,
1980, 242:74-94; promoter elements from yeast or other fungi such
as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter.
PGK (phosphoglycerol kinase) promoter, alkaline phosphatase
promoter; and the animal transcriptional control regions, which
exhibit tissue specificity and have been utilized in transgenic
animals: elastase I gene control region which is active in
pancreatic acinar cells (Swift et al., 1984. Cell 38:639-646;
Ornitz et al. 1986. Cold Spring Harbor Symp. Quant. Biol.
50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene
control region which is active in pancreatic beta cells (Hanahan,
1985, Nature 315:115-122), immunoglobulin gene control region which
is active in lymphoid cells (Grosschedl et al., 1984, Cell
38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et
al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus
control region which is active in testicular, breast, lymphoid and
mast cells (Leder et al., 1986. Cell 45:485-495), albumin gene
control region which is active in liver (Pinkert et al., 1987,
Genes and Devel. 1:268-276), alpha-fetoprotein gene control region
which is active in liver (Krumlauf et al. 1985, Mol. Cell. Biol.
5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha
1-antitrypsin gene control region which is active in the liver
(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene
control region which is active in myeloid cells (Mogram et al.,
1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94),
myelin basic protein gene control region which is active in
oligodendrocyte cells in the brain (Readhead et al., 1987. Cell
48:703-712), myosin light chain-2 gene control region which is
active in skeletal muscle (Sani, 1985, Nature 314:283-286), and
gonadotropic releasing hormone gene control region which is active
in the hypothalamus (Mason et al. 1986, Science 234:1372-1378).
[0385] Expression vectors comprising an isolated nucleic acid
molecule, or degenerate variants thereof, encoding a mutant cation
channel protein of the present invention, or an expression vector
comprising an isolated nucleic acid molecule hybridizable under
standard hybridization conditions to an isolated nucleic acid
molecule of the present invention, can be identified by four
general approaches: (a) PCR amplification of the desired plasmid
DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence
or absence of selection marker gene functions, and (d) expression
of inserted sequences. In the first approach, the nucleic acids can
be amplified by PCR to provide for detection of the amplified
product. In the second approach, the presence of a foreign gene
inserted in an expression vector can be detected by nucleic acid
hybridization using probes comprising sequences that are homologous
to an inserted marker gene. In the third approach, the recombinant
vector/host system can be identified and selected based upon the
presence or absence of certain "selection marker" gene functions
(e.g., .beta.-galactosidase activity, thymidine kinase activity,
resistance to antibiotics, transformation phenotype, occlusion body
formation in baculovirus, etc.) caused by the insertion of foreign
genes in the vector. In another example, if an isolated nucleic of
the present invention, or degenerate variants thereof, which encode
a mutant cation channel protein of the present invention or
conserved variants thereof, or an isolated nucleic acid molecule
hybridizable thereto under standard hybridization conditions, is
inserted within the "selection marker" gene sequence of the vector,
recombinants containing the insert can be identified by the absence
of the inserted gene function. In the fourth approach, recombinant
expression vectors can be identified by assaying for the activity,
biochemical, or immunological characteristics of the gene product
expressed by the recombinant, provided that the expressed protein
assumes a functionally active conformation.
Production of a Mutant Cation Channel Protein of the Present
Invention
[0386] Moreover, the present invention extends to a method of
producing a mutant cation channel protein comprising an amino acid
sequence of SEQ ID NO:16, or conserved variants thereof. More
specifically, a method of the present invention comprises the steps
of culturing a unicellular host either transformed or transfected
with an expression vector of the present invention explained above,
under conditions that provide for expression of the mutant cation
channel protein, and recovering the mutant cation channel protein
from the transformed or transfected unicellular host. As explained
above, the conditions which provide for expression of a mutant
channel protein of the present invention are dependent upon the
expression vector and promoter used to transform or transfect a
unicellular host of the invention. Since the conditions needed
relative to the promoter used are within the knowledge of one of
ordinary skill in this art, conditions for specific promoters are
not repeated here.
[0387] Moreover, collection of a cation channel protein of the
present invention produced pursuant to the method stated above, is
also within the knowledge of a skilled artisan.
Crystal of a Cation Channel Protein
[0388] As explained above, the present invention extends to a
crystal of a cation channel protein having a central pore, which is
found natively in a lipid bilayer membrane of an animal cell, such
that the central pore communicates with extracellular matrix and
cellular cytosol, wherein the crystal effectively diffracts x-rays
to a resolution of greater than 3.2 angstroms.
[0389] Moreover, the present invention extends to a crystal of a
cation channel protein as described above, wherein the cation
channel protein comprises a first layer of aromatic amino acid
residues positioned to extend into the lipid bilayer membrane
proximate to the interface an extracellular matrix and lipid
bilayer membrane, a second layer of aromatic amino acid residues
positioned to extend into the lipid bilayer membrane proximate to
the interface of cellular cytosol and said lipid bilayer membrane,
a tetramer of four identical transmembrane subunits, and a central
pore formed by the four identical transmembrane subunits.
[0390] Furthermore, each transmembrane subunit comprises an inner
transmembrane alpha-helix which has a kink therein, an outer
transmembrane alpha-helix, and a pore alpha-helix, wherein each
subunit is inserted into the tetramer of the cation channel protein
so that the outer transmembrane helix of each subunit contacts the
first and second layers of aromatic amino acid residues described
above, and abuts the lipid bilayer membrane. Moreover, the inner
transmembrane helix of each subunit abuts the central pore of the
cation channel protein, contacts the first and second layers of
aromatic amino acid residues, is tilted by about 25.degree. with
respect to the normal of the lipid bilayer membrane, and is packed
against inner transmembrane alpha helices of other transmembrane
subunits at the second layer of aromatic amino acid residues
forming a bundle at the second layer. The pore alpha-helix of each
subunit is located at the first layer of said aromatic amino acid
residues, and positioned between inner transmembrane alpha-helices
of adjacent subunits, and are directed, in an amino carboxyl sense,
towards a point near the center of the central pore.
[0391] It has been further determined, based on examination of a
crystal of the present invention, that the central pore of a cation
channel protein, comprises a pore region located at the first layer
of aromatic amino acid residues, and connected to the inner and
outer transmembrane alpha-helices of said subunits. More
particularly, the pore region comprises about 25-45 amino acid
residues, a turret connected to the pore alpha-helix and the outer
alpha-helix, wherein the turret is located at the interface of said
extracellular matrix and the lipid bilayer membrane. The pore
region further comprises an ion selectivity filter connected to the
pore alpha-helix and the inner transmembrane alpha-helix of each
subunit. The ion selectivity filter extends into the central pore
of the cation channel protein, and comprises a signature amino acid
residue sequence having main chain atoms which create a stack of
sequential oxygen atoms along the selectivity filter that extend
into the central pore, and amino acid residues having side chains
that interact with the pore helix. It is the signature sequence
which enables a cation channel protein to discriminate among the
cation intended to permeate the protein, and other cations, so that
only the cation intended to permeate the channel protein is
permitted to permeate.
[0392] The central pore further comprises a tunnel into the lipid
bilayer membrane which communicates with the cellular cytosol, and
a cavity located within the lipid bilayer membrane between the pore
region and the tunnel, and connected to the them, such that the
central pore crosses the membrane.
[0393] Furthermore, the structure of all ion channel proteins share
common features, which are set forth in the crystal of a cation
channel protein described above. Consequently, the present
invention extends to a crystal of a cation channel protein having a
central pore, which is described above, wherein the cation is
selected from the group consisting of: Na.sup.+, K.sup.+, and
Ca.sup.2+. Hence, the present invention extends to crystals of
potassium channel proteins, sodium channel proteins, and calcium
ion channels, to name only a few. In a preferred embodiment, the
crystal of a cation channel protein comprises a crystal of a
potassium ion channel protein.
[0394] In addition, a crystal of an ion channel protein of a
present invention can comprise an amino acid sequence of any
presently known, or subsequently discovered cation protein channel.
Consequently, the present invention extends to a crystal of a
cation channel protein having a central pore, which is found
natively in a lipid bilayer membrane of an animal cell, such that
the central pore communicates with extracellular matrix and
cellular cytosol, wherein the crystal comprises an amino acid
sequence of:
[0395] residues 23 to 119 of SEQ ID NO:1 (Streptomyces
lividans);
[0396] residues 61 to 119 of SEQ ID NO:2 (E. coli);
[0397] residues 61 to 119 of SEQ ID NO:3 (Clostridium
acetobutylicum);
[0398] residues 61 to 119 of SEQ ID NO:4 (Drosophila
melanogaster);
[0399] residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
[0400] residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
[0401] residues 61 to 119 of SEQ ID NO:7 (Paramecium
tetraaurelia);
[0402] residues 61 to 119 of SEQ ID NO:8 (C. elegans);
[0403] residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
[0404] residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
[0405] residues 61 to 119 of SEQ ID NO:11 (Arabidopsis
thaliana);
[0406] residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
[0407] residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus);
or
[0408] residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
[0409] or conserved variants thereof.
[0410] In a preferred embodiment, a crystal of the present
invention having a central pore, which is found natively in a lipid
bilayer membrane of an animal cell, such that the central pore
communicates with extracellular matrix and cellular cytosol,
comprises an amino sequence of amino acid residues 23 to 119 of SEQ
ID NO:1, has a space grouping of C2, and a unit cell of dimensions
of a=128.8 .ANG., b=68.9 .ANG., c=112.0 .ANG., and
.beta.=124.6.degree.. Moreover, preferably, the present invention
extends to a crystal as described above, wherein the cation
K.sup.+.
[0411] Furthermore, the present invention extends to a crystal of a
cation channel protein having a central pore, which is found
natively in a lipid bilayer membrane of an animal cell, such that
the central pore communicates with extracellular matrix and
cellular cytosol, wherein the channel protein comprises a signature
sequence comprising:
[0412] Thr-Val-Gly-Tyr-Gly-Asp (SEQ ID NO:15).
Method for Growing a Crystal of the Present Invention
[0413] The present invention further extends to a method for
growing a crystal of a cation channel protein having a central
pore, which is found natively in a lipid bilayer membrane of an
animal cell, such that the central pore communicates with
extracellular matrix and cellular cytosol, by sitting-drop vapor
diffusion. Such a method of the present invention comprises the
steps of providing the cation channel protein, removing a
predetermined number of carboxy terminal amino acid residues from
the cation channel protein to form a truncated cation channel
protein, dissolving the truncated cation channel protein in a
protein solution, such that the concentration of dissolved
truncated channel protein is about 5 to about 10 mg/ml, and mixing
equal volumes of protein solution with reservoir mixture at
20.degree. C. Preferably, the reservoir mixture comprises 200 mM
CaCl.sub.2, 100 mM Hepes, 48% PEG 400, pH 7.5, and the protein
solution comprises (150 mM KCl, 50 mM Tris, 2 mM DTT, pH 7.5).
[0414] Moreover, the present invention extends to a method of
growing a crystal of a cation channel protein as described above,
wherein a crystal can be grown comprising any type of cation
channel protein. In particular, the present invention can be used
to grow crystals of potassium channel proteins, sodium channel
proteins, or calcium channel proteins, to name only a few.
[0415] Furthermore, the present invention extends to a method of
growing a crystal of a cation channel protein, as described herein,
wherein the crystal comprises an amino acid sequence of:
[0416] residues 23 to 119 of SEQ ID NO:1 (Streptomyces
lividans);
[0417] residues 61 to 119 of SEQ ID NO:2 (E. coli);
[0418] residues 61 to 119 of SEQ ID NO:3 (Clostridium
acetobutylicum);
[0419] residues 61 to 119 of SEQ ID NO:4 (Drosophila
melanogaster);
[0420] residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
[0421] residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
[0422] residues 61 to 119 of SEQ ID NO:7 (Paramecium
tetraaurelia);
[0423] residues 61 to 119 of SEQ ID NO:8 (C. elegans);
[0424] residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
[0425] residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
[0426] residues 61 to 119 of SEQ ID NO:11 (Arabidopsis
thaliana);
[0427] residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
[0428] residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or
residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
[0429] or conserved variants thereof.
Use of Crystal of a Cation Channel Protein in Assay Systems for
Screening Drugs and Agents
[0430] In another embodiment, the present invention extends to a
method of using a crystal of a cation channel protein, as described
herein, in an assay system for screening drugs and other agents for
their ability to modulate the function of a cation channel protein,
comprising the steps of initially selecting a potential drug or
agent by performing rational drug design with the three-dimensional
structure determined for a crystal of the present invention,
wherein the selecting is performed in conjunction with computer
modeling. After potential drugs or agents have been selected, a
cation channel protein is contacted with the potential drug or
agent. If the drug or therapeutic agent has potential use for
modulating the function of a cation channel protein, a change in
the function of the cation channel after contact with the agent,
relative to the function of a similar cation channel protein not
contacted with the agent, or the function of the same cation
channel protein prior to contact with the agent. Hence, the change
in function is indicative of the ability of the drug or agent to
modulate the function of a cation channel protein.
[0431] Furthermore, the present invention extends to extends to a
method of using a crystal of a cation channel protein as described
herein, in an assay system for screening drugs and other agents for
their ability to modulate the function of a cation channel protein,
wherein the crystal comprises a Na.sup.+ channel protein, a K.sup.+
channel protein, or a Ca.sup.2+ channel protein.
[0432] The present invention further extends to a method of using a
crystal of a cation channel protein in an assay for screening drugs
or other agents for their ability to modulate the function of a
cation channel protein, wherein the crystal of the cation channel
protein comprises an amino acid sequence of:
[0433] residues 23 to 119 of SEQ ID NO:1 (Streptomyces
lividans);
[0434] residues 61 to 119 of SEQ ID NO:2 (E. coli);
[0435] residues 61 to 119 of SEQ ID NO:3 (Clostridium
acetobutylicum);
[0436] residues 61 to 119 of SEQ ID NO:4 (Drosophila
melanogaster);
[0437] residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
[0438] residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
[0439] residues 61 to 119 of SEQ ID NO:7 (Paramecium
tetraaurelia);
[0440] residues 61 to 119 of SEQ ID NO:8 (C. elegans);
[0441] residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
[0442] residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
[0443] residues 61 to 119 of SEQ ID NO:11 (Arabidopsis
thaliana);
[0444] residues 61 to 1119 of SEQ ID NO:12 (Homo sapiens);
[0445] residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus);
or
[0446] residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
[0447] or conserved variants thereof.
[0448] In a preferred embodiment of a method of using a crystal of
a cation channel protein in an assay for screening drugs or other
agents for their ability to modulate the function of a cation
channel protein, the crystal comprises a potassium channel protein,
comprising amino acid residues 23 to 119 of SEQ ID NO:1, a space
grouping of C2, and a unit cell of dimensions of a=128.8 .ANG.,
b=68.9 .ANG., c=112.0 .ANG., and .beta.=124.6.degree..
[0449] Moreover, it is important to note that a drug's or agent's
ability to modulate the function of a cation channel protein
includes, but is not limited to, increasing or decreasing the
cation channel protein's permeability to the specific cation
relative the permeability of the same or a similar not contacted
with the drug or agent, or the same cation channel protein prior to
contact with the drug or agent.
[0450] In a further embodiment, the present invention extends to a
method of using a crystal of a cation channel protein, as set forth
herein, in an assay system for screening drugs and other agents for
their ability to treat conditions related to the function of cation
channel proteins in vivo, and particularly in abnormal cellular
control processes related to the functioning of cation channel
protein. Such a method comprises the initial step of selecting a
potential drug or other agent by performing rational drug design
with the three-dimensional structure determined for a crystal of
the invention, wherein the selecting is performed in conjunction
with computer modeling. After potential drugs or therapeutic agents
are selected, a cation channel protein is contacted with the
potential drug or agent. If an interaction of the potential drug or
other agent with the cation channel is detected, it is indicative
of the potential use of the drug or agent to treat conditions
related the function of cation channel proteins in vivo. Examples
of such conditions include, but are not limited to, cardiac
arrhythmia, diabetes mellitus, seizure disorder, asthma or
hypertension, to name only a few.
[0451] Furthermore, a crystal of a cation channel protein used in
the method for screening drugs or agents for their ability to
interact with a cation channel comprises an Na.sup.+ channel
protein. K.sup.+ channel protein, or Ca.sup.2+ channel protein.
Hence, the method of the present invention can be used to screen
drugs or agents capable of treating conditions related to the
function of such channels.
[0452] Moreover, the present invention extends to a crystal used in
the method for screening drugs or agents for their ability to
interact with a cation channel protein comprising an amino acid
sequence of:
[0453] residues 23 to 119 of SEQ ID NO:1 (Streptomyces
lividans);
[0454] residues 61 to 119 of SEQ ID NO:2 (E. coli);
[0455] residues 61 to 119 of SEQ ID NO:3 (Clostridium
acetobutylicum);
[0456] residues 61 to 119 of SEQ ID NO:4 (Drosophila
melanogaster);
[0457] residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
[0458] residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
[0459] residues 61 to 119 of SEQ ID NO:7 (Paramecium
tetraaurelia);
[0460] residues 61 to 119 of SEQ ID NO:8 (C. elegans);
[0461] residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
[0462] residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
[0463] residues 61 to 119 of SEQ ID NO:11 (Arabidopsis
thaliana);
[0464] residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
[0465] residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or
residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
[0466] or conserved variants thereof.
[0467] In a preferred embodiment, a crystal used in a method for
screening drugs or agents for their ability to interact with a
cation channel, comprises amino acid residues 23 to 119 of SEQ ID
NO:1, has a space grouping of C2, and a unit cell of dimensions of
a=128.8 .ANG., b=68.9 .ANG., c=112.0 .ANG., and
.beta.=124.6.degree..
[0468] In yet another embodiment, the present invention extends to
a method of using a crystal of a cation channel protein described
herein, in an assay system for screening drugs and other agents for
their ability to permeate through a cation channel protein,
comprising an initial step of selecting a potential drug or other
agent by performing rational drug design with the three-dimensional
structure determined for the crystal, wherein the selecting of the
potential drug or agent is performed in conjunction with computer
modeling. After a potential drug or agent has been selected, a
cation channel protein can be prepared for use in the assay. For
example, preparing the cation channel protein can include isolating
the cation channel protein from the membrane of a cell, and then
inserting the cation channel protein into a membrane having a first
and second side which is impermeable to the potential drug or
agent. As a result, the cation channel protein traverses the
membrane, such that the extracellular portion of the cation channel
protein is located on the first side of the membrane, and the
intracellular portion of the cation channel protein is located on
the second side of the membrane. The extracellular portion of the
cation channel membrane can then be contacted with the potential
drug or agent. The presence of the drug or agent in the second side
of the membrane is indicative of the drug's or agent's potential to
permeate the cation channel protein, and the drug or agent is
selected based on its ability to permeate the cation channel
protein.
[0469] In addition, a crystal used in a method for screening drugs
or agents for their ability to permeate a cation channel can
comprise a Na.sup.+ channel protein, a K.sup.+ protein channel, or
a Ca.sup.2+ protein channel.
[0470] Furthermore, the present invention extends to the use of a
crystal in an assay system for screening drugs and other agents for
their ability to permeate through a cation channel protein, wherein
the crystal comprises an amino acid sequence of:
[0471] residues 23 to 119 of SEQ ID NO:1 (Streptomyces
lividans);
[0472] residues 61 to 119 of SEQ ID NO:2 (E. coli);
[0473] residues 61 to 119 of SEQ ID NO:3 (Clostridium
acetobutylicum);
[0474] residues 61 to 119 of SEQ ID NO:4 (Drosophila
melanogaster);
[0475] residues 61 to 119 of SEQ ID NO:5 (Homo sapiens);
[0476] residues 61 to 119 of SEQ ID NO:6 (Homo sapiens);
[0477] residues 61 to 119 of SEQ ID NO:7 (Paramecium
tetraaurelia);
[0478] residues 61 to 19 of SEQ ID NO:8 (C. elegans);
[0479] residues 61 to 119 of SEQ ID NO:9 (Mus musculus);
[0480] residues 61 to 119 of SEQ ID NO:10 (Homo sapiens);
[0481] residues 61 to 119 of SEQ ID NO:11 (Arabidopsis
thaliana);
[0482] residues 61 to 119 of SEQ ID NO:12 (Homo sapiens);
[0483] residues 61 to 119 of SEQ ID NO:13 (Rattus novegicus); or
residues 61 to 119 of SEQ ID NO:14 (Homo sapiens);
[0484] or conserved variants thereof.
[0485] In a preferred embodiment, the crystal used in an assay
system of the present invention for screening drugs and other
agents for their ability to permeate through a cation channel
protein comprises amino acid residues 23 to 119 of SEQ ID NO:1, has
a space grouping of C2, and a unit cell of dimensions of a=128.8
.ANG., b=68.9 .ANG., c=112.0 .ANG., and .beta.=124.6.degree..
[0486] In the assay systems disclosed herein, Once the
three-dimensional structure of a crystal comprising a cation
channel protein is determined, a potentia drugs and therapeutic
agents which may interact with a carrier channel protein, i.e. bind
or modulate the function thereof, or perhaps be able to permeate
through such a protein can be examined through the use of computer
modeling using a docking program such as GRAM, DOCK, or AUTODOCK
[Dunbrack et al., 1997, supra]. This procedure can include computer
fitting of potential drugs or agents to a cation channel protein to
ascertain how well the shape and the chemical structure of the
potential drug or agent will complement or interact with a cation
channel protein. [Bugg et al., Scientific American, Dec.:92-98
(1993); West et al., TIPS, 16:67-74 (1995)]. Computer programs can
also be employed to estimate the attraction, repulsion, and steric
hindrance of a potential drug or agent to a cation channel protein.
Generally the tighter the fit, the lower the steric hindrances, and
the greater the attractive forces, the more potent the potential
drug or agent, since these properties are consistent with a tighter
binding, and are clearly indicative of an interaction with a cation
channel protein. Furthermore, the more specificity in the design of
a potential drug the more likely that the drug will not interact as
well with other proteins. This will minimize potential side-effects
due to unwanted interactions with other proteins.
[0487] Furthermore, computer modeling programs based on the
structure of a cation channel protein in a crystal of the present
invention, can be used to modify potential drugs or agents in order
to identify potentially more promising drugs. Such analysis has
been shown to be effective in the development of HIV protease
inhibitors [Lam et al., Science 263:380-384 (1994): Wlodawer et
al., Ann. Rev. Biochem. 62:543-585 (1993); Appelt, Perspectives in
Drug Discovery and Design 1:23-48 (1993); Erickson, Perspectives in
Drug Discovery and Design 1:109-128 (1993)]. Alternatively a
potential drug or agent can be obtained by initially screening a
random peptide library produced by recombinant bacteriophage for
example, [Scott and Smith, Science, 249:386-390 (1990); Cwirla et
al., Proc. Natl. Acad. Sci., 87:6378-6382 (1990): Devlin et al.,
Science, 249:404-406 (1990)]. A peptide selected in this manner
would then be systematically modified by computer modeling programs
in odrer to enhance its potential interaction with a cation channel
protein.
[0488] Moreover, through the use of the three-dimensional structure
disclosed herein and computer modeling, a large number of these
compounds can be rapidly screened on the computer monitor screen,
and a few likely candidates can be determined without the laborious
synthesis of untold numbers of compounds.
[0489] Once a potential drug or agent is identified, it can be
either selected from a library of chemicals as are commercially
available from most large chemical companies including Merck,
GlaxoWelcome, Bristol Meyers Squib, Monsanto/Searle, Eli Lilly.
Novartis and Pharmacia UpJohn, alternatively the potential drug or
agent may be synthesized de novo. The de novo synthesis of one or
even a relatively small group of specific compounds is reasonable
in the art of drug design. The potential drug or agent can then be
placed into an assay of the present invention to determine whether
it binds with a cation channel protein.
[0490] When suitable potential drugs or agents are identified, a
supplemental crystal is grown which comprises a cation channel
protein. Preferably the crystal effectively diffracts X-rays for
the determination of the atomic coordinates of the protein-ligand
complex to a resolution of greater than 5.0 Angstroms, more
preferably greater than 3.0 Angstroms, and even more preferably
greater than 2.0 Angstroms. The three-dimensional structure of the
supplemental crystal is determined by Molecular Replacement
Analysis. Molecular replacement involves using a known
three-dimensional structure as a search model to determine the
structure of a closely related molecule or protein-ligand complex
in a new crystal form. The measured X-ray diffraction properties of
the new crystal are compared with the search model structure to
compute the position and orientation of the protein in the new
crystal. Computer programs that can be used include: X-PLOR and
AMORE [J. Navaza. Acta Crystallographics ASO, 157-163 (1994)]. Once
the position and orientation are known an electron density map can
be calculated using the search model to provide X-ray phases.
Thereafter, the electron density is inspected for structural
differences and the search model is modified to conform to the new
structure.
[0491] The present invention may be better understood by reference
to the following non-limiting Examples, which are provided as
exemplary of the invention. The following examples are presented in
order to more fully illustrate the preferred embodiments of the
invention.
[0492] They should in no way be construed, however, as limiting the
broad scope of the invention.
EXAMPLE I
[0493] Potassium Channel Structure: Molecular Basis of K.sup.+
Conduction and Selectivity
[0494] The K.sup.+ channel from Streptomyces lividans is an
integral membrane protein with sequence similarity to all known
K.sup.+ channels, particularly in the pore region. X-ray analysis
with data to 3.2 (reveals that four identical subunits create an
inverted tepee, or cone, cradling the selectivity filter of the
pore in its outer end. The narrow selectivity filter is only 12
.ANG. long, while the remainder of the pore is wider and lined with
hydrophobic amino acids. A large, water-filled cavity and helix
dipoles are positioned so as to overcome electrostatic
destabilization of an ion in the pore at the center of the bilayer.
Main-chain carbonyl oxygen atoms from the K.sup.+ channel signature
sequence line the selectivity filter, which is held open by
structural constraints to coordinate K.sup.+ ions but not smaller
Na.sup.+ ions. The selectivity filter contains two K.sup.+ ions
about 7.5 .ANG. apart. This configuration promotes ion conduction
by exploiting electrostatic repulsive forces to overcome attractive
forces between K.sup.+ ions and the selectivity filter. The
architecture of the pore establishes the physical principles
underlying selective K.sup.+ conduction.
[0495] More particularly, potassium ions diffuse rapidly across
cell membranes through proteins called K.sup.+ channels, which
underlie many fundamental biological processes including electrical
signaling in the nervous system. Potassium channels use diverse
mechanisms of gating (the processes by which the pore opens and
closes), but they all exhibit very similar ion permeability
characteristics (1). All K.sup.+ channels show a selectivity
sequence of K.sup.+ Rb.sup.+>Cs.sup.+, while permeability for
the smallest alkali metal ions Na.sup.+ and Li.sup.+ is
immeasurably low. Potassium is at least ten thousand times more
permeant than Na.sup.+, a feature that is essential to the function
of K.sup.+ channels. Potassium channels also share a constellation
of permeability characteristics that is indicative of a multi-ion
conduction mechanism. The flux of ions in one direction shows high
order coupling to flux in the opposite direction, and ionic
mixtures result in anomalous conduction behavior (2). Because of
these properties, K.sup.+ channels are classified as "long pore
channels", invoking the notion that multiple ions queue inside a
long, narrow pore in single-file fashion. In addition, the pores of
all K.sup.+ channels can be blocked by tetraethylammonium ions
(3).
[0496] Molecular cloning and mutagenesis experiments have
reinforced the conclusion that all K.sup.+ channels have
essentially the same pore constitution. Without exception, they
contain a critical amino acid sequence that has been termed the
K.sup.+ channel signature sequence. Mutation of these amino acids
disrupts the channel's ability to discriminate between K.sup.+ and
Na.sup.+ ions (4).
[0497] Biophysicists have been tantalized for the past quarter
century about chemical basis of the impressive fidelity with which
the channel distinguishes between K.sup.+ and Na.sup.+ ions, which
are featureless spheres of Pauling radius 1.33 .ANG. and 0.95 .ANG.
and the ability of K.sup.+ channels to be concurrently so highly
selective and exhibit a throughput rate approaching the diffusion
limit. The 10.sup.4 margin by which K.sup.+ is selected over
Na.sup.+ implies strong energetic interactions between K.sup.+ ions
and the pore. And yet strong energetic interactions seem
incongruent with throughput rates up to 10.sup.8 ions per
second.
[0498] Potassium Channel Architecture
[0499] Amino acid sequences show the relationship of the K.sup.+
channel from Streptomyces lividans (kcsa K.sup.+ channel) (5) to
other channels in biology, including vertebrate and invertebrate
voltage-dependent K.sup.+ channels, vertebrate inward rectifier and
Ca.sup.2+-activated K.sup.+ channels, K.sup.+ channels from plants
and bacteria, and cyclic nucleotide-gated cation channels (FIG. 1).
On the basis of hydrophobicity analysis, there are two closely
related varieties of K.sup.+ channels, those containing two
membrane-spanning segments per subunit and those containing six. In
all cases, the functional K.sup.+ channel protein is a tetramer
(6), typically of four identical subunits (7). Subunits of the two
membrane-spanning variety appear to be shortened versions of their
larger counterparts, as if they simply lack the first four
membrane-spanning segments. Though the kcsa K.sup.+ channel belongs
to the two membrane-spanning set of K.sup.+ channels, its amino
acid sequence is actually closer to those of eukaryotic six
membrane-spanning K.sup.+ channels. In particular, its sequence in
the pore region, located between the membrane-spanning stretches
and containing the K.sup.+ channel signature sequence, is nearly
identical to that found in the Drosophila (Shaker) and vertebrate
voltage-gated K.sup.+ channels (FIG. 1). Moreover, through a study
of the kcsa K.sup.+ channel interaction with eukaryotic K.sub.+
channel toxins, as described infra, it has been confirmed that the
kcsa K.sup.+ pore structure is indeed very similar to that of
eukaryotic K.sup.+ channels, and that its structure is maintained
when it is removed from the membrane using detergent (8).
[0500] Furthermore, the kcsa K.sup.+ channel structure from residue
position 23 to 119 of SEQ ID NO:1 has been determined with X-ray
crystallography (Table 1). The cytoplasmic carboxyl terminus
(residues 126 to 158 of SEQ ID NO:1) were removed in the
preparation and the remaining residues were disordered. The kcsa
K.sup.+ channel crystals are radiation sensitive and the
diffraction pattern is anisotropic, with reflections observed along
the best and worst directions at 2.5 .ANG. and 3.5 .ANG. Bragg
spacings, respectively. By careful, data selection, anisotropy
correction, introduction of heavy atom sites by site-directed
mutagenesis, averaging and solvent flattening, an interpretable
electron density map has been calculated (FIG. 2, A-C). This map
was without main chain breaks and showed strong side chain density
(FIG. 2C). The model was refined with data to 3.2 .ANG. (the data
set was 93% complete to 3.2 .ANG. with 67% completeness between 3.3
.ANG. and 3.2 .ANG.), maintaining highly restrained stereochemistry
and keeping tight noncrystallographic symmetry of cation channel
proteins, and that they all will have four inner helices arranged
like the poles of a tepee, four pore helices, and a selectivity
filter--tuned to select the appropriate cation--located close to
the extracellular surface.
[0501] Surprisingly, this structure of the kcsa K.sup.+ channel is
in excellent agreement with extensive functional and mutagenesis
studies on Shaker and other eukaryotic K.sup.+ channels (FIG.
4).
[0502] The pore-region of K.sup.+ channels was first discovered
with pore-blocking scorpion toxins (11). These inhibitors interact
with amino acids (white) comprising the broad extracellular-facing
entryway to the pore (12). The impermeant organic cation
tetraethylammonium (TEA) blocks K.sup.+ channels from both sides of
the membrane at distinct sites (13). Amino acids interacting with
externally and internally applied TEA are located just external to
(yellow) and internal to (mustard) the structure formed by the
signature sequence amino acids (14, 15). Alteration of the
signature sequence amino acids (red main chain atoms) disrupts
K.sup.+ selectivity (4). Amino acids close to the intracellular
opening on the Shaker K.sup.+ channel map to the inner helix on the
kcsa K.sup.+ channel (16). Interestingly, exposure to the cytoplasm
of the region above the inner helix bundle (pink side chains)
requires an open voltage-dependent gate, whereas the region at or
below the bundle (green side chains) is exposed whether or not the
gate was open. The correlation between the transition zone for gate
dependent exposure to the cytoplasm in the Shaker K.sup.+ channel
and the inner helix bundle in this structure has implications for
mechanisms of gating in K.sup.+ channels.
[0503] General Properties of the Ion Conduction Pore
[0504] Both the intracellular and extracellular entryways are
charged negative by acidic amino acids (FIG. 5A, red), an effect
that would raise the local concentration of cations while lowering
the concentration of anions. The overall length of the pore is
about 45 .ANG. and its diameter varies along its distance (FIG.
5B). From inside the cell (bottom) the pore begins as a tunnel
about 18 .ANG. in length (the internal pore) and then opens into a
wide cavity (about 10 .ANG. across) near the middle of the
membrane. A K.sup.+ ion could move throughout the internal pore and
cavity and still remain mostly hydrated. In contrast, the
selectivity filter separating the cavity from the extracellular
solution is so narrow that a K.sup.+ ion would have to shed its
hydrating waters to enter. The chemical composition of the wall
lining the internal pore and cavity is predominantly hydrophobic
(FIG. 5A, yellow). The selectivity filter, on the other hand, is
lined exclusively by polar main chain atoms belonging to the
signature sequence amino acids. The distinct mechanisms operating
in the cavity and internal pore versus the selectivity filter are
discussed below.
[0505] As explained above, potassium channel proteins exclude the
smaller alkali metal cations Li.sup.+, (radius 0.60 .ANG.) and
Na.sup.+ (0.95 .ANG.) but allow permeation of the larger members of
the series Rb.sup.- (1.48 .ANG.) and Cs.sup.+ (1.69 .ANG.). In fact
Rb.sup.+ is nearly the perfect K.sup.+ (1.33 .ANG.) analog as its
size and permeability characteristics are very similar to those of
K.sup.+. Because they are more electron dense than K.sup.+,
Rb.sup.+ and Cs.sup.+ allow visualization of the locations of
permeant ions in the pore. By difference electron density maps
calculated with data from crystals transferred into
Rb.sup.+-containing (FIG. 6A) or Cs.sup.+-containing (FIG. 6B)
solutions, multiple ions are well-defined in the pore. The
selectivity filter contains two ions (inner and outer ions) located
at opposite ends, about 7.5 .ANG. apart (center to center). In the
Rb.sup.+ difference map, there actually are two partially separated
peaks at the inner aspect of the selectivity filter. These peaks
are too close to each other (2.6 .ANG.) to represent two
simultaneously occupied ion binding sites. Although Applicant ise
under no obligation to explain such peaks, and is not to be bound
by any explanations. Applicant merely postulates these peaks may
represent a single ion (on average) in rapid equilibrium between
adjacent sites. The single inner ion peak in the Cs.sup.+
difference map undoubtedly reflects the lower resolution at which
the map was calculated (to 5 .ANG. for Cs.sup.+ versus 4.0 .ANG.
for Rb.sup.+) since the Rb.sup.+ difference map, when calculated at
the same lower resolution, also shows only a single peak at the
Cs.sup.+ position. The Rb.sup.+ positions correspond to strong
peaks (presumably K.sup.+ ions) in a high contour native electron
density map (not shown). Thus, the selectivity filter may contain
two K.sup.+ ions. A third weaker peak is located below the
selectivity fitter at the center of the large cavity in the
Rb.sup.+ difference map (FIG. 6A, tower peak) and in the Cs.sup.+
difference map at lower contour (not shown). Electron density at
the cavity center is prominent in MIR maps even prior to averaging
(FIG. 6C, lower diffuse peak). The difference electron density maps
show this to be related to the presence of one or more poorly
localized cations situated at least 4 .ANG. away from the closest
protein groups.
[0506] The Cavity and Internal Pore
[0507] FIGS. 5B and 6 indicate that surprisingly, a 10 .ANG.
diameter cavity is in the center of the channel protein with an ion
in it. Electrostatic calculations indicate that when an ion is
moved alone a narrow pore through a membrane it must cross an
energy barrier that is maximum at the membrane center (17). The
electrostatic field emanating from a cation polarizes its
environment, bringing the negative ends of dipoles closer to it and
thereby stabilizing it. At the bilayer center, the polarizability
of the surrounding medium is minimal and therefore the energy of
the cation is highest. Thus, simple electrostatic considerations
allow an understanding of the functional significance of the cavity
and its strategic location. The cavity will serve to overcome the
electrostatic destabilization resulting from the low dielectric
bilayer by simply surrounding an ion with polarizable water. A
second feature of the K.sup.+ channel structure will also stabilize
a cation at the bilayer center. The four pore helices point
directly at the center of the cavity (FIG. 3, A, B and D). The
amino to carboxyl orientation of these helices will impose a
negative electrostatic (cation attractive) potential via the helix
dipole effect (18). The ends of the helices are rather far (about 8
.ANG.) from the cavity center, but all four contribute to the
effect. Therefore, two properties of the structure, the aqueous
cavity and the oriented helices, help to solve a very fundamental
physical problem in biology--how to lower the electrostatic barrier
facing a cation crossing a lipid bilayer. Thus, the diffuse
electron density in the cavity center most likely reflects not an
ion binding site, but rather a hydrated cation cloud (FIG. 7).
[0508] In summary, the inner pore and cavity lower electrostatic
barriers without creating deep energy wells. The structural and
chemical design of this part of the pore ensure a low resistance
pathway from the cytoplasm to the selectivity filter, facilitating
a high throughput. Functional experiments on K.sup.+ channels
support this conclusion. When TEA from the cytoplasm migrates to
its binding site at the top of the cavity, >50% of the physical
distance across the membrane (FIG. 4 and FIG. 5), it traverses only
about 20% of the transmembrane voltage difference (15). Thus, 80%
of the transmembrane voltage is imposed across the relatively short
selectivity filter. The rate limiting steps for a K.sup.+ ion
traversing the channel are thereby limited to this short distance.
In effect, the K.sup.+ channel has thinned the relevant
transmembrane diffusion distance to a mere 12 .ANG..
[0509] The Selectivity Filter
[0510] Construction of the atomic model for the K.sup.+ channel
selectivity filter was based on the experimental electron density
map which showed a continuous ridge of electron density
attributable to the main chain, as well as strong valine and
tyrosine side chain density directed away from the pore (FIG. 8A).
K.sup.+ ion positions defined by difference Fourier analysis (FIG.
6 and FIG. 8A, yellow density) along with knowledge of alkali metal
cation coordination in small molecules were also used in the
construction. The side chain locations preclude their direct
participation in ion coordination, leaving this function to the
main chain atoms. The precise orientation of individual carbonyl
oxygens can not be discerned at the resolution of this X-ray
analysis. Although Applicant is under no obligation to explain the
orientation of individual carbonyl atoms, and are not to be bound
by such explanations. Applicant merely proposes they are directed
inward to account for K.sup.+ ion coordination (FIG. 8B). A single
water molecule (the only one modeled in the structure) located
between the two K.sup.+ ions in the selectivity filter was
justified by the presence of a strong electron density peak in the
experimental map which was never associated with an ion peak in the
difference Fourier maps (19).
[0511] The structure of the selectivity filter exhibits two
essential features. First, the use of main chain atoms creates a
stack of sequential oxygen rings and thus affords numerous closely
spaced sites of suitable dimensions for coordinating a dehydrated
K.sup.+ ion. The K.sup.+ ion thus has only a very small distance to
diffuse from one site to the next within the selectivity filter.
The second important structural feature of the selectivity filter
is the protein packing around it. The Val and Tyr side chains from
the V-G-Y-G sequence point away from the pore and make specific
interactions with amino acids from the tilted pore helix. In
collusion with the pore helix Trp residues, the four Tyr side
chains form a massive sheet of aromatic amino acids, twelve in
total, that is positioned like a cuff around the selectivity filter
(FIG. 8C). The hydrogen bonding, for example between the Tyr
hydroxyls and Trp nitrogens, and the extensive van der Waals
contacts within the sheet, offer the immediate impression that this
structure behaves like a layer of springs stretched radially
outward to hold the pore open at its proper diameter.
[0512] Applicant postulates, although under no obligation to do so,
and not to be bound thereby, that when an ion enters the
selectivity filter it evidently dehydrates (nearly completely). To
compensate for the energetic cost of dehydration, the carbonyl
oxygen atoms must take the place of the water oxygen atoms. That
is, they must come in very close contact with the ion and act like
surrogate water (20, 21). The structure reveals that the
selectivity filter is being held open as if to prevent it from
accommodating a Na.sup.+ ion with its smaller radius.
[0513] Therefore, Applicant postulates that a K.sup.+ ion fits in
the filter just right, so that the energetic costs and gains are
well balanced. Sodium on the other hand is too small. The structure
of the selectivity filter with its molecular springs holding it
open prevents the carbonyl oxygen atoms from approaching close
enough to compensate for the cost of dehydration of a Na.sup.+
ion.
[0514] This analysis shows that the selectivity filter contains two
K.sup.+ ions in the presence of about 150 mM K.sup.- (FIG. 6 and
FIG. 8). The ions are located at opposite ends of the selectivity
filter, separated by about 7.5 .ANG.. That is roughly the average
distance between K.sup.+ ions in a 4 Molar KCl solution, and in the
selectivity filter there are no intervening Cl.sup.- anions to
balance the charge. Although under no obligation to explain such
results, and without intending to be bound by any explanation,
Applicant postulates, that the selectivity filter attracts and
concentrates K.sup.+ ions. The structure implies that a single
K.sup.+ ion would be held very tightly, but that the presence of
two K.sup.+ ions results in mutual repulsion, hence their locations
near opposite ends of the selectivity filter. Thus, when a second
ion enters, the attractive force between a K.sup.+ ion and the
selectivity filter becomes perfectly balanced by the repulsive
force between ions, and this is what allows conduction to occur.
This picture accounts for both a strong interaction between K.sup.+
ions and the selectivity filter and a high throughput mediated by
electrostatic repulsion. On the basis of functional measurements,
the same concept of destabilization by multiple ion occupancy has
been proposed for Ca.sup.2+ channels (22) and for K.sup.+ channels
(23) and perhaps is a general property of all selective ion
channels.
[0515] Experimental Procedures
[0516] Cloning and Expression of the kcsa Gene
[0517] The kcsa gene was subcloned into pQE60 (Qiagen) vector and
expressed in E. coli XL-1 Blue cells upon induction with
1-.beta.-D-thiogalactopyranoside. The carboxy-terminal histidine
tagged protein was extracted by homogenization and solubilization
in 40 mM decylmaltoside (Antrace). The kcsa K.sup.+ channel was
purified on a cobalt affinity column. Thirty-five carboxyl terminal
amino acids were cleaved by chymotripsin proteolysis. The truncated
channel was purified to homogeneity by gel filtration and the
detergent exchanged in a final dialysis step against 5 mM
N,N,-dimethyldodecylamine-N-oxide (LDAO). Crystals were grown at
20.degree. C. by using the sitting drop method by mixing equal
volumes of a solubilizing solution with reservoir mixture. Through
the entire preparation, the channel protein was maintained in
solutions containing 150 mM KCl. For definition of K.sup.+ sites,
crystals were transferred into solutions where 150 mM KCl was
replaced by 150 mM RbCl or 150 mM CsCl.
[0518] X-Ray Crystallography
[0519] Crystals (space group C2: .alpha.=128.8 .ANG., b=68.9 .ANG.,
c=112.0 .ANG., .beta.=124.6.degree. were flash-frozen by
transferring directly from the crystal mother liquor to a stream of
boiled-off nitrogen (24). Since crystals of the mutant L90C
diffracted significantly better than wild type protein crystals,
the former were used for native data collection. Data were
collected from multiple crystals and six sets were selected and
merged to form the native data set used for structure
determination. Mercury derivatives were obtained by direct addition
of methyl mercury to the crystallization solution of cysteine
mutant crystals. MALDI-TOF mass spectrometry confirmed 60-90%
derivatization of crystals prior to data collection. All data were
collected at Cornell High Energy Synchrotron Source (CHESS),
station A1, using the Princeton 2K CCD (25). Data were processed
with DENZO and SCALEPACK (26) and the CCP4 package (27). Heavy atom
positions were determined with SHELX-97 (28) and cross-difference
Fourier analysis. These positions confirmed the four-fold
noncrystallographic symmetry observed in the self-rotation
Patterson function and allowed the determination of initial
orientation matrices. An initial model (90% complete) was built
into a solvent flattened (64% solvent content), four-fold averaged
electron density map using the program O (29). The tracing of the
model was facilitated by the use of the mercury atom positions as
residue markers. L86C was used solely for this purpose. After
torsional refinement (with strict four-fold noncrystallographic
symmetry constraints) using XPLOR 3.851 (30), this model was used
in the anisotropic scaling (sharpening (31)) of the native data
with XPLOR. The structure factor sigma values were also rescaled
appropriately and the corrected data were used for all subsequent
procedures. Four-fold averaging, solvent flattening and phase
extension were applied in DM (32), resulting in a marked
improvement of the electron density that allowed correction of the
model and the building of additional residues. Refinement consisted
of rounds of positional (in the initial tages phase information was
also included as a restraint) and grouped B-factor refinement in
XPLOR. Four-fold noncrystallographic symmetry was highly restrained
with the force constant for positional restraints set as 1000
kcal/mol/.ANG..sup.2. The diffuse ion cloud described in the text
was initially modeled as one or more K.sup.+ ions and several water
molecules, however the results were unsatisfactory. Therefore, this
and other strong unmodeled density present in solvent flattened
maps (no averaging included) was Fourier back-transformed, scaled
and included in the refinement procedure, as partial structure
factors. The final model includes amino acids 23 to 119 of each
chain. The following residues were truncated: Arg27 to C.beta.,
Ile60, to C.gamma., Arg64 to C.beta., Glu71 to B.beta. and Arg117
to N.epsilon.. The stereochemistry is strongly restrained, with no
outliers on the Ramachandran plot. The high B-factor values reflect
the intensity decay of the data beyond 4 .ANG..
SUMMARY
[0520] Without intending to be bound by such proposals, and with no
obligation to explain these results, Applicant proposes the
following principles underlying the structure and operation of
K.sup.+ channels. (i) The pore structure defines an inverted tepee
architecture with the selectivity filter held at its wide end. This
architecture also describes the pore of cyclic nucleotide-gated
channels and probably Na.sup.+ and Ca.sup.2+ channels as well. (ii)
The narrow selectivity filter is only 12 .ANG. long, while
surprisingly, the remainder of the pore is wider and has a
relatively inert hydrophobic lining. These structural and chemical
properties favor a high K.sup.+ throughput by minimizing the
distance over which K.sup.+ interacts strongly with the channel.
(iii) A large water-filled cavity and helix dipoles help to
overcome the high electrostatic energy barrier facing a cation in
the low dielectric membrane center. (iv) The K.sup.+ selectivity
filter is lined by carbonyl oxygen atoms providing multiple closely
spaced sites. The filter is constrained in an optimal geometry so
that a dehydrated K.sup.+ ion fits with proper coordination while
the Na.sup.+ ion is too small. (v) Two K.sup.+ ions at close
proximity in the selectivity filter repel each other. The repulsion
overcomes the otherwise strong interaction between ion and protein
and allows rapid conduction in the setting of high selectivity.
3TABLE 1 Summary of data collection and refinement statistics. Data
Collection and Phasing: Resolution Completeness Phasing Dataset
(.ANG.) Redudancy Overall/outer Rmerge # Power .paragraph. R-Cullis
+ L90C-a 15.0-3.7 3.5 91.3/93.3% 0.071 1.61 0.70 L90C-b 15.0-3.7
7.0 91.5/94.1% 0.083 1.87 0.50 V93C 15.0-3.7 4.1 98.3/99.1% 0.075
1.35 0.63 A32C 15.0-4.0 2.3 84.1/83.8% 0.076 1.45 0.66 A29C
15.0-5.0 2.7 73.9/74.0% 0.063 1.03 0.85 A42C 15.0-6.5 2.0
90.7/90.3% 0.057 0.97 0.81 L86C 30.0-6.0 2.3 58.7/58.9% 0.057 --
--
[0521]
4 % of measured I/.sigma.I data with I/.sigma.I>2 Native
30.0-3.2 6.1 93.3% 0.086 15.8 79 Outer Shell 3.3-3.2 2.3 66.6%
0.286 3.9 50 Anisotropic correction: Average F.O.M* Average F.O.M*
(30.0-3.2 .ANG.) (3.4-3.2 .ANG.) Before Sharpening .differential.
0.76 0.55 After sharpening .differential. 0.83 0.64 Refinement:
Root-mean-square deviation of Resolution 10.0-3.2 .ANG. bond
angles: 1.096.degree. R-cryst. &: 28.0% bond lengths 0.005
.ANG. R-free &: 29.0% ncs related atoms: 0.006 .ANG. No. of
reflections with.vertline.F.vertline./.sigma..v-
ertline.F.vertline. > 2: 12054 related atoms: 10 .ANG..sup.2 No.
of protein atoms: 710 per subunit B-factor for non- No. of ligand
atoms: 1 water, 3 K.sup.+atoms bonded atoms: 36 .ANG..sup.2 Mean
B-factor for 90 .ANG..sup.2 side-chain atoms: Mean B-factor for 110
.ANG..sup.2 side-chain atoms: # Rmerge =
.SIGMA..SIGMA.I-Ij/.SIGMA.I.: .paragraph. Phasing power =
<.vertline.Fh.vertline.>/<E>: -R-Cullis =
.SIGMA..vertline..vertline.Fph .+-.
Fp.vertline.-.vertline.Fhc.vertline..-
vertline./.SIGMA..vertline.Fph .+-. Fp.vertline.. only for centric
data: & R-cryst. =
.SIGMA..vertline.Fp-Fp(calc).vertline./.SIGMA..vertline.Fp.ver-
tline.. r-free the same for R-cryst., but calculated on 10% of data
selected in thin resolution shells and excluded from refinement:
*F.O.M.: figure of merit; .sigma. in both cases four-fold averaging
and solvent flattening were applied: Ij is the observed intensity.
I is the average intensity. Fh is the root-mean-square heavy-atom
structure factor. E is the lack of closure error, Fph is the
structure factor for the derivative, Fp is the structure factor for
the native. Fhc is the calculated structure factor for the
heavy-atom, Fp(calc) is the calculated native structure value.
[0522] 10. J. Deisenhofer et at. Nature 318, 618 (1985); S. W.
Cowan et at. Nature 358, 727 (1992); A. Kreusch and G. E. Schulz,
J. Mol. Biol. 243, 891 (1994).
[0523] 11. R. MacKinnon and C. Miller, Science 245, 1382
(1989).
[0524] 12. R. MacKinnon, L. Heginbotham, T. Abramson, Neuron 5, 767
(1990); M. Stocker and C. Miller, Proc. Natl. Acad. Sci. U.S.A. 91,
9509 (1994); S. A. N. Goldstein, D. J. Pheasant, C. Miller, Neuron
12, 1377 (1994); P. Hidalgo and R. MacKinnon, Science 268, 307
(1995); J. Aiyar et al., Neuron 15, 1169 (1995); D. Naranjo and C.
Miller, Neuron 16, 123 (1996); R. Ranganathan, J. H. Lewis, R.
MacKinnon, Neuron 16, 131 (1996); A. Gross and R. MacKinnon, Neuron
16, 399 (1996).
[0525] 13. C. M. Armstrong and B. Hille. J. Gen. Physiol. 59, 388
(1972).
[0526] 14. R. MacKinnon and G. Yellen, Science 250, 276 (1990).
[0527] 15. G. Yellen, M. E. Jurman, T. Abramson, R. MacKinnon,
ibid. 251, 939 (1991).
[0528] 16. Y. Liu. M. Holmgren, M. E. Jurman, G. Yellen, Neuron 19,
175 (1997).
[0529] 17. V. A. Parsegian, Annals NY Acad. Sciences 264, 161
(1975).
[0530] 18. D. Sali, M. Bycroft, A R. Fersht, Nature 335, 740
(1988); J. Aqvist, H. Luecke, F. A. Quiocho, A. Warshel, Proc.
Natl. Acad. Sci. U.S.A. 88, 2026 (1991); D. J. Lockhart and P. S.
Kim, Science 257, 947 (1992); D. J. Lockhart and P. S. Kim, Science
260, 198 (1993).
[0531] 19. The temperature factors for Val76 and Gly77 main chain
atoms (but not side chain atoms) refined to higher values than for
neighboring atoms. This result is explicable based on the
difference Fourier analysis showing alternative positions of the
inner K.sup.+ ion in the selectivity filter and therefore, by
inference, alternative conformations of the coordinating main chain
atoms, depending on the location of the K.sup.+ ion.
[0532] 20. F. Bezanilla and C. M. Armstrong, J. Gen. Physiol. 60,
588 (1972).
[0533] 21. Hille, ibid. 61, 669 (1973).
[0534] 22. W. Almers and E. W. McCleskey, J. Physiol, (Lond.) 353,
585 (1984); P. Hess and R. W. Tsien, Nature 309, 453 (1984).
[0535] 23. J. Neyton and C. Miller, J. Gen. Physiol. 92, 569
(1988).
[0536] 24. The kcsa gene was subcloned into pQE60 (Qiagen) vector
and expressed in E. coli XL-1 Blue cells upon induction with
1-(-D-thiogalactopyranoside. The carboxyl-terminal histidine tagged
protein was extracted by homogenization and solubilization in 40 mM
decylmaltoside (Anatrace). The kcsa channel was purified on a
cobalt affinity column. Thirty-five carboxyl terminal amino acids
were cleaved by chymotrypsin proteolysis. The truncated channel was
purified to homogeneity by gel filtration and the detergent
exchanged in a final dialysis step against 5 mM
N,N-dimethyldodecylamine-N-oxide (LDAO). Crystals were grown at 20
(C by using the sitting drop method by mixing equal volumes of
protein solution (5-10 mg/ml. 150 mM KCl. 50 mM Tris pH 7.5, 2 mM
DTT) with reservoir mixture (200 mM CaCl.sub.2, 100 mM Hepes pH 7.5
and 48% PEG 400). Through the entire preparation the channel
protein was maintained in solutions containing 150 mM KCl. For
definition of K.sup.+ sites, crystals were transferred into
solutions where 150 mM KCl was replaced by 150 mM RbCl or 150 mM
CsCl.
[0537] 25. M. W. Tate et al., J. Appl. Cryst. 28, 196 (1995); D. J.
Thiel, et al., Rev. Sci. Instrum. 67, 1 (1996).
[0538] 26. Z. Otwinowski, in Data Collection and Processing, L.
Sawyer and S. Bailey, Eds. (Science and Engineering Research
Council Daresbury Laboratory, Daresbury, UK, 1993), pp. 56-62.
[0539] 27. Collaborative Computational Project 4 (CCP4), Acta
Cryst. DS50, 760(1994).
[0540] 28. G. M. Sheldrick, Acta Cryst. 46, 467 (1990).
[0541] 29. T. A. Jones. J. Y. Zou. J. Y. Cowan, M. Kjeldgaard,
ibid. A47, 110 (1991).
[0542] 30. A. T. Brunger. X-Plor (Version 3.851) Manual (New Haven,
Conn.: The Howard Hughes Medical Institute and Department of
Molecular Biophysics and Biochemistry, Yale University).
[0543] 31. S. J. Gamblin, D. W. Rodgers, T. Stehle, Proceedings of
the CCP4 Study weekend, Daresbury Laboratory, (1996) pp.
163-169.
[0544] 32. K. Y. J. Zhang and P. Main, Acta Cryst. A46, 377.
[0545] 33. P. J. Kraulis, J. Appl. Cryst. 24, 946 (1991).
[0546] 34. O. S. Smart. J. G. Neduvelil, X. Wang, B. A. Wallace, M.
P. Sansom, J. Mol. Graphics 14, 354 (1996).
EXAMPLE II
Structural Conservation in Prokaryotic and Eukaryotic K.sup.-
Channels Revealed by Scorpion Toxins
[0547] Scorpion toxins inhibit ion conduction through K.sup.+
channels by occluding the pore at their extracellular opening. A
single toxin protein binds very specifically to a single K.sup.+
channel to cause inhibition. The toxins are 35 to 40 amino acids in
length and have a characteristic fold that is held rigidly by three
disulfide bridges (1). They are active site inhibitors, because
when they bind to the channel they interact energetically with
K.sup.+ ions in the pore (2-4). The intimate interaction between
these inhibitors and the pore of K.sup.+ channels has been
exploited to gain insights into the structure and function of
K.sup.+ channels.
[0548] Studies employing site-directed mutagenesis of the Shaker
K.sup.+ channel have mapped the scorpion toxin binding site to
regions corresponding to the extracellular entryway of the kcsa
K.sup.+ channel (4-9). Although the K.sup.+ channel selectivity
filter amino acids are highly conserved, the residues lining the
entryway are quite variable. As if to mirror the amino acid
variation at the binding site, the toxins are also highly variable
in their amino acid composition. A given scorpion venom is a
veritable library of toxins, apparently ensuring that a scorpion
will inhibit a large fraction of K.sup.+ channel types in its
victim. Studies on the specificity of toxin-channel interactions
have led to the following understanding. The extracellular entryway
to the K.sup.+ channel is relatively conserved in its
three-dimensional structure but the precise amino acid composition
is not conserved. The scorpion toxins have a shape, dictated by
their conserved fold, that enables them to fit snugly into the
entryway, but the affinity of a given toxin-channel pair depends on
the residue match (or mismatch) on both interaction surfaces.
[0549] A study of the interaction between the kcsa K.sup.+ channel
(5) and the scorpion toxin agitoxin2 has been undertaken (10). By
producing, through mutagenesis, a competent toxin binding site, it
is shown that the kcsa K.sup.+ channel pore structure and
extracellular entryway is very similar to that of eukaryotic
voltage-gated K.sup.+ channels such as the Shaker K.sup.+ channel
from Drosophila and the vertebrate voltage-gated K.sup.+ channels,
and that mutated potassium channel proteins of prokaryotic
organisms mimic the physiological functions and chemical properties
of eukaryotic cation binding proteins. By combining functional data
collected on the toxin-channel interaction with the structures of
both proteins Applicant proposes, without intending to be bound by
such proposals, a highly-restrained model of the complex
structure.
[0550] Experimental Procedures
[0551] Three mutations (Q58A, T61 S, R64D) were introduced into the
kcsa K.sup.+ channel gene to modify its pore region sequence using
PCR mutagenesis and confirmed by DNA sequencing. The gene also
contained a mutation at the second residue (P2A) to introduce an
ncol restriction endonuclease site and it was lacking the last two
carboxyl terminal residues (both Arg) to avoid proteolysis during
the protein preparation. This gene was cloned into the pQE60 vector
for expression with a carboxyl terminal thrombin and hexahistidine
fusion. Channel protein was expressed in XL-1 Blue strain of E.
coli (Stratagene) by induction with
l-.beta.-D-thiogalactopyranoside at a concentration of 1.0 mM.
Three hours following induction bacteria were sonicated in 50 mM
Tris buffer (7.5), 100 mM KCl. 10 mM Mg.sub.2SO.sub.4, 25 mg DNAse
1, 250 mM sucrose, in addition to pepstatin, leupeptin, and PMSF.
The channel was extracted in the same solution containg 40 mM
decylmaltoside (Anatrace) at room temperature. Following
centrifugation the supernatant was bound to cobalt resin (Talon) at
a protein to resin ratio that will saturate the resin. The resin
was washed, and detergent concentration was lowered to 10.0 mM. One
mL columns were prepared. The control resin (no channel) was
handled in the same manner. The resin preparation was the same for
mass spectrometry and binding studies.
[0552] Forty mg of Leiurus quinquestriatus hebraeus venom, (Alomone
Labs) was suspended in buffer identical to that of the channel
(10.0 mM declymaltoside) and applied to the column. After washing,
channel was eluted with 1.0 M imidazole in the same buffer.
[0553] Wild type and mutant agitoxin2 were prepared (10). Tritiated
N-ethylmaleimide (NEN Life Sciences) was conjugated to agitoxin2
D20C (14). Binding, was performed in a 300 .mu.L volume containing
50 mM Tris (7.5), 100 mM KCl, 10 mM declymaltoside, and 0.3 .mu.L
of cobalt resin saturated with the mutant kcsa K.sup.+ channel for
30 minutes at room temperature. Following brief centrifugation the
supernatant was removed, resin was applied to a filter, rinsed
briefly with ice cold buffer, and then counted in a scintillation
counter. All binding measurements were made with a paired control
containing a saturating concentration (200 times K.sub.D) of
unlabeled wild type agitoxin2 to determine nonspecific binding. The
competition assay was carried out under the same conditions.
Labeled Agitoxin2 at 0.06 .mu.M was always present and unlabeled
toxin was added to compete with bound labeled toxin.
Discussion
[0554] Guided by knowledge of the toxin receptor on the Shaker
K.sup.+ channel, set forth in SEQ ID NO:4, three point mutations
were introduced into the kcsa K.sup.+ channel (SEQ ID NO:1) that
should render it sensitive to scorpion toxins (FIG. 9). The amino
acid sequence of the mutated kcsa K.sup.+ is set forth in SEQ ID
NO:16. Amino acids 61 and 64 of SEQ ID NO:1 were changed to their
Shaker K.sup.+ channel counterpart, and 58 was changed to alanine
since a small side chain at this latter position favors binding (4,
7). The mutant kcsa K.sup.+ channel protein was expressed in
Escherichia coli, extracted from the membrane with the detergent
decylmaltoside, and bound to cobalt resin through a carboxyl
terminal hexahistidine tag (11). A 1 mL column, prepared with the
K.sup.+ channel-containing resin, was used to screen the venom of
the Middle East scorpion Leiurus quinquestriatus hebraeus, the
source of numerous well-characterized ion channel toxins. Forty
milligrams of venom was added to the column and after washing, the
K.sup.+ channel protein was eluted with an imidazole solution (12).
The eluate was analyzed with MALDI-TOF mass spectrometry, focusing
on the low mass range appropriate for scorpion toxins (about 4000
Da). The K.sup.+ channel column resulted in a dramatic enhancement
of specific peaks (FIG. 10, A-C). Three of these peaks corresponded
in mass to the known K.sup.+ channel toxins agitoxin2,
charybdotoxin, and Lq2 (FIG. 10, C and D). A fourth peak (FIG. 10C,
asterisk) may represent a novel toxin, which is currently under
study. However, Applicant is under no obligation to explain this
peak, and is not bound by any theories set forth herein regarding
this peak. The peak corresponding to chlorotoxin, a reported
chloride channel inhibitor (13), did not bind and provides an
indication of the degree to which the K.sup.+ channel toxins are
purified by the mutant kcsa K.sup.+ channel column (FIG. 10, A and
C).
[0555] Further quantitative analysis was carried out with
agitoxin2. Radiolabeled agitoxin2 was prepared by producing the
mutation D20C in the toxin (located far from its channel binding
surface) and conjugating it with tritiated N-ethylmaleimide (14). A
filter assay showed that labeled agitoxin2 binds to the mutant kcsa
K.sup.+ channel with an equilibrium dissociation constant, K.sub.D,
of about 0.6 mM (FIG. 11A). In contrast, no binding to the wild
type channel could be detected (not shown). The total capacity of
resin saturated with mutant channel protein, based on the specific
activity of radiolabeled toxin and the known 1:1 stoichiometry (one
toxin per tetrameric channel), is nearly 50 pmoles of channel per
.mu.L of resin. This value approximates the expected capacity of
the resin and therefore implies that all of the channel in the
preparation must have a correct conformation.
[0556] Amino acids in a well-defined region of agitoxin2 form its
functional interaction surface, as determined by the effects of
alanine substitution on binding to the Shaker K.sup.+ channel [FIG.
11C (4, 8)]. Mutation of Lys 27 and Asn 30 had the largest
destabilizing effects. It is noteworthy that Lys 27 is conserved in
all members of this toxin family because its side chain apparently
plugs the pore of K.sup.+ channels (3). To confirm that agitoxin2
uses the same amino acids to interact with the mutant kcsa K.sup.+
channel, the effects of the K27A and N30A toxin mutations with a
competition binding assay were studied (FIG. 11B). These mutations
decreased the affinity for the toxin significantly (130-fold and
45-fold, respectively), as anticipated from the Shaker K.sup.+
channel studies. In contrast, the D20C mutation (predicted to be on
the back side of the toxin), even with a bulky N-ethylmaleimide
adduct, did not influence affinity (FIG. 11. A and B). These
results indicate that agitoxin2 binds in the same manner to both
the mutant prokaryotic kcsa K.sup.+ channel protein and the
eukaryotic Shaker K.sup.+ channel protein. The affinity for the
Shaker K.sup.+ channel is considerably higher (K.sub.D.sup.+1 nM),
but only three amino acids have been mutated in the prokaryotic
cation channel protein to mimic the site on the Shaker K.sup.+
channel (FIG. 9).
[0557] These results demonstrate that the overall structure of the
agitoxin2 receptor site is very similar on both the kcsa and Shaker
K.sup.+ channels. This conclusion justifies the use of energetic
data borrowed from Shaker K.sup.+ channel studies to assist in the
docking of agitoxin2 onto the kcsa K.sup.+ channel structure.
Thermodynamic mutant cycle analysis has allowed the identification
of numerous energetically coupled residue pairs on the interface
[pairs of residues that are related by the fact that mutating one
influences the effect (on equilibrium binding) of mutating the
other (8)]. The four best defined of these residue pairs are
displayed in matched colors on the kcsa K.sup.+ channel and
agitoxin2 surfaces (FIG. 12 A). The three off-center residue pairs
(blue, green, yellow) have the strongest mutant cycle coupling
energies [>3 kT (4, 8)]. The central residue pair (red) is
coupled by 1.7 kT and independent information places Lys 27 (red
residue on agitoxin2, FIG. 11 A) over the pore (3, 4). More visual
inspection suggests a unique orientation for the toxin on the
channel (FIG. 12 B). If the toxin is placed with its functionally
defined interaction surface face-down in the groove formed by the
turrets (5), with Lys 27 at the center, the colors match well in
three dimensions. The toxin seems to fit perfectly into the
vestibule of a K.sup.+ channel. The four-fold symmetry of the
K.sup.+ channel provides four statistically distinguishable but
energetically identical orientations available for a toxin to bind
[(FIG. 12 A) (15)].
[0558] In summary, through a combination of structural and
functional data, it is shown that prokaryotic channel proteins can
be mutated to mimic the physiological functions and chemical
properties of eukaryotic channel proteins. Furthermore, disclosed
herein is a view of a K.sup.+ channel in complex with a neurotoxin
from scorpion venom. The kcsa K.sup.+ channel is structurally very
similar to eukaryotic K.sup.+ channels. This unexpected structural
conservation, determined through application of techniques
developed here, can be exploited to advance our understanding of
K.sup.+ channel pharmacology, and prepare mutant prokaryotic
channel proteins that can be used to screen potential drugs or
agents that may interact with eukaryotic cation channel proteins in
vivo, and treat conditions related to the function of proteins.
REFERENCES
[0559] The following references, along with other relevant
information was cited in Example II, and set forth below. All
references cited in Example II are hereby incorporated by reference
in their entirety.
[0560] 1. M. L. Garcia et al., J. Bioenerg. Biomemb. 23, 615
(1991); C. Miller, Neuron 15, 5 (1995).
[0561] 2. R. MacKinnon and C. Miller, J. Gen. Physiol. 91, 335
(1988); K. M.
[0562] 3. C. S. Park and C. Miller, Neuron 9, 307 (1992).
[0563] 4. R. Ranganathan. J. H. Lewis. R. MacKinnon. Neuron 16, 131
(1996).
[0564] 5. D. A. Doyle et al., Science xxxx (1998).
[0565] 6. R. MacKinnon and C. Miller, Science 245, 1382 (1989); R.
MacKinnon, L. Heginbotham, T. Abramson, Neuron 5, 767 (1990); M.
Stocker and C. Miller, Proc. Natl. Acad. Sci. USA 91, 9509 (1994);
D. Naranjo and C. Miller, Neuron 16, 123 (1996).
[0566] 7. S. Goldstein, D. J. Pheasant, C. Miller, Neuron 12, 1377
(1994).
[0567] 8. P. Hidalgo and R. MacKinnon, Science 268, 307 (1995).
[0568] 9. A. Gross and R. MacKinnon, Neuron 16, 399 (1996).
[0569] 10. M. L. Garcia et al., Biochemistry 33, 6834 (1994).
[0570] 11. Three mutations (Q58A, T61S, R64D) were introduced into
the kcsa K.sup.+ channel gene to modify its pore region sequence
using PCR mutagenesis and confirmed by DNA sequencing. The gene
also contained a mutation at the second residue (P2A) to introduce
an ncol restriction endonuclease site and it was lacking the last
two carboxyl terminal residues. This gene was cloned into the pQE60
vector for expression with a carboxyl terminal thrombin and
hexahistidine fusion. Channel protein was expressed in XL-1 Blue
strain of E. coli (Stratagene) by induction with
1-.beta.-D-thiogalactopyranoside at a concentration of 1.0 mM.
Three hours following induction bacteria were sonicated in 50 mM
Tris buffer (7.5), 100 mM KCl, 10 mM Mg.sub.2SO.sub.4, 25 mg DNAse
1, 250 mM sucrose, in addition to pepstatin, leupeptin, and PMSF.
The channel was extracted in the same solution containg 40 mM
decylmaltoside (Anatrace) at room temperature. Following
centrifugation the supernatant was bound to cobalt resin (Talon) at
a protein to resin ratio that will saturate the resin. The resin
was washed, and detergent concentration was lowered to 10.0 mM. One
mL columns were prepared. The control resin (no channel) was
handled in the same manner. The resin preparation was the same for
mass spectrometry and binding studies.
[0571] 12. Forty mg of Leiurus quinquestriatus hebraeus venom
(Alomone Labs) was suspended in buffer identical to that of the
channel (10.0 mM declymaltoside) and applied to the column. After
washing, channel was eluted with 1.0 M imidazole in the same
buffer, 13. J. A. Debin, J. E. Maggio, G. R. Strichartz, Am. J.
Physiol. Soc. 264, C369 (1993); G. Lippens, J. Najib, S. J. Wodak,
A. Tartar, Biochemistry 34, 13 (1995).
[0572] 14. S. K. Aggarwal and R. MacKinnon, Neuron 16, 1169
(1996).
[0573] 15. R. MacKinnon, Nature 350, 232 (1991).
[0574] 16. S. L. Cohen and B. T. Chait, Anal. Chem. 68, 31
(1996).
[0575] 17. Wild type and mutant agitoxin2 were prepared (10).
Tritiated N-ethylmaleimide (NEN Life Sciences) was conjugated to
agitoxin2 D20C (14). Binding was performed in a 300 .mu.L volume
containing 50 mM Tris (7.5), 100 mM KCl, 10 mM declymaltoside, and
0.3 .mu.L of cobalt resin saturated with the mutant kcsa K.sup.+
channel for 30 minutes at room temperature. Following brief
centrifugation the supernatant was removed, resin was applied to a
filter, rinsed briefly with ice cold buffer, and then counted in a
scintillation counter. All binding measurements were made with a
paired control containing a saturating concentration (200 times
K.sub.d) of unlabeled wild type agitoxin2 to determine nonspecific
binding. The competition assay was carried out under the same
conditions. Labeled Agitoxin2 at 0.06 .mu.M was always present and
unlabeled toxin was added to compete with bound labeled toxin.
[0576] 18. A. M. Krezel et al., Prot. Sci. 4 1478 (1995).
[0577] 19. A. Nicholls. K. A. Sharp, B. Honig, Proteins 11, 281
(1991).
[0578] 20. T. A. Jones, J. Y. Zou, J. Y. Cowan, M. Kjeldgaard, Acta
Cryst. A47, 110 (1991).
[0579] The present invention is not to be limited in scope by the
specific embodiments describe herein. Indeed, various modifications
of the invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and the accompanying figures. Such modifications are intended to
fall within the scope of the appended claims.
[0580] It is further to be understood that all base sizes or amino
acid sizes, and all molecular weight or molecular mass values,
given for nucleic acids or polypeptides are approximate, and are
provided for description.
[0581] Various publications are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 0
0
* * * * *