U.S. patent application number 10/743381 was filed with the patent office on 2004-06-24 for enhancers of cftr chloride channel function.
This patent application is currently assigned to Case Western Reserve University. Invention is credited to Adams, Lynn, Davis, Pamela B., Ma, Jian Jie.
Application Number | 20040121957 10/743381 |
Document ID | / |
Family ID | 32599521 |
Filed Date | 2004-06-24 |
United States Patent
Application |
20040121957 |
Kind Code |
A1 |
Adams, Lynn ; et
al. |
June 24, 2004 |
Enhancers of CFTR chloride channel function
Abstract
Phosphorylation of the cystic fibrosis transmembrane conductance
regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) is
essential for opening the CFTR chloride channel. A short segment
containing many negatively charged amino acids (817-838, NEG2)
within the regulatory (R) domain of CFTR is a critical regulator of
the chloride channel activity. Deletion of NEG2 from CFTR
completely eliminates the PKA dependence of the chloride channel.
Exogenous NEG2 peptide interacts with the CFTR molecule and
exhibits stimulatory effects on CFTR function. Our data suggest
that NEG2 interacts with other cytosolic domains of CFTR to control
the opening transitions of the chloride channel.
Inventors: |
Adams, Lynn; (Cleveland
Heights, OH) ; Davis, Pamela B.; (Cleveland Heights,
OH) ; Ma, Jian Jie; (Highland Heights, OH) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
Case Western Reserve
University
10900 Euclid Avenue
Cleveland
OH
44106-4959
|
Family ID: |
32599521 |
Appl. No.: |
10/743381 |
Filed: |
December 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10743381 |
Dec 23, 2003 |
|
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09512260 |
Feb 24, 2000 |
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60121495 |
Feb 24, 1999 |
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Current U.S.
Class: |
435/6.16 ;
514/1.8; 514/17.4; 530/350 |
Current CPC
Class: |
C07K 14/4712
20130101 |
Class at
Publication: |
514/012 ;
514/013; 514/014; 514/015; 530/350 |
International
Class: |
A61K 038/17; C07K
014/705 |
Goverment Interests
[0001] This invention was made with government support under RO1
HL/DK 49003, P30 DK27651 and RO1 DK51770 awarded by the National
Institute of Health. The government has certain rights in the
invention.
Claims
1. An isolated polypeptide comprising a portion of CFTR (cystic
fibrosis transmembrane conductance regulator) protein of between 10
and 100 amino acids, said portion comprising 18 amino acids as
shown in SEQ ID NO: 1.
2. The polypeptide of claim 1 which comprises 22 amino acids as
shown in SEQ ID NO: 2.
3. The polypeptide of claim 1 wherein the polypeptide is fused to a
membrane-penetrating peptide.
4. The polypeptide of claim 2 wherein the polypeptide is fused to a
membrane-penetrating peptide.
5. The polypeptide of claim 3 wherein the membrane-penetrating
peptide is selected from the group consisting of: VP-22 (SEQ ID NO:
3), (SEQ ID NO: 4), and (SEQ ID NO: 5).
6. The polypeptide of claim 4 wherein the membrane-penetrating
peptide is selected from the group consisting of: VP-22 (SEQ ID NO:
3), (SEQ ID NO: 4), and (SEQ ID NO: 5).
7. The polypeptide of claim 1 which is free of phosphorylation.
8. A method of activating a CFTR protein comprising: applying a
polypeptide to a CFTR protein which forms a cAMP regulated chloride
channel, said polypeptide comprising a portion of CFTR protein of
between about 10 and 100 amino acids, said portion comprising 18
amino acids as shown in SEQ ID NO: 1, whereby the open probability
of the channel formed by the CFTR increases by at least 25%.
9. The method of claim 8 wherein the open probability of the
channel formed by the CFTR increases by at least 50%.
10. The method of claim 8 wherein the open probability of the
channel formed by the CFTR increases by at least 75%.
11. The method of claim 8 wherein the open probability of the
channel formed by the CFTR increases by at least 100%.
12. The method of claim 8 wherein the open probability of the
channel formed by the CFTR increases by at least 125%.
13. The method of claim 8 wherein the open probability of the
channel formed by the CFTR increases by at least 150%.
14. The method of claim 8 wherein the open probability of the
channel formed by the CFTR increases by at least 200%.
15. The method of claim 8 wherein the CFTR protein is a mutant
which reaches a cell's plasma membrane but fails to undergo full
activation.
16. The method of claim 15 wherein the CFTR protein is listed at
http://www.genet.sickkids.on.ca/cftr-cgi-bin/fulltable.
17. The method of claim 8 wherein the step of applying is performed
by administering an aerosolized polypeptide to a patient with a
mutant CFTR protein.
18. The method of claim 8 wherein the CFTR protein is in a lipid
bilayer and a change in conductance is measured upon applying the
polypeptide.
19. The method of claim 8 wherein the step of applying the
polypeptide is accomplished by administering a nucleic acid
encoding the polypeptide to a patient who expresses the CFTR
protein, whereby the polypeptide is expressed
20. The method of claim 19 wherein the nucleic acid is administered
as an aerosol to the patient's airways.
21. A method of activating a CFTR protein comprising: applying a
polypeptide to a CFTR protein which forms a cAMP regulated chloride
channel, said polypeptide comprising a portion of CFTR protein of
between 10 and 100 amino acids, said portion comprising 22 amino
acids as shown in SEQ ID NO: 1, whereby the open probability of the
channel formed by the CFTR increases by at least 25%.
22. The method of claim 21 wherein the open probability of the
channel formed by the CFTR increases by at least 50%.
23. The method of claim 21 wherein the open probability of the
channel formed by the CFTR increases by at least 75%.
24. The method of claim 21 wherein the open probability of the
channel formed by the CFTR increases by at least 100%.
25. The method of claim 21 wherein the open probability of the
channel formed by the CFTR increases by at least 125%.
26. The method of claim 21 wherein the open probability of the
channel formed by the CFTR increases by at least 150%.
27. The method of claim 21 wherein the open probability of the
channel formed by the CFTR increases by at least 200%.
28. The method of claim 21 wherein the CFTR protein is a mutant
which reaches a cell's plasma membrane but fails to undergo full
activation.
29. The method of claim 28 wherein the CFTR protein is listed at
http://www.genet.sickkids.on.ca/cftr-cgi-bin/fulltable.
30. The method of claim 21 wherein the step of applying is
performed by administering an aerosolized polypeptide to a patient
with a mutant CFTR protein.
31. The method of claim 21 wherein the CFTR protein is in a lipid
bilayer and a change in conductance is measured upon applying the
polypeptide.
32. The method of claim 21 wherein the step of applying the
polypeptide is accomplished by administering a nucleic acid
encoding the polypeptide to a patient who expresses the CFTR
protein, whereby the polypeptide is expressed.
33. The method of claim 32 wherein the nucleic acid is administered
as an aerosol to the patient's airways.
34. The method of claim 8 or 21 wherein the polypeptide is free of
phosphorylation.
Description
TECHNICAL FIELD OF THE INVENTION
[0002] This invention is related to the field of cystic fibrosis.
More particularly, it is related to the area of therapeutic
treatments and drug discovery for treating cystic fibrosis.
BACKGROUND OF THE INVENTION
[0003] Defects in CFTR, a chloride channel located in the apical
membrane of epithelial cells, are associated with the common
genetic disease, cystic fibrosis (Quinton, 1986, Welsh and Smith,
1993, Zielenski and Tsui, 1995). CFTR is a 1480 amino acid protein
that is a member of the ATP binding cassette (ABC) transporter
family (Riordan et al., 1989, Higgins, 1992). Each half of CFTR
contains a transmembrane domain and a nucleotide binding fold
(NBF), and the two halves are connected by a regulatory, or R
domain. The R domain is unique to CFTR and contains several
consensus PKA phosphorylation sites (Cheng et al., 1991, Picciotto
et al., 1992).
[0004] Opening of the CFTR channel is controlled by PKA
phosphorylation of serine residues in the R domain (Tabcharani et
al., 1991, Bear et al., 1992) and ATP binding and hydrolysis at the
NBFs (Anderson et al., 1991, Gunderson and Kopito, 1995).
Phosphorylation adds negative charges to the R domain, and
introduces global conformational changes reflected by the reduction
in the .alpha.-helical content of the R domain protein (Dulhanty
and Riordan, 1994). Thus, electrostatic and/or allosteric changes
mediated by phosphorylation are likely to be responsible for
interactions between the R domain and other CFTR domains that
regulate channel function (Rich et al., 1993, Gadsby and Naim,
1994).
[0005] Rich et al., 1991 showed that deletion of amino acids
708-835 from the R domain (.DELTA.R-CFTR), which removes most of
the PKA consensus sites, renders the CFTR channel PKA independent,
but the open probability of .DELTA.R-CFTR is one-third that of the
wild type channel and does not increase upon PKA phosphorylation
(Ma et al., 1997, Winter and Welsh, 1997). Thus, it is possible
that deletion of the R domain removes both inhibitory and
stimulatory effects conferred by the R domain on CFTR chloride
channel function. This conclusion is supported by studies that show
that addition of exogenous unphosphorylated R domain protein (amino
acids 588-858) to wt-CFTR blocks the chloride channel (Ma et al.,
1996), suggesting that the unphosphorylated R domain is inhibitory.
Conversely, exogenous phosphorylated R domain protein (amino acids
588-855 or 645-834) stimulated the .DELTA.R-CFTR channel,
suggesting that the phosphorylated R domain is stimulatory (Ma et
al., 1997, Winter and Welsh, 1997). Therefore, it appears that the
manifest activity (stimulatory or inhibitory) depends on the
phosphorylation state of the R domain.
[0006] About 25% of the known 700 mutations in CFTR produce a
mutant CFTR protein which is properly transported to the apical
membrane of epithelial cells but have only low level, residual
channel activity. There is a need in the art for agents which can
boost the level of channel activity in those mutants having low
level activity.
SUMMARY OF THE INVENTION
[0007] It is an object of the present invention to provide an
isolated polypeptide useful for enhancing the open probability of
CFTR chloride channels.
[0008] It is another object of the present invention to provide a
method of activating a CFTR protein to enhance its open
probability.
[0009] These and other objects of the invention are achieved by
providing one or more of the embodiments described below. In one
embodiment of the invention an isolated polypeptide is provided.
The polypeptide comprises a portion of CFTR (cystic fibrosis
transmembrane conductance regulator) protein of between 10 and 100
amino acids, said portion comprising 18 amino acids as shown in SEQ
ID NO: 1.
[0010] In another embodiment of the invention a method is provided
for activating a CFTR protein. A polypeptide is applied to a CFTR
protein which forms a cAMP regulated chloride channel. The
polypeptide consists of a portion of CFTR protein which comprises
18 amino acids as shown in SEQ ID NO: 1, whereby the open
probability of the channel formed by the CFTR increases by at least
25%.
[0011] According to another aspect of the invention a method is
provided for activating a CFTR protein. A polypeptide is applied to
a CFTR protein which forms a cAMP regulated chloride channel. The
polypeptide consists of a portion of CFTR protein which comprises
22 amino acids as shown in SEQ ID NO: 2, whereby the open
probability of the channel formed by the CFTR increases by at least
25%.
[0012] The present invention thus provides the art with reagents
and tools for enhancing function of channels which are defective in
cystic fibrosis patients.
DETAILED DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. Deletion of Negatively Charged Regions from the R
Domain Results in Expression of Mature Glycosylated,
Phosphorylatable CFTR Proteins
[0014] (FIG. 1A) Sequences of NEG1 and NEG2 within the R domain.
Residues where, mutations have been identified in the CFTR cDNA are
underlined (E822K, E826K, D836Y).
[0015] (FIG. 1B) NEG2 is predicted to form an amphipathic
.alpha.-helix as determined by secondary structure determination
(Geourion and Deleage, 1995, Rost and Sander, 1993, Rost and
Sander, 1994) and illustrated in this space filling model.
Negatively charged residues are colored pink, and the positively
charged lysine is colored green.
[0016] (FIG. 1C) In vitro phosphorylation of wt--(lane 1),
.DELTA.NEG1--(lane 2) and .DELTA.NEG2-CFTR (lane 3) by PKA in the
presence of .gamma.-.sup.32P-ATP. Both the core (band B) and fully
glycosylated (band C) forms of all three CFTR molecules are
phosphorylated.
[0017] FIG. 2. .DELTA.NEG2-CFTR Forms a Chloride Channel that is
Unregulated by PKA
[0018] (FIG. 2A) Single channel currents of wt, .DELTA.NEG1- and
.DELTA.NEG2-CFTR incorporated into the lipid bilayer. While
activities of wt- and .DELTA.NEG1-CFTR absolutely require the
presence of PKA in the cis-intracellular solution, the
.DELTA.NEG2-CFTR channel opens without PKA phosphorylation.
[0019] (FIG. 2B) Diary plot of .DELTA.NEG2-CFTR channel open
probability versus time shows that addition of 200 units/ml of PKA,
a maximally stimulating concentration, does not affect channel
activity. The dashed line indicates the average open probability
for each segment of the experiment. Channels were recorded at -100
mV.
[0020] FIG. 3. The Synthetic NEG2 Peptide both Stimulates and
Inhibits CFTR
[0021] (FIG. 3A) Diary plot (open probability versus time) of a
wt-CFTR channel illustrating the effect of the NEG2 peptide on the
open probability of the channel in the planar lipid bilayer. The
concentration of synthetic NEG2 in the cis-intracellular solution
is indicated above the plot.
[0022] (FIG. 3B) Single channel currents from the wt-CFTR channel
were acquired at -80 mV at the points indicated in A. The
cis-intracellular solution contained 2 mM ATP and 50 units
PKA/ml.
[0023] (FIG. 3C) Single channel trace from .DELTA.NEG2-CFTR
incorporated into the lipid bilayer membrane. Traces were acquired
at -80 mV. The cis-solution contained 2 mM ATP and no PKA. The top
two traces were acquired before synthetic NEG2 peptide addition,
with the second trace being an expansion of the first. In the
bottom two traces, 0.44 .mu.M of the NEG2 peptide has been added
and stimulation is observed. The closed time visibly decreases
after peptide addition.
[0024] FIG. 4. NEG2 Enhances CFTR Channel Activity by Increasing
the Opening Rate of the Channel
[0025] Histograms of open and closed events of the wt-CFTR channel
at -80 mV were generated without peptide (control, left panel) and
with 4.4 .mu.M NEG2 peptide in the cis-solution (right panel).
[0026] (FIG. 4A) The open time histograms contain a single
exponential component with a time constant of 124 ms (control) and
105 ms (peptide-stimulated).
[0027] (FIG. 4B) The closed time histograms contain a fast
component and multiple slow components.
[0028] (FIG. 4C) The closed-burst duration histograms were
constructed using a delimiter of 40 ms (represented by the arrow in
B). The solid lines in C represent the fit according to the double
exponential equation y=P.sub.2* exp [t-.alpha.-exp
.alpha.t-.alpha.)].sub.2+P.sub.3* exp
[t-.alpha..sub.3-exp(t-.alpha..sub.3)] where .alpha..sub.2=log
.tau..sub..sigma.2.alpha..sub.3=log
.tau..sub..tau.3P.sub.2=probability of the intermediate closed
component, and P.sub.3=probability of the long closed component.
The best fit parameters are P.sub.2=0.811, .tau..sub.c2=459 ms,
P.sub.3=0.189, .tau..sub.c3=2494 ms (control); P.sub.2=0.957,
.tau..sub.c2=105 ms, P.sub.3=0.043, .tau..sub.c3=1652 ms
(peptide-stimulated).
DETAILED DESCRIPTION OF THE INVENTION
[0029] It is a discovery of the present inventors that negatively
charged amino acids at the carboxyl terminal of the R domain
(817-838, NEG2) is involved in both the stimulatory and inhibitory
functions of the R domain on the chloride channel. Moreover, a
polypeptide which contains this portion of the CFTR amino acid
sequence can be used to enhance the open probability of both
wild-type and minimally active mutant CFTR protein.
[0030] The isolated polypeptide according to the invention consists
of a portion of CFTR (cystic fibrosis transmembrane conductance
regulator) protein. The portion preferably contains at least 18
amino acids as shown in SEQ ID NO: 1. However, fewer amino acid
residues of the sequence may be used if they retain the channel
enhancing function described herein for the 18 and 22 residue
polypeptides. See also SEQ ID NO: 2. Thus the polypeptide may be
from about 10 or 15 amino acid residues up to about 30 or even 100
amino acid residues. An isolated polypeptide may be synthetic or
made in a recombinant organism. It may be a proteolytic cleavage
product of a larger primary expression product, including
full-length, wild-type CFTR. Preferably the polypeptide will be
free of full-length CFTR. The polypeptide will preferably be free
of other proteins and polypeptides as well. However, it may be
desirable that the polypeptide be fused to another polypeptide to
provide additional functional properties. For example, fusion to
another protein such as keyhole limpet hemocyanin would be used to
increase immunogenicity. Another desirable fusion partner is a
membrane-penetrating peptide. Such peptides include VP-22 (SEQ ID
NO: 3), as well as the peptides shown in SEQ ID NO: 4 and SEQ ID
NO: 5. Such peptides can be used to facilitate the uptake by target
cells of the polypeptide.
[0031] The polypeptides of the present invention can be used to
enhance the function of wild-type or minimally active mutant CFTR
proteins. The polypeptide functions to decrease the closed time of
the channels formed by CFTR. A polypeptide can be applied to the
CFTR protein in any context. It can be applied in vitro or in vivo.
If in vitro it can be to CFTR in cultured cells or to planar
bilayer membranes containing CFTR. If in vivo, the polypeptide can
be applied directly to airway epithelial cells. Such application
can be by any means known in the art, including but not limited to
using a gargle or a nebulizer to deliver aerosolized polypeptide to
the target cells. In addition, the peptide can be delivered in an
indirect mode, by delivering a gene construct to the airway
epithelial cells, which when taken up by the cells causes them to
express the polypeptide. The delivery of the polypeptide to the
CFTR preferably increases the open probability of the channel
formed by the CFTR by at least 25%. More preferably it increases
the open probability by at least 50%, at least 75%, at least 100%,
at least 125%, at least 150%, or at least 200%.
[0032] A CFTR construct comprises a nucleic acid sequence encoding
the amino acid sequence shown in SEQ ID NO: 1. A suitable promoter
for expression in lung epithelia is also desirable. Many such
promoters are known in the art, and any can be used as appropriate
for a particular application.
[0033] It is believed that the administration of the polypeptide of
the present invention will be the most useful in treatment of a
class of mutants which produce CFTR proteins which are properly
delivered to the plasma membrane but which are only residually or
minimally active. Known mutants of CFTR are listed at
http://www.genet.sickkids.on.ca/cftr-cgi-bi- n/fulltable. One can
determine that a particular CFTR mutant is fully processed and
reaches the plasma membrane in a Western blot assay using antibody
against CFTR. Fully processed mutants achieve mature glycosylation
status and appear on the gel as "band C and band B" whereas mutants
that are retained in the endoplasmic reticulum are not fully
glycosylated and show only "band B". See Example 2, below and FIG.
1C.
[0034] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples which are provided
herein for purposes of illustration only, and are not intended to
limit the scope of the invention.
EXAMPLES
Example 1
[0035] Deletion of a Negatively Charged Region (a.a. 817-838) From
the R Domain of CFTR Alters PKA-Dependent Regulation of the CFTR
Channel.
[0036] CFTR contains a large intracellular regulatory (R) domain
where multiple PKA phosphorylation sites are located. There are two
regions within the R domain that contain a high proportion of
negatively charged amino acids, a.a. 725-733 (NEG1) and a.a.
817-838 (NEG2). It is possible that these two regions could have
allosteric or electrostatic interactions with other regions of CFTR
and thus affect its chloride channel function. To test the role of
NEG1 and NEG2, two deletion mutants, NEG1 -CFTR and NEG2-CFTR, were
created. The CFTR mutants were transiently expressed in HEK 293
cells, and their single channel functions were studied using the
bilayer reconstitution system. Western blots indicate that both
NEG1-CFTR and NEG2-CFTR process normally and traffic to the plasma
membrane of HEK 293 cells. Both mutants form functional chloride
channels in the bilayer membrane, with single channel conductances
similar to the wt-CFTR channel. Like wt-CFTR, opening of NEG1-CFTR
requires absolutely PKA phosphorylation and ATP binding/hydrolysis.
In contrast to wt-CFTR, opening of NEG2-CFTR does not require PKA
phosphorylation. Thus, deletion of NEG2, but not NEG1, alters
PKA-dependent regulation of the CFTR chloride channel. Our data
suggest that NEG2 could form a `putative gating particle` of the
CFTR channel possibly through electrostatic and/or allosteric
interactions with other domains of CFTR.
Example 2
[0037] .DELTA.NEG1- and .DELTA.NEG2-CFTR are Glycosylated.
[0038] The R domain of CFTR contains two negatively charged
regions, amino acids 725-733 (NEG1) and amino acids 817-838 (NEG2),
that reside in close proximity to two PKA phosphorylation sites,
S737 and S813, used in vivo (FIG. 1A) (Cheng, et al. 1991). NEG2 is
predicted to form an amphipathic (-helical structure with a
negatively charged face (FIG. 1B) (Geoudjon and Deleage, 1995, Rost
and Sander, 1993, Rost and Sander, 1994). Three mutations (E822K,
E826K, D836Y), two of which were clearly obtained from patients
with CF (E822K and D836Y). have been identified within the NEG2
region that result in the removal of negative charges
(www.genet.sickkids.on.ca). The E822K CFTR channel has a low open
probability relative to wt-CFTR (wild type-CFTR), but the E826K
CFTR channel has single channel properties similar to wt-CFTR
(Vankeerberghen et al., 1998). The presence of these
disease-causing mutations suggests the potential importance of the
NEG2 region. To investigate the roles of NEG1 and NEG2 in CFTR
function, these regions were deleted from CFTR using mutagenesis
and subcloning. The .DELTA.NEG1- and .DELTA.NEG2-CFTR proteins were
transiently expressed in human embryonic kidney 293 cells. Membrane
vesicles containing the CFTR proteins were isolated and subjected
to SDS-PAGE. Like wt-CFTR, both .DELTA.NEG1- and .DELTA.NEG2-CFTR
are present both in the core glycosylated (band B) and the fully
glycosylated form (band C) (FIG. 1C).
Example 3
[0039] The Open Probability of the .DELTA.NEG2-CFTR Chloride
Channel is Much Less Than That of Wild Type but is Independent PKA,
Although it Contains all PKA Phosphorylation Sites.
[0040] Single channel measurements indicate that the
.DELTA.NEG1-CFTR channel is similar to wt-CFTR in its PKA
dependence. No chloride channels are observed in the absence of PKA
(FIG. 2A) and the open probability in the presence of PKA and ATP
is similar to wt-CFTR. In contrast, the .DELTA.NEG2-CFTR channel
opens without PKA (FIG. 2A). The constitutive activity of the
.DELTA.NEG2-CFTR channel is unlikely to be due to the endogenous
phosphorylation of the .DELTA.NEG2-CFTR protein, since protein
phosphatase 2A, which decreases activity of the wt-CFTR opened by
PKA and ATP (Ma et al., 1997), has no effect on the
.DELTA.NEG2-CFTR channel (n=4). Moreover, addition of PKA up to 200
units/ml, four times the concentration required to fully activate
wt-CFTR (Ma et al., 1997), does not increase the open probability
of the channel (FIG. 2B). .DELTA.NEG2-CFTR has conductance
properties similar to wild type (Tao et al., 1996). However, the
open probability of the .DELTA.NEG2-CFTR chloride channel is much
less than that of wild type and cannot be increased by PKA
(P.sub.o=0.035 (0.012 and P.sub.o=0.026 (0.013 without and with PKA
respectively, n=5). While NEG2 may represent an inhibitory region,
removal of these amino acids does not result in a fully activated
channel. The failure of the .DELTA.NEG2-CFTR channel to respond to
PKA does not result from inability of the channel to be
phosphorylated, for an in vitro assay using (-.sup.32P-ATP showed
comparable phosphorylation of wt-CFTR and .DELTA.NEG2-CFTR (FIG.
1C). Thus, it appears that removal of NEG2 from CFTR completely
eliminates the PKA dependence of the chloride channel, although the
.DELTA.NEG2-CFTR channel still contains all ten PKA sites and can
be phosphorylated.
Example 4
[0041] NEG2 Polypeptide Stimulates Both Wild-Type and a NEG2 CFTR
Proteins at Concentrations Greater than 0.44 .mu.M.
[0042] To test whether the NEG2 region is responsible for both
stimulatory and inhibitory interactions between the R domain and
other domains, synthetic NEG2, a 22 amino acid peptide, was added
to the cis-intracellular side of single CFTR channels captured in
the planar lipid bilayer (FIG. 3). The diary plot of open
probability as a function of time shows the activity of a single
wt-CFTR channel during the course of the experiment (FIG. 3A).
After peptide addition, there are periods of intense stimulation
that last 4 to 8 minutes. These stimulatory periods are followed by
either a return to the basal level of activity before peptide
addition, or by an almost complete inhibition of the channel, where
only a flickery 3 pS conductance is observed. During stimulation,
the open probability more than doubles and more transitions are
observed between the open and closed states (FIG. 3B). The
stimulatory response was observed in 6 of 7 experiments at
concentrations .gtoreq.0.44 .mu.M (the remaining channel was
inhibited upon peptide addition (4.4 .mu.M) and no stimulation was
seen). Profound inhibition was observed in three channels at
concentrations .gtoreq.4.4 .mu.M. When the NEG2 peptide was added
to the intracellular side of the .DELTA.NEG2-CFTR channel, which
lacks its own endogenous NEG2 sequence, a similar stimulatory
response was observed (FIG. 3C).
Example 5
[0043] The NEG2 Peptide Decreases the Closed Time of the Wild-Type
CFTR Protein.
[0044] In order to understand the mechanism responsible for the
increase in open probability, the gating kinetics of wt-CFTR
without peptide and during stimulation by synthetic NEG2 were
analyzed. The open time distributions of the wt-CFTR did not change
during peptide stimulation, as both control (without NEG2 peptide)
and peptide-stimulated channels had an open lifetime of
approximately 120 ms (FIG. 4A). Thus, the increase in the open
probability is not due to a change in the closing rate of the
channel. However, the closed time distribution for the stimulated
channel is clearly shifted to the left compared to the control
channel (FIG. 4B). There are three components to the closed state,
a fast (.tau..sub.c1), an intermediate (.tau..sub.c2), and a long
(.tau..sub.c3) closed component. The fast closed component is
probably due to closings within a burst (Carson et al., 1995).
Therefore, to identify better the closed times between bursts, a
delimiter of .tau..sub.c=40 ms was set at the nadir between the
fast and intermediate closed times (illustrated by the arrow in
FIG. 4B) to generate the closed-burst duration histograms. As shown
in FIG. 4C, following peptide stimulation, the intermediate closed
time was reduced from 459 ms to 105 ms, whereas the long closed
time remained relatively unchanged. Thus, the interaction of NEG2
with CFTR increased the intermediate-opening rate of the channel.
This increase in opening rate is similar to that observed when the
phosphorylated R domain protein (amino acids 645-834) was added to
CFTR-.DELTA.R/S660A in excised inside-out patches (Winter and
Welsh, 1997). Additionally, modification of C832, which resides
within NEG2, by N-ethylmaleimide (NEM) results in irreversible
stimulation of PKA-phosphorylated CFTR chloride channel activity
(Cotten and Welsh, 1997), further emphasizing the importance of
NEG2 in CFTR regulation.
[0045] These data, taken together, show that the NEG2 region
confers both stimulatory and inhibitory functions of the R domain
on the CFTR channel. When this region is deleted from CFTR, the
resultant channel opens without PKA (loss of inhibitory function),
but it never achieves open probability comparable to wild type even
when phosphorylated with PKA (loss of stimulatory function). This
same sequence, expressed as a peptide, results in stimulation of
channel openings at lower concentrations and profound inhibition of
channel activity at higher concentrations, when added to the
intracellular side of CFTR channels. It seems likely that this
sequence interacts with CFTR at different sites on the nucleotide
binding domains to either stimulate or inhibit channel openings.
Phosphorylation of the R domain, in this model, changes its
conformation and thus presents the NEG2 sequence better to the
stimulatory than the inhibitory site. A current model for channel
opening is that phosphorylated channels open in response to ATP
binding and hydrolysis at the first nucleotide binding fold (NBF1)
(Gadsby and Nairn, 1994, Ma and Davis, 1998). Since stimulation by
NEG2 occurs by increasing channel openings, a likely site of
stimulation is NBF1, though other models are possible.
METHODS USED IN EXAMPLES 1-5
[0046] Subcloning of CFTR Gene
[0047] The wt, .DELTA.NEG1-, and .DELTA.NEG2-CFTR cDNAs were
subcloned into an Epstein-Barr virus-based episomal eukaryotic
expression vector, pCEP4 (Invitrogen, San Diego, Calif.), between
the Nhel and Xhol restriction sites. The .DELTA.NEG1 and
.DELTA.NEG2 deletion mutants were created using the pALTER
mutagenesis system and shuttled from pALTER into pCEP4 by
substituting the corresponding fragment in pCEP4 wt-CFTR with the
mutant fragment between the Xhol and BstZ171 restriction sites. The
.DELTA.NEG1-CFTR cDNA has 27 bases deleted (amino acids 725-733).
The .DELTA.NEG2-CFTR cDNA has 66 bases deleted (amino acids
817-838).
[0048] Expression of CFTR in HEK 293 Cells
[0049] A human embryonic kidney cell line (293-EBNA HEK;
Invitrogen) was used for transfection and expression of the CFTR
proteins (Ma et al., 1997, Ma et al., 1996, Xie et al., 1995). The
HEK-293 cell line contains a pCMV-EBNA vector, which constitutively
expresses the Epstein-Barr virus nuclear antigen-1 (EBNA-1) gene
product and increases the transfection efficiency of Epstein-Barr
virus-based vectors. The cells were maintained in Dulbecco's
Modified Eagle Medium with 10% FBS and 1% L-glutamine. Geneticin
(G418, 250 (g/ml) was added to the cell culture medium to maintain
selection of the cells containing the PCMV-EBNA vector.
Lipofectamine reagent (Life Technologies, Inc) in Optimem media
(serum-free) was used to transfect the HEK-293 cells with
pCEP4(wt), pCEP4(.DELTA.NEG1), or pCEP4(.DELTA.NEG2). After 5
hours, serum was added to the media (10% final serum
concentration). Twenty-four hours after transfection, the
transfection media was replaced with fresh media. The cells were
harvested two days after transfection and microsomal membrane
vesicles were prepared for single channel measurements in the lipid
bilayer reconstitution system.
[0050] Vesicle Preparation From Transfected HEK 293 Cells
[0051] HEK-293 cells transfected with pCEP4(CFTR) were harvested
and homogenized using a combination of hypotonic lysis and Dounce
homogenization in the presence of protease inhibitors (Ma et al.,
1997, Ma et al., 1996, Xie et al., 1995). Microsomes were collected
by centrifugation of postnuclear supernatant (4500.times.g, 15 min)
at 100,000.times.g for 20 min and resuspended in a buffer
containing 250 mM sucrose, 10 mM HEPES, pH 7.2. The membrane
vesicles were stored at -75.degree. C. until use.
[0052] In Vitro Phosphorylation of CFTR Proteins
[0053] CFTR proteins isolated in membrane vesicles were bound to
protein G agarose using a mouse monoclonal anti-human CFTR antibody
(Genzyme). The protein G agarose was washed, and (-.sup.32P-ATP (10
(Ci) and protein kinase A (.about.10 units/50(l) was added. Samples
were incubated at 30(C. for one hour during phosphorylation. Excess
(.gamma.-.sup.32P-ATP was removed, and SDS-PAGE sample buffer (200
mM Tris-Cl, pH 6.7,9% SDS, 6% beta-mercaptoethanol, 15% glycerol,
and 0.01% bromophenol blue) was added to denature CFTR and release
it from the protein G agarose. The samples were subjected to
electrophoresis on a 5% SDS-polyacrylamide gel, transferred to a
polyvinylidene difluoride membrane, and exposed to film.
[0054] Preparation of NEG2 Peptides
[0055] The 22 amino acid peptide corresponding to NEG2 was custom
made by Quality Controlled Biochemicals. Inc. The peptide was
resuspended in water to a concentration of 1 mg/ml and pH was
adjusted to a physiological range (7.2-7.4) using KOH and HCl. The
space filling model of the NEG2 peptide was generated, based on
secondary structure predictions (Geou jon and Deleage, 1995, Rost
and Sander, 1993, Rost and Sander, 1994), using the Insight II
program from Molecular Simulations Incorporated.
[0056] Reconstitution of CFTR Channels in Lipid Bilayer
Membranes
[0057] Lipid bilayer membranes were formed across an aperture of
.about.200 (m diameter with a mixture of
phosphatidylethanolamine:phospha- tidylserine:cholesterol in a
ratio of 5:5:1. The lipids were dissolved in decane at a
concentration of 33 mg/ml. The recording solutions contained: cis
(intracellular), 200 mM CsCl, 1 mM MgCl.sub.2, 2 mM ATP, and 10 mM
HEPES-Tris (pH 7.4); trans (extracellular), 50 mM CsCl, 10 mM
HEPES-Tris (pH 7.4). Vesicles (1-4 (l) containing either wild-type,
.DELTA.NEG1-, or .DELTA.NEG2-CFTR were added to the cis solution.
The PKA catalytic subunit was present at a concentration of 50
units/ml in the cis solution unless noted otherwise. Single channel
currents were recorded with an Axopatch 200A patch clamp unit (Axon
Instruments). The currents were sampled at 1-2.5 ms/point. Single
channel data analyses were performed with pClamp and TIPS
softwares.
[0058] References
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[0061] Carson, M. R., Travis, S. M., and Welsh, M. J. (1995). The
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[0073] Quinton, P. M. (1986). Missing Cl.sup.- conductance in
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Sequence CWU 1
1
5 1 18 PRT Homo sapiens 1 Gly Leu Glu Ile Ser Glu Glu Ile Asn Glu
Glu Asp Leu Lys Glu Cys 1 5 10 15 Phe Phe 2 22 PRT Homo sapiens 2
Gly Leu Glu Ile Ser Glu Glu Ile Asn Glu Glu Asp Leu Lys Glu Cys 1 5
10 15 Phe Phe Asp Asp Met Glu 20 3 559 PRT HSV-1 3 Met Ala Arg Phe
His Arg Pro Ser Glu Asp Glu Asp Asp Tyr Glu Tyr 1 5 10 15 Ser Asp
Leu Trp Val Arg Glu Asn Ser Leu Tyr Asp Tyr Glu Ser Gly 20 25 30
Ser Asp Asp His Val Tyr Glu Glu Leu Arg Ala Ala Thr Ser Gly Pro 35
40 45 Glu Pro Ser Gly Arg Arg Ala Ser Val Arg Ala Cys Ala Ser Ala
Ala 50 55 60 Ala Val Gln Pro Ala Ala Arg Gly Arg Asp Arg Ala Ala
Ala Ala Gly 65 70 75 80 Thr Thr Val Ala Ala Pro Ala Ala Ala Pro Ala
Arg Arg Ser Ser Ser 85 90 95 Arg Ala Ser Ser Arg Pro Pro Arg Ala
Ala Ala Asp Pro Pro Val Leu 100 105 110 Arg Pro Ala Thr Arg Gly Ser
Ser Gly Gly Ala Gly Ala Val Ala Val 115 120 125 Gly Pro Pro Arg Pro
Arg Ala Pro Pro Gly Ala Asn Ala Val Ala Ser 130 135 140 Gly Arg Pro
Leu Ala Phe Ser Ala Ala Pro Lys Thr Pro Lys Ala Pro 145 150 155 160
Trp Cys Gly Pro Thr His Ala Tyr Asn Arg Thr Ile Phe Cys Glu Ala 165
170 175 Val Ala Leu Val Ala Ala Glu Tyr Ala Arg Gln Ala Ala Ala Ser
Val 180 185 190 Trp Asp Ser Asp Pro Pro Lys Ser Asn Glu Arg Leu Asp
Arg Met Leu 195 200 205 Lys Ser Ala Ala Ile Arg Ile Leu Val Cys Glu
Gly Ser Gly Leu Leu 210 215 220 Ala Ala Ala Asn Asp Ile Leu Ala Ala
Arg Ala Gln Arg Pro Ala Ala 225 230 235 240 Arg Gly Ser Thr Ser Gly
Gly Glu Ser Arg Leu Arg Gly Glu Arg Ala 245 250 255 Arg Pro Met Thr
Ser Arg Arg Ser Val Lys Ser Gly Pro Arg Glu Val 260 265 270 Pro Arg
Asp Glu Tyr Glu Asp Leu Tyr Tyr Thr Pro Ser Ser Gly Met 275 280 285
Ala Ser Pro Asp Ser Pro Pro Asp Thr Ser Arg Arg Gly Ala Leu Gln 290
295 300 Thr Arg Ser Arg Gln Arg Gly Glu Val Arg Phe Val Gln Tyr Asp
Glu 305 310 315 320 Ser Asp Tyr Ala Leu Tyr Gly Gly Ser Ser Ser Glu
Asp Asp Glu His 325 330 335 Pro Glu Val Pro Arg Thr Arg Arg Pro Val
Ser Gly Ala Val Leu Ser 340 345 350 Gly Pro Gly Pro Ala Arg Ala Pro
Pro Pro Pro Ala Gly Ser Gly Gly 355 360 365 Ala Gly Arg Thr Pro Thr
Thr Ala Pro Arg Ala Pro Arg Thr Gln Arg 370 375 380 Val Ala Thr Lys
Ala Pro Ala Ala Pro Ala Ala Glu Thr Thr Arg Gly 385 390 395 400 Arg
Lys Ser Ala Gln Pro Glu Ser Ala Ala Leu Pro Asp Ala Pro Ala 405 410
415 Ser Thr Ala Pro Thr Arg Ser Lys Thr Pro Ala Gln Gly Leu Ala Arg
420 425 430 Lys Leu His Phe Ser Thr Ala Pro Pro Asn Pro Asp Ala Pro
Trp Thr 435 440 445 Pro Arg Val Ala Gly Phe Asn Lys Arg Val Phe Cys
Ala Ala Val Gly 450 455 460 Arg Leu Ala Ala Met His Ala Arg Met Ala
Ala Val Gln Leu Trp Asp 465 470 475 480 Met Ser Arg Pro Arg Thr Asp
Glu Asp Leu Asn Glu Leu Leu Gly Ile 485 490 495 Thr Thr Ile Arg Val
Thr Val Cys Glu Gly Lys Asn Leu Leu Gln Arg 500 505 510 Ala Asn Glu
Leu Val Asn Pro Asp Val Val Gln Asp Val Asp Ala Ala 515 520 525 Thr
Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr Glu Arg Pro 530 535
540 Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro Val Glu 545
550 555 4 27 PRT Artificial Sequence membrane permeating peptide 4
Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5
10 15 Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 5 16 PRT
Artificial Sequence membrane permeating peptide 5 Arg Gln Ile Lys
Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15
* * * * *
References