Biological Bypass Bridge with Sodium Channels, Calcium Channels and/or Potassium Channels to Compensate for Conduction Block in the Heart

Abstract

This invention provides a bypass bridge comprising a tract of gap junction-coupled cells having a first end and a second end, both ends capable of being attached to two selected sites in a heart so as to allow the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites, wherein the cells functionally express a sodium channel. The invention also provides related methods of making the bypass bridge, methods of implanting same in a heart, and methods of treating a disorder associated with an impaired conduction in a subject's heart.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 60/704,210, filed Jul. 29, 2005; 60/701,312, filed Jul. 21, 2005; and 60/781,723 filed Mar. 14, 2006, the entire contents of which are incorporated herein by reference.

[0003] Throughout this application, various publications are referenced in parentheses by author name and date, patent number, patent application or publication number. Full citations for these publications may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to those skilled therein as of the date of the invention described and claimed herein. However, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention.

FIELD OF THE INVENTION

[0004] The present invention relates to a bypass bridge comprising a tract of gap junction-coupled cells that functionally express a sodium channel, the two ends of the tract being attachable to two selected sites in a heart so as to allow the conduction of a pacemaker and/or pacemaker and/or electrical signal/current across the tract between the two sites.

BACKGROUND OF THE INVENTION

[0005] Electronic pacemakers are lifesaving devices that provide a regular heartbeat in settings where the sinoatrial node, atrioventricular conduction, or both, have failed. Thus, one of the major indications for electronic pacemaker therapy is high degree heart block, such that a normally functioning sinus node impulse cannot propagate to the ventricle. The result is ventricular arrest and/or fibrillation, and death.

[0006] Malfunction or loss of pacemaker cells can occur due to disease or aging. For example, acute myocardial infarction (MI) kills millions of people each year and generally induces in survivors marked reductions in myocyte number and cardiac pump function. Adult cardiac myocytes divide only rarely, and the usual responses to myocyte cell loss include compensatory hypertrophy and/or congestive heart failure, a disease with a significant annual mortality. There have been recent reports of the delivery of bone marrow-derived and/or circulating human mesenchymal stem cells (hMSCs) to the hearts of post-myocardial infarct patients resulting in some improvement of mechanical performance (Strauer et al., 2002; Perin et al., 2003) in the absence of overt toxicity. The presumption in these and other animal studies (Orlic et al., 2001) is that the hMSC integrate into the cardiac syncytium and then differentiate into new heart cells restoring mechanical function.

[0007] An alternative application of cell therapy, described herein, involves growing cells such as hMSCs into a bypass bridge comprising a tract of gap junction-coupled cells that can be used to conduct pacemaker and/or electrical current/signals across a region of the heart exhibiting impaired electrical conduction.

SUMMARY OF THE INVENTION

[0008] The invention disclosed herein provides a bypass bridge comprising a tract of gap junction-coupled cells having a first end and a second end, both ends capable of being attached to two selected sites in a heart so as to allow the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites, wherein the cells functionally express a sodium channel. In certain embodiments of the bypass bridge, cells in the tract further functionally express a pacemaker ion channel which induces a pacemaker current so as to induce a pacemaker current in the cells. In preferred embodiments, the first end of the tract of cells is capable of being attached to the atrium and the second end is capable of being attached to the ventricle, so as to form an atrioventricular (AV) bridge that allows conduction of a pacemaker and/or electrical signal/current across the tract from the atrium to the ventricle.

[0009] The invention also provides a method of making a bypass bridge for implantation in a heart comprising (a) transfecting a cell with, and functionally expressing therein, a nucleic acid encoding a sodium channel and optionally a nucleic acid encoding a pacemaker ion channel, and (b) growing the transfected cell into a tract of cells having a first and a second end capable of being attached to two selected sites in the heart, wherein the cells are physically interconnected via electrically conductive gap junctions.

[0010] The invention further provides a method of implanting a bypass bridge in a heart comprising (a) making a bypass bridge by any of the methods disclosed herein, (b) selecting a first and a second site in the heart, and (c) attaching the first end of the tract to the first site and the second end of the tract to the second site, so as to thereby implant a bypass bridge in the heart that allows the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites.

[0011] The present invention also provides a method of treating a disorder associated with an impaired conduction in a subject's heart comprising (a) transfecting a cell with a nucleic acid encoding a sodium channel, wherein the cell functionally expresses the sodium channel, (b) growing the transfected cell into a tract of cells having a first end and a second end, wherein the cells are physically interconnected via electrically conductive gap junctions, (c) selecting a first site and a second site in the heart between which sites conduction is impaired, and (d) attaching the first end of the tract to the first site and the second end of the tract to the second site, so as to allow the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites and thereby treat the subject.

[0012] This invention further provides a method of treating a disorder associated with an impaired conduction and impaired sinus node activity in a subject's heart comprising (a) transfecting a cell with at least one nucleic acid encoding a sodium channel and a pacemaker ion channel, wherein the cell functionally expresses the sodium channel and the pacemaker ion channel, (b) growing the transfected cell into a tract of cells having a first end and a second end, wherein the cells are physically interconnected via electrically conductive gap junctions, (c) selecting a first site in the left atrium of the heart and a second site, between which sites conduction is impaired, and (d) attaching the first end of the tract to the first site and the second end of the tract to the second site, so as to allow the propagation of a pacemaker and/or electrical signal/current generated by the sinus node and/or tract of cells between the two sites and thereby treat the subject.

BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1. Alignment of Mouse and Human Hcn2 Polypeptide Sequences.

[0014] The mouse and human HCN2 polypeptide sequences are aligned for maximum correspondence. The amino acid sequences of mHCN2 and hHCN2 are set forth in SEQ ID NO: 14 and SEQ ID NO: 16, respectively.

[0015] FIG. 2. Amino Acid Sequence of the Human HCN212 Chimeric Channel.

[0016] The shaded N-terminal portion of the sequence is derived from hHCN2; the underlined intramembranous portion from hHCN1; and the C-terminal portion (without shading or underline) from hHCN2. The amino acid sequence of the hHCN212 chimeric channel is set forth in SEQ ID NO: 2. This 889-amino acid long chimeric hHCN212 sequence shows 91.2% identity with the 863-amino acid long mHCN212 sequence in 893 residues overlap when aligned for maximum correspondence.

[0017] FIG. 3. Amino Acid Sequence of the Mouse HCN212 Chimeric Channel.

[0018] The shaded N-terminal portion of the sequence is derived from mouse HCN2; the underlined intramembranous portion from mouse HCN1; and the C-terminal portion (without shading or underline) from mouse HCN2. The amino acid sequence of the mouse HCN212 chimeric channel is set forth in SEQ ID NO: 6. This 863-amino acid long chimeric mHCN212 sequence shows 91.2% identity with the 889-amino acid long hHCN212 sequence in 893 residues overlap when aligned for maximum correspondence.

[0019] FIG. 4. A-D. Identification of Connexins in Gap Junctions of Human Mesenchymal Stem Cells (hMSCs).

[0020] Immunostaining of Cx43 (A), Cx40 (B) and Cx45 (C). D, Immunoblot analysis of Cx43 in canine ventricle myocytes and hMSCs. Whole cell lysates (120 .mu.g) from ventricle cells or hMSCs were resolved by SDS, transferred to membranes, and blotted with Cx43 antibodies. Molecular weight markers are indicated.

[0021] FIG. 5. A-E. Macroscopic and Single Channel Properties of Gap Junctions Between hMSC Pairs.

[0022] Gap junction currents (k) elicited from hMSCs using a symmetrical bipolar pulse protocol (10 s, from .+-.10 mV to .+-.110 mV, V.sub.h=0 mV) showed two types of voltage-dependent current deactivation: symmetrical (A) and asymmetrical (B). C, summary plots of normalized instantaneous (o) and steady-state (.cndot.) g.sub.i versus V.sub.j. Left panel, quasi-symmetrical relationship from 5 pairs; continuous line, Boltzmann fit: V.sub.j,o=-70/65 mV, g.sub.j,min=0.29/0.34, g.sub.j,max=0.99/1.00, z=2.2/2.3 for negative/positive V.sub.j. Right panel, asymmetrical relationship from 6 pairs; Boltzmann fit for negative V.sub.j: V.sub.j,0=-72 mV, g.sub.j,min=0.25, g.sub.j,max=0.99, z=1.5. D and E, single channel recordings from pairs of hMSCs. Pulse protocol (V.sub.1 and V.sub.2) and associated multichannel currents (I.sub.2) recorded from a cell pair during maintained V.sub.j of .+-.80 mV. The discrete current steps indicate the opening andclosing of single channels. Dashed line: zero current level. The all points current histograms on the right-hand side reveal a conductance of .about.50 pS.

[0023] FIG. 6. A-E. Macroscopic Properties of Junctions in Cell Pairs Between a hMSC and HeLa Cell Expressing Only Cx40, Cx43 or Cx45.

[0024] In all cases hMSC to Hela cell coupling was tested 6 to 12 after hours initiating co-culture. A, Ij elicited in response to a series of 5-s voltage steps (V.sub.j) in hMSC-HeLaCx43 pairs. Top, symmetrical current deactivation; bottom, asymmetrical current voltage dependence. B, Macroscopic Ij recordings from hMSC-HelaCx40 pairs exhibit symmetrical (top panel) and asymmetrical (bottom panel) voltage dependent deactivation. C, Asymmetric Ij from hMSC-HeLaCx43 pair exhibits voltage dependent gating when Cx45 side is relatively negative. Ij recorded from hMSC. D, g.sub.j,ss plots versus V.sub.j from pairs between hMSC and transfected HeLa cells. Left panel, hMSC-HeLaCx43 pairs, quasi-symmetrical relationship (.cndot.) and asymmetrical relationship (o); continuous and dashed lines are Boltzmann fits (see text for details). Middle panel, symmetrical (.cndot.) and asymmetrical (o) relationships from hMSC-HeLaCx40 pairs; the continuous and dashed lines correspond to Boltzmann fits (see text for details). Right panel, asymmetrical relationship from hMSC-HeLaCx45 cell pairs; continuous line, Boltzmann fit for positive V.sub.j (see text for details). E, Cell-to-cell Lucifer Yellow (LY) spread in cell pairs: from an hMSC to an hMSC (upper panel), from a HeLaCx43 to an hMSC (middle panel), and from an hMSC to a HeLaCx43 (bottom panel). In all cases a pipette containing 2 mM LY was attached to the left-hand cell in the whole-cell configuration. Epifluorescent micrographs taken at 12 min after dye injection show LY spread to the adjacent (right-hand) cell. The simultaneously measured junctional conductance revealed gj of .about.13 nS, .about.16 nS, and .about.18 nS of the pairs, respectively. Cell Tracker green was used to distinguish hMSCs from HeLa cells or vice versa in all experiments.

[0025] FIG. 7. A-C. Macroscopic and Single Channel Properties of Gap Junctions Between hMSC-Canine Ventricle Cell Pairs.

[0026] Myocytes were plated between 12 and 72 h and co-cultured with hMSCs for 6 to 12 h before measuring coupling. A, Localization of Cx43 for hMSC-canine ventricle cell pairs. Most of Cx43 was localized to the ventricular cell ends and a small amount of Cx43 was present along the lateral borders. The intensive Cx43 staining was detected between the end of the rod-shaped ventricular cell (middle cell) and the hMSC (right cell). There is no detectable Cx43 staining between the ventricular cell and the hMSC on the left side. B, Top, phase-contrast micrograph of a hMSC-canine ventricular myocyte pair. Bottom, monopolar pulse protocol (V.sub.1 and V.sub.2) and associated macroscopic junctional currents (I.sub.2) exhibiting asymmetrical voltage dependence. C, Top, multichannel current elicited by symmetrical biphasic 60 mV pulse. Dashed line, zero current level; dotted lines, represent discrete current steps indicative of opening and closing of channels. The current histograms yielded a conductance of .about.40-50 pS. Bottom, multichannel recording during maintained V.sub.j of 60 mV. The current histograms revealed several conductances of 48-64 pS with several events with conductance of 84 pS to 99 pS (arrows) which resemble operation of Cx43, heterotypic Cx40-Cx43 and/or homotypic Cx40 channels.

[0027] FIG. 8. A-B. Inactivation Properties of a Sodium Channel Suitable for Incorporation into a Cardiac Bypass Bridge.

[0028] The inactivation properties of the SKM-1 sodium channel measured in Xenopus oocytes are shown. Left, (A) The holding potential ranged from -100 mV to -55 mV and the test potential was +30 mV. Right, (B) averaged inactivation curves for 11 oocytes.

[0029] FIG. 9. A-B. I-V Relationship of a Sodium Channel Suitable for Incorporation into a Cardiac Bypass Bridge.

[0030] Shown here is I-V relationship for the SKM-1 channel recorded in Xenopus oocytes. Left, (A) Raw data recorded from a holding potential of -80 mV to potentials between -70 mV and +50 mV in 10 mV increments. Right, (B) Peak inward 1-V relationship.

[0031] FIG. 10. Biophysical Properties of a Calcium Channel Suitable for Incorporation into a Cardiac Bypass Bridge.

[0032] CaV1.2, alpha2 and gamma, Pl.b and red fluorescent reporter were co-expressed in HEK293 cells. Ca.sup.2+ current was recorded in the red fluorescent cells. The cells were held at -50 mV. Test pulses (60 ms) were from -40 mV to +50 mV with a 10 mV interval.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention provides a bypass bridge comprising a tract of gap junction-coupled cells having a first end and a second end, both ends capable of being attached to two selected sites in a heart so as to allow the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites, wherein the cells functionally express a sodium channel. In certain embodiments of the bypass bridge, cells in the tract further functionally express a pacemaker ion channel which induces a pacemaker current so as to induce a pacemaker current in the cells. In preferred embodiments, the first endof the tract of cells is capable of being attached to the atrium and the second end is capable of being attached to the ventricle, so as to form an atrioventricular ("AV") bridge that allows conduction of a pacemaker and/or electrical signal/current across the tract from the atrium to the ventricle and thereby stimulate contraction of the ventricle.

[0034] The invention also provides a bypass bridge comprising a tract of gap junction-coupled cells having a first end and a second end, both ends capable of being attached to two selected sites in a heart so as to allow the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites, wherein the cells functionally express a potassium channel or calcium channel instead of a sodium channel. The embodiments disclosed herein for the bypass bridge expressing a sodium channel are equally applicable to the bypass bridge expressing a potassium or calcium channel.

[0035] Exemplary cells that may be used for growing the tract include, but are not limited to, stem cells, cardiomyocytes, fibroblasts or skeletal muscle cells engineered to express at least one cardiac connexin, or endothelial cells. In preferred embodiments, the stem cells are adult mesenchymal stem cell (MSCs) or embryonic stem cells (ESCs), wherein said stem cells are substantially incapable of differentiation. In various embodiments, the MSCs are autologous, allogeneic or heterogenic relative to the subject into whose heart the bypass bridge is to be introduced, and the subject is a human being. As used herein, a "subject" shall mean any animal or artificially modified animal. Animals include, but are not limited to, humans, non-human primates, dogs, cats, cows, horses, sheep, pigs, rabbits, ferrets, rodents such as mice, rats and guinea pigs, and birds such as chickens and turkeys. Artificially modified animals include, but are not limited to, SCID mice with human immune systems. In a preferred embodiment, the subject is a human.

[0036] In more preferred embodiments of the instant bypass bridge, the stem cells are human adult mesenchymal stem cells (hMSCs) or human embryonic stem cells (hESCs), wherein the stem cells are substantially incapable of differentiation. In other preferred embodiments, the hMSCs (a) express CD29, CD44, CD54, and HLA class I surface markers; and (b) do not express CD 14, CD34, CD45, and HLA class II surface markers. In additional embodiments, the hMSCs have been passaged at least 9 times, preferably 9-12 times. In further embodiments, the cells further express at least one cardiac connexin. In still further embodiments, the at least one cardiac connexin is Cx43, Cx40, or Cx45.

[0037] Thus, the present invention is based on the use of cell therapy to construct in the heart a bypass tract incorporating functional sodium ion channels, and optionally pacemaker channels, and optionally or alternatively potassium or calcium channels. The bypass bridge can be used to conduct a pacemaker and/or electrical signal/current between any two sites in the heart between which there is impaired pacemaker and/or electrical conduction. For example, the bypass may be used as an AV bridge to take over or supplement the function of a diseased AV node. Human MSCs may be grown in culture on a non-bioreactive matrix into a strip or tract of cells containing two ends. Once growth is complete, one end of the tract may be attached, e.g., by sutures, to a first selected site in the heart such as an atrium, and the other end may attached to a second selected site such as a ventricle. In a bypass bridge used as an AV bridge, for example, pacemaker and/or electrical signals/current generated by the sinus node to activate the atria will propagate across the artificially constructed tract to excite the ventricle. In this way the normal sequence of atrioventricular activation will be maintained.

[0038] Human MSCs may be prepared in several ways including, but not limited to, the following:

[0039] 1: In culture without incorporation of additional molecular determinants of conduction. Here the cells' own ability to form gap junctions that communicate pacemaker and/or electrical signals are used as a means to conduct an electrotonic wave from one site in the heart to another.

[0040] 2: In culture following transfection of the cells, such as by electroporation or viral infection, to introduce a gene for at least one of the cardiac connexins Cx43, Cx40 or Cx45, to enhance formation of gap junctions and thereby facilitate cell-to-cell propagation of pacemaker/electrical signals.

[0041] 3: In culture following transfection of the cells to introduce a nucleic acid encoding the alpha and the accessory subunits of an L-type calcium channel, thereby increasing the likelihood of not just electrotonic propagation of a wavefront, but its active propagation by an action potential.

[0042] 4: In culture following transfection of the cells to introduce a nucleic acid encoding the alpha subunit, with or without the accessory subunits, of a sodium channel, thereby increasing the likelihood of not just electrotonic propagation or calcium-dependent propagation of a wavefront, but its active propagation by a sodium-dependent action potential.

[0043] 5: In culture following transfection of the cells to introduce a nucleic acid encoding the calcium and/or sodium channel subunits as in 3 and/or 4, and a nucleic acid encoding an alpha subunit, with or without the accessory subunits of a potassium channel, thereby increasing the likelihood of not just active propagation by an action potential, but additional control of the initial resting potential and its voltage-time course of repolarization and refractoriness.

[0044] 6: Combinations of steps 2 through 5.

[0045] The preparation of a bypass bridge from hMSCs thus prepared allows conduction of pacemaker and/or electrical signals/current between any two selected sites in the heart. In the case of an AV bridge, it will not only facilitate propagation of signals from atrium to ventricle, but provides sufficient delay from atrial to ventricular contraction to maximize ventricular filling and emptying, thus mimicking the normal activation and contractile sequence of the heart. Moreover, the cells in the bypass bridge, preferably cells at one end of the tract, may also be further transfected with a nucleic acid encoding a pacemaker current channel, wherein said transfected cells express a pacemaker current. Increases or decreases in calcium current, sodium current, potassium current or pacemaker current (I.sub.f) may be obtained by increasing or reducing in the cells the expression of the ion channels carrying these currents. These approaches, when used with gene therapy and stem cell technology to improve atrial impulse initiation in the setting of sinus node disease offer a completely physiologic system rather than its electronic replacement. Thus, in embodiments of this invention, the bypass bridge expresses one or more genes encoding a pacemaker ion channel and thereby induces a pacemaker current.

[0046] In different embodiments of this invention, the nucleic acid encoding the ion channel or connexin gene is introduced into the cell by infection with a viral vector, plasmid transformation, cosmid transformation, electroporation, lipofection, transfection using a chemical transfection reagent, heat shock transfection, or microinjection. In further embodiments, the viral vector is an adenoviral, an adeno-associated viral (AAV), or a retroviral vector.

[0047] In embodiments of the instant bypass bridge, the sodium channel is a SKM-1 channel. In certain embodiments, the SKM-1 channel comprises an alpha subunit. In other embodiments, the SKM-1 channel further comprises an accessory subunit. In additional embodiments, the tract further functionally expresses a potassium channel. The potassium channel may comprise a Kir2. 1 or Kir2.2 alpha subunit, and may further comprise an accessory subunit. In different embodiments of the bypass bridge, cells in the tract further functionally express an L-type calcium channel, which may comprise an alpha subunit and accessory subunits. In further embodiments, cells in the tract forming the bypass bridge further functionally express one or more of at least one cardiac connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel, so as to change the voltage-time course of repolarization and/or refractoriness of the heart. In various embodiments, the at least one cardiac connexin is Cx43, Cx40, or Cx45.

[0048] In various embodiments of the bypass bridge that expresses a pacemaker ion channel, said pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or a mutant or chimera thereof, and (b) a MiRP1 beta subunit. The HCN channel may be any of HCN1, HCN2, HCN3 or HCN4. In embodiments of this invention, the pacemaker channels are engineered to operate at progressively slower rates, so as to equate to primary and subsidiary pacemakers similar to the sinus node and Purkinje system. In preferred embodiments, the pacemaker ion channel is expressed in cells in the first end of the tract. In more preferred embodiments, the cells expressing the pacemaker ion channel are located in a region extending 0.5 mm from the first end.

[0049] Hyperpolarization-activated cation currents, termed I.sub.f, I.sub.h, or I.sub.q, were initially discovered in heart and nerve cells over 20 years ago (for review, see DiFrancesco, 1993; Pape, 1996). These currents, carried by Na+ and K+ ions, contribute to a wide range of physiological functions, including cardiac and neuronal pacemaker activity, the setting of resting potentials, input conductance and length constants, and dendritic integration (see Robinson and Siegelbaum, 2003; Biel et al., 2002). The hyperpolarization-activated, cyclic nucleotide-gated (HCN) family of ion channel subunits has been identified by molecular cloning (for review, see Clapham, 1998; Santoro and Tibbs, 1999; Biel et al., 2002), and when heterologously expressed, each of the four different HCN isoforms generates channels with the principal properties of native If, confirming that HCN channels are the molecular correlate of this current.

[0050] As used herein, a "HCN channel" shall mean a hyperpolarization-activated, cyclic nucleotide-gated ion channel responsible for the hyperpolarization-activated cation currents that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. There are four HCN isoforms: HCN1, HCN2, HCN3 and HCN4. All four isoforms are expressed in brain; HCN1, HCN2 and HCN4 are also prominently expressed in heart, with HCN4 and HCN1 predominating in sinoatrial node and HCN2 in the ventricular specialized conducting system. "mHCN" designates murine or mouse HCN; "hHCN" designates human HCN.

[0051] HCN channels, similar to voltage-gatedK+ (Kv) channels, have four subunits, each of which has six transmembrane segments, S1-S6: the positively charged S4 domain forms the major voltage sensor, whereas S5 and S6, together with the S5-S6 linker connecting the two, form the pore domain containing the ion permeation pathway and the gates that control the flow of ions (Larsson, 2002). Mutational studies on HCN channels indicate that mutations in the S4 voltage sensor, the S4-S5 linker implicated in the coupling of voltage sensing to pore opening and closing, the S5, S6 and S5-S6 linker which form the pore, the C-linker, and the C-terminal cyclic nucleotide binding domain (CNBD), may be particularly important in affecting HCN channel activity. In embodiments of the bypass bridge expressing a mutant HCN, said mutant HCN channel provides an improved characteristic, as compared to a wild-type HCN channel, selected from the group consisting of faster kinetics, more positive activation, increased expression, increased stability, preserved or enhanced cAMP responsiveness, and preserved or enhanced neurohumoral response. Mutant HCN channels for inducing pacemaker activity in cells is also described in U.S. Provisional Application Nos. 60/781,723 (filed Mar. 14, 2006) and 60/832,515, entitled "Chimeric HCN Channels," which is being filed concurrently with the subject application (Jul. 21, 2006). Mutant HCN channels are also discussed in U.S. application Ser. No. 10/342,506. The preceding applications are herein incorporated by reference in their entirety.

[0052] In certain embodiments of the present invention, the mutant HCN channel carries at least one mutation in S4 voltage sensor, the S4-S5 linker, S5, S6, the S5-S6 linker, and/or the C-linker, and the CNBD which mutations result in one or more of the above discussed improved characteristics. In other embodiments, the HCN mutant is E324A-HCN2, Y331A-HCN2, R339A-HCN2, or Y331A, E324A-HCN2. In preferred embodiments, the mutant HCN channel is E324A-HCN2.

[0053] In addition to the mutations noted above, many mutations in different HCN isoforms have been reported. These include R318Q, W323A, E324A, E324D, E324K, E324Q, F327A, T330A and Y331A, Y331D, Y331F, Y331K, D332A, M338A, R339A, R339C, R339D, R339E and R339Q in HCN2 made by Chen et al. (2001a) to investigate in greater detail the role of the E324, Y331 and 8339 residues in voltage sensing and activation. Chen et al. (2001b) have also reported the R538E and R591E mutations in mHCN1; Tsang et al. (2004) have reported G231A and M232A in mHCN1; Vemana et al (2004) have reported R247C, T249C, K250C, 1251C, L252C, S253C, L254C, L258C, R259C, L260C, S261C, C318S, S338C in mHCN2; Macri and Accili (2004) have reported S306Q, Y331D AND G404S in mHCN2; and Decher et al. (2004) have reported Y331A, Y331D, Y331S, R331FD, R339E, R339Q, 1439A, S441A, S441T, D443A, D443C, D443E, D443K, D443N, D443R, R447A, R447D, R447E, R447Y, Y449A, Y449D, Y449F, Y449G, Y449W, Y453A, Y453D, Y453F, Y453L, Y453W, P466Q, P466V, Y476A, Y477A and Y481A in mHCN2. The entire contents of all of the above publications are incorporated herein by reference. Certain of the reported mutations listed above may confer, singly or in combination, beneficial characteristics on the HCN channel with regard to creating a biological pacemaker. The invention disclosed herein encompasses all mutations in HCN channels, singly or in combinations, which improve pacemaker activity of the channel such as by providing faster kinetics, more positive activation, increased expression and/or stability, preserved cyclic interval responsiveness, and/or preserved or enhanced neurohumoral response.

[0054] Mutations are identified herein by a designation with provides the single letter abbreviation of the amino acid residue that underwent mutation, the position of that residue within a polypeptide, and the single letter abbreviation of the amino acid residue to which the residue was mutated. Thus, for example, E324A identifies a mutant polypeptide in which the glutamate residue (E) at position 324 was mutated to alanine (A). Y331A, E324A-HCN2 indicates a mouse HCN2 having a double mutation, one in which tyrosine (Y) at position 331 was mutated to alanine (A), and the other in which the glutamate residue at position 324 was mutated to alanine.

[0055] In general terms, HCN polypeptides can be divided into three major domains: (1) a cytoplasmic amino terminal domain; (2) the membrane spanning domains and their linking regions; and (3) a cytoplasmic carboxy-terminal domain. The N-terminal domain does not appear to play a major role in channel activation (Biel et al., 2002). However, the membrane spanning domains with their linking regions play an important role in determining the kinetics of gating, whereas the CNBD is largely responsible for the ability of the channel to respond to the sympathetic and parasympathetic nervous systems that respectively raise and lower cellular cAMP levels.

[0056] In embodiments of the bypass bridge expressing a HCN chimera, the chimeric HCN channel preferably provides an improved characteristic, as compared to a wild-type HCN channel, selected from the group consisting of faster kinetics, more positive activation, increased expression, increased stability, preserved or enhanced cAMP responsiveness, and preserved or enhanced neurohumoral response. HCN chimeras for inducing pacemaker activity in cells is described in detail in U.S. Provisional Application No. 60/715,934 (filed Sep. 9, 2005) and 60/832,515 entitled "Chimeric HCN Channels," which is being filed concurrently with the subject application (Jul. 21, 2006), both of which are herein incorporated by reference in their entirety.

[0057] As used herein, a "HCN chimera" or "chimeric HCN channel" shall mean a HCN c channel comprising portions of more than one HCN channel isoform. Thus, a chimera may comprise portions of HCN1 and HCN2 or HCN3 or HCN4, and so forth. In preferred embodiments, the portions are an amino terminal portion, an intramembranous portion, and a carboxy terminal portion. In other preferred embodiments, the portions are derived from human HCN isoforms. In addition, an HCN chimera encompasses an ion channel comprising portions of HCN channels derived from different animal species. Accordingly, in various embodiments of the bypass bridge, at least one portion of the HCN chimera is derived from an animal species which is different from the animal species from which at least one of the other two portions is derived. For example, one portion of the channel may be derived from a human and another portion may be derived from a non-human.

[0058] In additional embodiments of the instant bypass bridge, the HCN chimera is mHCN112, mHCN212, mHCN312, mHCN412, mHCN114, mHCN214, mHCN314, mHCN414, hHCN112, hHCN212, hHCN312, hHCN412, hHCN114, hHCN214, hHCN314, or hHCN414. In different embodiments, the HCN chimera is mHCN112, mHCN212, mHCN312, mHCN412, mHCN114, mHCN214, mHCN314, mHCN414, hHCN112, hHCN212, hHCN312, hHCN412, hHCN114, hHCN214, hHCN314, or hHCN414. In a preferred embodiment, the HCN chimera is hHCN212 having the sequence set forth in SEQ ID NO: 2 (see FIG. 2). In yet another preferred embodiment, the HCN chimera is mHCN212 having the sequence set forth in SEQ ID NO: 6 (see FIG. 3).

[0059] As used herein, the term "HCNXYZ" (wherein X, Y and Z are any one of the integers 1, 2, 3 or 4, with the proviso that at least one of X, Y and Z is a different number from at least one of the other numbers) shall mean a chimeric HCN channel polypeptide comprising three contiguous portions in the order XYZ, wherein X is an N-terminal portion, Y is an intramembrane portion, and Z is a C-terminal portion, and wherein the number X, Y or Z designates the HCN channel from which that portion is derived. For example, HCN112 is an HCN chimera with a N-terminal portion and intramembrane portion from HCN1 and a C-terminal portion from HCN2.

[0060] In certain embodiments of the bypass bridge, the HCN chimera comprises an amino terminal portion contiguous with an intramembrane portion contiguous with a carboxy terminal portion, wherein each portion is a portion of an HCN channel or a portion of a mutant thereof, and wherein one portion derives from an HCN channel or a mutant thereof which is different from the HCN channel or mutant thereof from which at least one of the other two portions derive. Thus, in various embodiments, at least one portion of the chimera is derived from a HCN channel containing a mutation which provides an improved characteristic, as compared to a portion from a wild-type HCN channel, selected from the group consisting of faster kinetics, more positive activation, increased expression, increased stability, preserved or enhanced cAMP responsiveness, and preserved or enhanced neurohumoral response. In certain embodiments, the mutant HCN channel contains a mutation in a region of the channel selected from the group consisting of the S4 voltage sensor, the S4-S5 linker, SS, S6 and S5-S6 linker, the C-linker, and the CNBD. In other embodiments, the mutant portion is derived from mHCN2 having the sequence set forth in SEQ ID NO: 14 (see FIG. 1) and comprises E324A-mHCN2, Y331A-mHCN2, R339A-mHCN2, or Y331A, E324A-mHCN2. In preferred embodiments, the mutant portion comprises E324A-mHCN2.

[0061] The pacemaker activity of a HCN channel may be enhanced by co-expressing the HCN channel with its beta subunit, MiRP 1, which increases the magnitude of the current expressed and/or speeds its kinetics of activation. See U.S. Pat. No. 6,783,979 and Qu et al. (2004), the entire contents of which are incorporated herein by reference.

[0062] In certain embodiments of the bypass bridge disclosed herein, the pacemaker current is conducted by electrotonic conduction. In other embodiments, the pacemaker current is actively propagated by an action potential. In further embodiments, the action potential is a sodium-dependent action potential. In additional embodiments, cells in the tract further functionally express an L-type calcium channel and the action potential is a calcium-dependent action potential.

[0063] The present invention also provides a kit comprising a packaging material containing therein (a) any of the bypass bridges disclosed herein. The kit may also contain labeling and instructions for implanting the bypass bridge in a heart, so as to thereby treat a disorder associated with impaired conduction, or a disorder associated with both impaired conduction and impaired sinus node activity, in a subject's heart in a subject's heart.

[0064] This invention also provides a tandem pacemaker system comprising (1) any of the bypass bridges disclosed herein, and (2) an electronic pacemaker and/or a biological pacemaker, wherein the bypass bridge operates in tandem with the electronic and/or biological pacemaker to more effectively treat a cardiac rhythm condition compared to the use of the biological or electronic pacemaker alone. In various embodiments, the bypass bridge is an AV bridge. The biological pacemaker or electronic pacemaker may have a pacing level at, around, above or below the normal resting sinus node pacemaker level. In embodiments where a biological pacemaker or electronic pacemaker is used in tandem with an AV bridge described herein, the natural AV node may be ablated. Further details of tandem pacemaker systems comprising a bypass bridge may be found in U.S. Provisional Application Nos. 60/701,312 (filed Jul. 21, 2005) and 60/781,723 (filed Mar. 14, 2006) and U.S. Ser. No. 11/490,997, filed concurrently herewith on Jul. 21, 2006, entitled "Tandem Cardiac Pacemaker System." The proceeding applications are herein incorporated by reference in their entirety.

[0065] The invention also provides a method of making a bypass bridge for implantation in a heart comprising (a) transfecting a cell with, and functionally expressing therein, a nucleic acid encoding a sodium channel, and (b) growing the transfected cell into a tract of cells having a first and a second end capable of being attached to two selected sites in the heart, wherein the cells are physically interconnected via electrically conductive gap junctions. An embodiment of this method further comprises transfecting cells in the tract with a nucleic acid encoding a pacemaker ion channel, wherein the nucleic acid is functionally expressed so as to induce a pacemaker current in the cells. In preferred embodiments, the pacemaker ion channel is expressed in cells in the first end of the tract. In more preferred embodiments, the cells expressing the pacemaker ion channel are located in a region extending 0.5 mm from the first end. In these embodiments, the transfection method used to introduce the pacemaker channel gene(s) to the first end may be locally applied, or separated from the distal portions of the tract using physical barriers, in order to restrict transfection to first end portion.

[0066] In various embodiments, the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or a mutant or chimera thereof, and (b) a MiRP 1 beta subunit. In preferred embodiments of the instant methods, the cells are hMSCs or hESCs, wherein said cells are substantially incapable of differentiation. In further embodiments, the human adult mesenchymal stem cells (a) express CD29, CD44, CD54, and HLA class I surface markers; and (b) do not express CD14, CD34, CD45, and HLA class II surface markers. In still further embodiments, the human adult mesenchymal stem cells have been passaged at least 9 times.

[0067] Additional embodiments of the instant methods for making a bypass bridge further comprise transfecting the cell with, and expressing therein, at least one nucleic acid encoding one or more of at least one cardiac connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel, such that implantation of a bypass bridge in a heart changes the voltage-time course of repolarization and/or refractoriness of the heart.

[0068] Variations of the instant methods for making a bypass bridge comprise (a) growing a cell into a tract of cells having a first and a second end capable of being attached to two selected sites in the heart, wherein the cells are physically interconnected via electrically conductive gap junctions, before (b) transfecting the cells in the tract with, and functionally expressing therein, a nucleic acid encoding a sodium channel, and optionally other ion channel genes and/or a pacemaker channel gene.

[0069] This invention further provides a method of implanting a bypass bridge in a heart comprising (a) making a bypass bridge comprising a tract of cells by any of the methods disclosed herein, (b) selecting a first and a second site in the heart, and (c) attaching the first end of the tract to the first site and the second end of the tract to the second site, so as to thereby implant a bypass bridge in the heart that allows the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites. Other embodiments further comprise transfecting cells in the tract with a nucleic acid encoding a pacemaker ion channel, wherein the nucleic acid is functionally expressed so as to induce a pacemaker current in the cells. In preferred embodiments, the pacemaker ion channel is expressed in cells in the first end of the tract. In more preferred embodiments, the cells expressing the pacemaker ion channel are located in a region extending 0.5 mm from the first end. In other preferred embodiments, the first site is in an atrium and the second site is in a ventricle, so as to allow propagation of a pacemaker and/or electrical signal/current across the tract from the atrium to the ventricle. In different embodiments of the instant methods, the a pacemaker and/or electrical signal/current is generated in the atrium by the sinus node, an electronic pacemaker, a biological pacemaker, or cells within the bypass bridge expressing a pacemaker current. In further embodiments, the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or a mutant or chimera thereof, and (b) a MiRP1 beta subunit.

[0070] In additional embodiments of the instant methods, the cells in the tract of the bypass bridge are stem cells, cardiomyocytes, fibroblasts or skeletal muscle cells engineered to express at least one cardiac connexins, or endothelial cells. In various embodiments, the stem cells are adult MSCs or ESCs, wherein said cells are substantially incapable of differentiation. In preferred embodiments, the stem cells are hMSCs or hESCs, wherein said stem cells are substantially incapable of differentiation. Other embodiments further comprise transfecting the cells with, and expressing therein, at least one nucleic acid encoding one or more of at least one cardiac connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel, so as to change the voltage-time course of repolarization and/or refractoriness of the heart. In further embodiments, the at least one connexin is Cx43, Cx40, or Cx45.

[0071] The present invention also provides a method of treating a disorder associated with an impaired conduction in a subject's heart comprising (a) transfecting a cell with a nucleic acid encoding a sodium channel, wherein the cell functionally expresses the sodium channel, (b) growing the transfected cell into a tract of cells having a first end and a second end, wherein the cells are physically interconnected via electrically conductive gap junctions, (c) selecting a first site and a second site in the subject's heart between which sites conduction is impaired, and (d) attaching the first end of the tract to the first site and the second end of the tract to the second site, so as to allow the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites and thereby treat the subject.

[0072] As used herein, "treating" a disorder shall mean causing the subject afflicted with the disorder to experience a reduction, remission or regression of the disorder and/or its symptoms. In various embodiments, recurrence of the disorder and/or its symptoms is prevented. In preferred embodiments, the subject is cured of the disorder and/or its symptoms. To "functionally express" a nucleic acid shall mean that a cell or other biological system into which the nucleic acid has been introduced produces a functional polypeptide encoded by the nucleic acid. The encoded polypeptide itself may also be said to be functionally expressed.

[0073] This invention further provides a method of treating a disorder associated with an impaired conduction and impaired sinus node activity in a subject's heart comprising (a) transfecting a cell with at least one nucleic acid encoding a sodium channel and a pacemaker ion channel, wherein the cell functionally expresses the sodium channel and the pacemaker ion channel, (b) growing the transfected cell into a tract of cells having a first end and a second end, wherein the cells are physically interconnected via electrically conductive gap junctions, (c) selecting a first site in the left atrium of the heart and a second site, between which sites conduction is impaired, and (d) attaching the first end of the tract to the first site and the second end of the tract to the second site, so as to allow the propagation of a pacemaker and/or electrical signal/current generated by the sinus node and/or tract of cells between the two sites and thereby treat the subject.

[0074] In embodiments of the instant methods, the cells are hMSCs or hESCs, wherein said stem cells are substantially incapable of differentiation. Various embodiments further comprise transfecting the cells in the tract with, and expressing therein, at least one nucleic acid encoding one or more of at least one cardiac connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel, so as to change the voltage-time course of repolarization and/or refractoriness of the heart. In further embodiments, the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or a mutant or chimera thereof, and (b) a MiRP1 beta subunit.

[0075] The present invention encompasses a variety of kits comprising the bypass bridge of the instant invention, as well as methods for making a bypass bridge, methods of implanting a bypass bridge in a heart, and methods of treating a disorder, corresponding to each of the different embodiments of the bypass bridge disclosed herein. These variant kits and methods are not all individually described in detail herein but will be readily evident and understood to one of ordinary skill in the art based on the disclosure of the different embodiments of the bypass bridge. Accordingly, the various embodiments described herein may be equally applied as appropriate to the bypass bridges, kits and methods of this invention.

[0076] The following Examples are presented to aid in understanding the invention, and are not intended, and should not be construed, to limit in any way the invention set forth in the claims which follow thereafter. These Examples do not include detailed descriptions of experimental methods that are well known to those of ordinary skill in the art, such as methods used in the construction of recombinant nucleic acid vectors, transfection of host cells with such recombinant vectors, and the functional expression of genes in transfected cells. Detailed descriptions of such conventional methods are provided in numerous publications, including Sambrook et al. (1989), the contents of which are hereby incorporated herein in their entirety.

Example 1

Culture of Gap Junction-Coupled Human Mesenchymal Stem Cells

[0077] Cell Cultures

[0078] Human mesenchymal stem cells (hMSCs; mesenchymal stem cells, human bone marrow; Poietics.TM.) were purchased from Clonetics/BioWhittaker (Walkersville, Md., USA), cultured in mesenchymal stem cell (MCS) growth medium and used from passages 2-4. Isolated and purified hMSCs can be cultured for many passages (12) without losing their unique properties, i.e., normal karyotype and telomerase activity (van den Bos et al., 1997; Pittenger et al., 1999).

[0079] HeLa cells transfected with rat Cx40, rat Cx43 or mouse Cx45 were cocultured with hMSCs. Production, characterization and culture conditions of transfected HeLa cells have been previously described (Elfgang et al., 1995; Valiunas et al., 2000; 2002).

[0080] Anti-Connexin Antibodies, Immunofluorescent Labeling, and Immunoblot Analysis

[0081] Commercially available mouse anticonnexin monoclonal and polyclonal antibodies (Chemicon International, Temecula, Calif.) of Cx40, Cx43 and Cx45 were used for immunostaining and immunoblots as described earlier (Laing and Beyer, 1995). Fluorescein-conjugated goat antimouse or antirabbit IgG (ICN Biomedicals, Inc., Costa Mesa, Calif.) was used as secondary antibody.

[0082] Electrophysiological Measurements Across Gap Junctions

[0083] Glass coverslips with adherent cells were transferred to an experimental chamber perfused at room temperature (.about.22.degree. C.) with bath solution containing (mM): NaCl, 150; KCl, 10; CaCl2, 2; Hepes, 5 (pH 7.4); glucose, 5. The patch pipettes were filled with solution containing (mM): potassium aspartate, 120; NaCl, 10; MgATP, 3; Hepes, 5 (pH 7.2); EGTA, 10 (pCa .about.8); filtered through 0.22 .mu.m pores. When filled, the resistance of the pipettes measured 1-2 M.OMEGA.. Experiments were carried out on cell pairs using a double voltage-clamp. This method permitted control of the membrane potential (Vm) and measurement of the associated junctional currents (Ij).

[0084] Dye Flux Studies

[0085] Dye transfer through gap junction channels was investigated using cell pairs. Lucifer Yellow (LY; Molecular Probes) was dissolved in the pipette solution to reach a concentration of 2 mM. Fluorescent dye cell-to-cell spread was imaged using a 16 bit 64 000 pixel grey scale digital CCD-camera (LYNXX 2000T, SpectraSource Instruments, Westlake Village, Calif.) (Valiunas et al., 2002). In experiments with heterologous pairs, LY was always injected into the cells which were tagged with Cell Tracker Green. The injected cell fluorescence intensity derived from LY is 10-15 times higher than the initial fluorescence from Cell Tracker Green.

[0086] Human MSCs Express Connexins

[0087] The connexins, Cx43 and Cx40, were immunolocalized, as evidenced by typical punctate staining, along regions of intimate cell-to-cell contact and within regions of the cytoplasm of the hMSCs grown in culture as monolayers (FIGS. 4A, B). Cx45 staining was also detected, but unlike that of Cx43 or Cx40, was not typical of connexin distribution in cells. Rather, it was characterized by fine granular cytoplasmic and reticular-like staining with no readily observed membrane-associated plaques (FIG. 4C). This does not exclude the possibility that Cx45 channels exist but does imply that their number relative to Cx43 and Cx40 homotypic, heterotypic and heteromeric channels is low. FIG. 4D illustrates Western blot analysis for canine ventricle myocytes and hMSCs with a Cx43 polyclonal antibody which adds further proof of Cx43 presence in hMSCs.

[0088] Gapjunctional Coupling Between hMSCs and Various Cell Lines

[0089] Gap junctional coupling among hMSCs is demonstrated in FIG. 5. Junctional currents recorded between hMSC pairs show quasi-symmetrical (FIG. 5A) and asymmetrical (FIG. 5B) voltage dependency arising in response to symmetrical 10-s transjunctional voltage steps (V.sub.j) of equal amplitude but opposite sign starting from .+-.10 mV to .+-.110 mV using increments of 20 mV. These behaviors are typically observed in cells which co-express Cx43 and Cx40 (Valiunas et al., 2001).

[0090] FIG. 5C summarizes the data obtained from hMSC pairs. The values of normalized instantaneous (g.sub.j,inst, o) and steady state conductances (g.sub.j,ss, .cndot.) (determined at the beginning and at the end of each V.sub.j step, respectively) were plotted versus V.sub.j. The left panel shows a quasi-symmetrical relationship from five hMSC pairs. The continuous curves represent the best fit of data to the Boltzmann equation with the following parameters: half-deactivation voltage, V.sub.j,o=-70/65 mV; minimum g.sub.j, g.sub.j,min=0.29/0.34; maximum g.sub.j, g.sub.j,max=0.99/1.00; gating charge, z=2.2/2.3 for negative/positive V, respectively. Summarized plots from six asymmetrical cases are shown in the right panel. The g.sub.j,ss declined in sigmoidal fashion at negative V.sub.j and showed a reduced voltage sensitivity to positive V.sub.j. Boltzman fitting for negative V.sub.j revealed the following values: V.sub.j,o=-72 mV, g.sub.j,min=0.25, g.sub.j,max=0.99, z=1.5.

[0091] FIGS. 5D and E illustrate typical multichannel recordings from a hMSC pair. Using 120 mM K aspartate as a pipette solution, channels were observed with unitary conductances of 28-80 pS range. Operation of channels with .about.50 pS conductance (see FIG. 5D) is consistent with previously published values (Valiunas et al., 1997; 2002) for Cx43 homotypic channels. This does not preclude the presence of other channel types, it merely suggests that Cx43 forms functional channels in hMSCs.

[0092] To further define the nature of the coupling, hMSCs were co-cultured with human HeLa cells stably transfected with Cx43, Cx40, and Cx45 (Elfgang et al., 1995) and it was found that hMSCs were able to couple to all these transfectants. FIG. 6A shows an example of junctional currents recorded between an hMSC and HeLaCx43 cell pairs that manifested symmetrically and asymmetrically voltage dependent currents in response to a series (from .+-.10 mV to .+-.110 mV) of symmetrical transjunctional voltage steps (V.sub.j). The quasi-symmetric record suggests that the dominant functional channel is homotypic Cx43 while the asymmetric record suggests the activity of another connexin in the hMSC (presumably Cx40 as shown by immunohistochemistry, see FIG. 4) that could be either a heterotypic or heteromeric form or both. These records are similar to those published for transfected cells: heterotypic and mixed (heteromeric) forms of Cx40 and Cx43 (Valiunas et al., 2000; 2001). Co-culture of hMSCs with HeLa cells transfected with Cx40 (FIG. 6B) also revealed symmetric and asymmetric voltage dependent junctional currents consistent with the co-expression of Cx43 and Cx40 in the hMSCs similar to the data for Cx43 HeLa-hMSC pairs. HeLa cells transfected with Cx45 coupled to hMSCs always produced asymmetric junctional currents with pronounced voltage gating when Cx45 (HeLa) side was negative (FIG. 6C). This is consistent with the dominant channel forms in the hMSC being Cx43 and Cx40 as both produce asymmetric currents when they form heterotypic channels with Cx45 (Valiunas et al., 2000; 2001). This does not exclude Cx45 as a functioning channel in hMSCs but it does indicate that Cx45 is a minor contributor to cell to cell coupling in hMSCs. The lack of visualized plaques in the immunostaining for Cx45 (FIG. 7) further supports this interpretation.

[0093] The summarized plots of g.sub.j,ss versus V.sub.j from pairs between hMSC and transfected HeLa cells are shown in FIG. 6D. The left panel shows the results from hMSC-HeLaCx43 pairs. For symmetrical data (.cndot., four preparations), Boltzmann fits (continuous lines) yielded the following parameters: V.sub.j,0=-61/65 mV, g.sub.j,min=0.24/0.33, g.sub.j,max=0.99/0.99, z=2.4/3.8 for negative/positive Vj. For asymmetrical data (o, three preparations), the Boltzmann fit (dashed line) at negative .sub.Vj values revealed the following parameter values: V.sub.j,o=-70 mV, g.sub.j,min=0.31, g.sub.j,max=1.00, z=2.2. The middle panel shows data from hMSC-HeLaCx40 pairs including three symmetrical (.cndot.) and two asymmetrical (o) g.sub.j,ss-V.sub.j-relationships. The continuous lines correspond to a Boltzmann fit to symmetrical data (V.sub.j,o=-57/76 mV, g.sub.j,min=0.22/0.29, g.sub.j,max=1.1/1.0, z=1.4/2.3; negative/positive Vj) and the dashed line is a fit to the asymmetrical data (Vj,o=-57/85 mV, gj,min=0.22/0.65, g.sub.j,max=1.1/1.0, z=1.3/2.2; negative/positive V.sub.j). The data from the six complete experiments from hMSC-HeLaCx45 cell pairs are shown on the right panel. The g.sub.j,ss plot versus V.sub.j was strongly asymmetrical and the best fit of the data to the Boltzmann equation at positive V.sub.j values revealed following parameter values: V.sub.j,0=31 mV, g.sub.j,min=0.07, g.sub.j,max=1.2, z=1.8.

[0094] FIG. 6E shows Lucifer Yellow transfer from an hMSC to an hMSC (upper panel), from a HeLaCx43 to an hMSC (middle panel), and from an hMSC to a HeLaCx43 (bottom panel). The junctional conductance of the cell pairs was simultaneously measured by methods described earlier (Valiunas et al., 2002) and revealed conductances of .about.13, .about.16 and .about.18 nS, respectively. The transfer of Lucifer Yellow was similar to that previously reported for homotypic Cx43 or co-expressed Cx43 and Cx40 in HeLa cells (Valiunas et al., 2002). Cell Tracker Green (Molecular Probes) was always used in one of the two populations of cells to allow heterologous pairs to be identified (Valiunas et al., 2000). Lucifer Yellow was always delivered to the cell containing cell tracker. The fluorescence intensity generated by the Cell Tracker Green was 10-15 times less than fluorescence intensity produced by the concentration of Lucifer Yellow delivered to the source cell.

[0095] Human MSCs were also co-cultured with adult canine ventricular myocytes as shown in FIG. 7. Immunostaining for Cx43 was detected between the rod-shaped ventricular myocytes and hMSCs as shown in FIG. 7A. The hMSCs couple electrically with cardiac myocytes. Both macroscopic (FIG. 7B) and multichannel (FIG. 7C) records were obtained. Junctional currents in FIG. 7B are asymmetrical while those in FIG. 7C show unitary events of the size range typically resulting from the operation of homotypic Cx43 or heterotypic Cx43-Cx40 or homotypic Cx40 channels (Valiunas et al., 2000; 2001). Heteromeric forms are also possible whose conductances are the same or similar to homotypic or heterotypic forms.

[0096] The studies of cell pairs have demonstrated effective coupling of hMSC to other hMSC (13.8.+-.2.4 nS, n=14), to HeLaCx43 (7.9.+-.2.1 nS, n=7), to HeLaCx40 (4.6.+-.2.6 nS, n=5), to HeLaCx45 (11.+-.2.6 nS, n=5.), and to ventricular myocytes (1.5.+-.1.3 nS, n=4).

[0097] These data suggest that MSCs should readily integrate into electrical syncytia of many tissues, promoting repair or serving as the substrate for a therapeutic delivery system. In particular, the data support the possibility of using hMSCs as a therapeutic substrate for repair of cardiac tissue. Other syncytia such as vascular smooth muscle or endothelial cells should also be able to couple to the hMSCs because of the ubiquity of Cx43 and Cx40 (Wang et al., 2001; Beyer, 1993). Thus, they may also be amenable to hMSCs-based therapeutics. For example, hMSCs can be transfected to express ion channels which then can influence the surrounding synctial tissue. Alternatively, the hMSCs can be transfected to express genes that produce small therapeutic molecules capable of permeating gap junctions and influencing recipient cells. Further, for short term therapy, small molecules can be directly loaded into hMSCs for delivery to recipient cells. The success of such approaches is dependent on gap junction channels as the final conduit for delivery of the therapeutic agent to the recipient cells. The feasibility of the first approach has been demonstrated herein by delivering HCN2-transfected hMSCs to the canine heart where they generate a spontaneous rhythm

Example 2

Ion channels Suitable for Incorporation into a Cardiac Bypass Bridge

[0098] As previously described herein, hMSCs form gap junctions that permit a tract of physically connected cells to conduct electrical signals by electrotonic conduction. Cell-to-cell propagation of electrical signals may be facilitated by functionally expressing in the cells one or more nucleic acids encoding at least one of the cardiac connexins Cx43, Cx40 or Cx45 in order to enhance formation of gap junctions. The expression in the cells of a nucleic acid(s) encoding the alpha subunit, with or without the accessory subunits, of a sodium channel, or the alpha and accessory subunits of an L-type calcium channel, also increases the likelihood of not just electrotonic propagation of a wavefront, but its active propagation by a sodium-dependent or calcium-dependent action potential. In addition, expression of a potassium channel in the cells both increases the likelihood of active propagation by an action potential and provides a means of controlling the initial resting potential and its voltage-time course of repolarization and refractoriness.

[0099] The biophysical properties of a SKM-1 sodium channel and a L-type calcium channel were assayed to investigate their suitability for providing an action potential in acardiac bypass bridge. The inactivation properties of the SKM-1 sodium channel measured in Xenopus oocytes are shown in FIG. 8, and the 1-V relationship for the SKM-1 channel in Xenopus oocytes is shown in FIG. 9. The CaV1.2, alpha2&gamma, Pl.b and red fluorescent reporter were co-expressed in HEK293 cells. FIG. 10 shows the Ca2+ current recorded in the red fluorescent cells.

[0100] The SKM-1 sodium channel and L-type calcium channel are used to "fine-tune" the conductivity of a bypass bridge to the extent that this is needed. One embodiment of the bypass bridge described herein is an AV bridge. Incorporation of sodium channels in an AV bridge can be viewed as counterintuitive in that the sodium channel is usually associated with very rapid conduction, whereas an AV bypass should conduct more slowly to operate most efficiently. However, because the degree of coupling among the cells can be manipulated it is possible to use a channel that routinely propagates rapidly and expect this to conduct more slowly. Incorporation of calcium channels, for inward calcium current, will elevate the plateau of the cardiac action potential and prolong repolarization and refractoriness. In addition, functionally expressing potassium channels would accelerate repolarization and can thus shorten refractoriness. Therefore, by manipulating the sodium, calcium and potassium currents and cell coupling, the characteristics of the cardiac electrical activity can be correspondingly manipulated.

REFERENCES

[0101] U.S. Provisional Application No. 60/704,210, filed Jul. 29, 2005 by Brink P R et al. [0102] U.S. Pat. No. 6,783,979, issued Aug. 31, 2004 to Rosen MR et al. [0103] U.S. Provisional Application No. 60/832,515, entitled "Chimeric HCN Channels," filed Jul. 21, 2006. [0104] U.S. Provisional application Ser. No. 11/490,997, entitled "Tandem Cardiac Pacemaker System," filed Jul. 21, 2006. [0105] Biel M, Schneider A, Wahl C (2002) Cardiac HCN channels: Structure, function, and modulation. Trends Cardiovasc Med 12: 202-216. [0106] Beyer E C (1993) Gap junctions. IntRevCytol 137C: 1-371993. [0107] Clapham D E (1998) Not so funny anymore: pacing channels are cloned. Neuron 21: 5-7. [0108] DiFrancesco D (1993) Pacemaker mechanisms in cardiac tissue. Annu Rev Physiol 55: 455-472. [0109] Elfgang C, Eckert R, Lichtenberg-Frate H, Butterweck A, Traub 0, Klein R A, Hulser D F, Willecke K. (1995) Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells. J Cell Bioi 129: 805-817. [0110] Larsson H P (2002) The Search Is on for the Voltage Sensor-to-gate Coupling. J Gen Physiol 120: 475-481. [0111] Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson S M, LiB, Pickel J, McKay R, Nadal-Ginard B, Bodine D M, Leri A, Anversa P (2001) Bone marrow cells regenerate infarcted myocardium. Nature 410: 701-705. [0112] Pape H C (1996) Queer current and pacemaker: the hyperpolarization-activated cation current in neurons. Annu Rev Physiol 58: 299-327. [0113] Perin E C, Geng Y J, Willerson J T (2003) Adult stem cell therapy in perspective. Circulation 107: 935-938. [0114] Qu J, Kryukova Y, Potapova I A, Doronin S V, Larsen M, Krishnamurthy G, Cohen I S, Robinson R B (2004) MiRP 1 modulates HCN2 channel expression and gating in cardiac myocytes. J Bioi Chem 279: 43497-43502. [0115] Robinson R B, Siegelbaum S A (2003) Hyperpolarization-activated cation currents: from molecules to physiological function. Annu Rev Physiol 65: 453-480. [0116] Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2''d ed., Cold Spring Harbor Laboratory Press, New York. [0117] Santoro B, Tibbs G R (1999) The HCN Gene Family: Molecular Basis of the Hyperpolarization-Activated Pacemaker Channels. Ann NY Acad Sci 868: 741-764. [0118] Strauer B E, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg R V, Kogler G, Wernet P (2002) Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 106; 1913-1918. [0119] Valiunas V, Beyer E C, Brink P R (2002) Cardiac gap junction channels show quantitative differences in selectivity. Circ Res 91: 104-111. [0120] Valiunas V, Bukauskas F F, Weingart R (1997) Conductances and selective permeability of connexin43 gap junction channels examined in neonatal rat heart cells. Circ Res 80: 708-719. [0121] Valiunas V, Gemel J, Brink P R, Beyer E C (2001) Gap junction channels formed by coexpressed connexin40 and connexin43. Am J Physiol Heart Circ Physiol 281: H1675-H1689. [0122] Valiunas V, Weingart R, Brink P R (2000) Formation of heterotypic gap junction channels by connexins 40 and 43. Circ Res 86: E42-E49. [0123] Wang H Z, Day N, Valcic M, Hsieh K, Serels S, Brink P R, Christ G J (2001a) Intercellular communication in cultured human vascular smooth muscle cells. Am J Physiol Cell Physiol 281: C75-C88.

Sequence CWU 1

1

2812670DNAHomo sapiens 1atggacgcgc gcgggggcgg cgggcggccc ggggagagcc cgggcgcgag ccccacgacc 60gggccgccgc cgccgccgcc gcccgcgccc ccccaacagc agccgccgcc gccgccgccg 120cccgcgcccc ccccgggccc cgggcccgcg cccccccagc acccgccccg ggccgaggcg 180ttgcccccgg aggcggcgga tgagggcggc ccgcggggcc ggctccgcag ccgcgacagc 240tcgtgcggcc gccccggcac cccgggcgcg gcgagcacgg ccaagggcag cccgaacggc 300gagtgcgggc gcggcgagcc gcagtgcagc cccgcggggc ccgagggccc ggcgcggggg 360cccaaggtgt cgttctcgtg ccgcggggcg gcctcggggc ccgcgccggg gccggggccg 420gcggaggagg cgggcagcga ggaggcgggc ccggcggggg agccgcgcgg cagccaggcc 480agcttcatgc agcgccagtt cggcgcgctc ctgcagccgg gcgtcaacaa gttctcgctg 540cggatgttcg gcagccagaa ggccgtggag cgcgagcagg agcgcgtcaa gtcggcgggg 600gcctggatca tccacccgta cagcgacttc aggttttact gggatttaat aatgcttata 660atgatggttg gaaatctagt catcatacca gttggaatca cattctttac agagcaaaca 720acaacaccat ggattatttt caatgtggca tcagatacag ttttcctatt ggacctgatc 780atgaatttta ggactgggac tgtcaatgaa gacagttctg aaatcatcct ggaccccaaa 840gtgatcaaga tgaattattt aaaaagctgg tctgtggttg acttcatctc atccatccca 900gtggattata tctttcttat tgtagaaaaa ggaatggatt ctgaagttta caagacagcc 960agggcacttc gcattgtgag gtttacaaaa attctcagtc tcttgcgttt attacgactt 1020tcaaggttaa ttagatacat acatcaatgg gaagagatat tccacatgac atatgatctc 1080gccagtgcag tggtgagaat ttttaatctc atcggcatga tgctgctcct gtgccactgg 1140gatggttgtc ttcagttctt agtaccacta ctgcaggact tcccaccaga ttgctgggtg 1200tctttaaatg aaatggttaa tgattcttgg ggaaagcagt attcatacgc actcttcaaa 1260gctatgagtc acatgctgtg cattgggtat ggagcccaag ccccagtcag catgtctgac 1320ctctggatta ccatgctgag catgatcgtc ggggccacct gctatgccat gtttgtcggc 1380catgccaccg ctttaatcca gtctctggac tcctcgcggc gccagtacca ggagaagtac 1440aagcaggtgg agcagtacat gtccttccac aagctgccag ctgacttccg ccagaagatc 1500cacgactact atgagcaccg ttaccagggc aagatgtttg acgaggacag catcctgggc 1560gagctcaacg ggcccctgcg ggaggagatc gtcaacttca actgccggaa gctggtggcc 1620tccatgccgc tgttcgccaa cgccgacccc aacttcgtca cggccatgct gaccaagctc 1680aagttcgagg tcttccagcc gggtgactac atcatccgcg aaggcaccat cgggaagaag 1740atgtacttca tccagcacgg cgtggtcagc gtgctcacta agggcaacaa ggagatgaag 1800ctgtccgatg gctcctactt cggggagatc tgcctgctca cccggggccg ccgcacggcg 1860agcgtgcggg ctgacaccta ctgccgcctc tattcgctga gcgtggacaa cttcaacgag 1920gtgctggagg agtaccccat gatgcggcgc gccttcgaga cggtggccat cgaccgcctg 1980gaccgcatcg gcaagaagaa ttccatcctc ctgcacaagg tgcagcatga cctcaactcg 2040ggcgtattca acaaccagga gaacgccatc atccaggaga tcgtcaagta cgaccgcgag 2100atggtgcagc aggccgagct gggtcagcgc gtgggcctct tcccgccgcc gccgccgccg 2160ccgcaggtca cctcggccat cgccacgctg cagcaggcgg cggccatgag cttctgcccg 2220caggtggcgc ggccgctcgt ggggccgctg gcgctcggct cgccgcgcct cgtgcgccgc 2280ccgcccccgg ggcccgcacc tgccgccgcc tcacccgggc ccccgccccc cgccagcccc 2340ccgggcgcgc ccgccagccc ccgggcaccg cggacctcgc cctacggcgg cctgcccgcc 2400gccccccttg ctgggcccgc cctgcccgcg cgccgcctga gccgcgcgtc gcgcccactg 2460tccgcctcgc agccctcgct gcctcacggc gcccccggcc ccgcggcctc cacacgcccg 2520gccagcagct ccacaccgcg cttggggccc acgcccgctg cccgggccgc cgcgcccagc 2580ccggaccgca gggactcggc ctcacccggc gccgccggcg gcctggaccc ccaggactcc 2640gcgcgctcgc gcctctcgtc caacttgtga 26702889PRTHomo sapiens 2Met Asp Ala Arg Gly Gly Gly Gly Arg Pro Gly Glu Ser Pro Gly Ala1 5 10 15Ser Pro Thr Thr Gly Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro Gln 20 25 30Gln Gln Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro Pro Gly Pro Gly 35 40 45Pro Ala Pro Pro Gln His Pro Pro Arg Ala Glu Ala Leu Pro Pro Glu 50 55 60Ala Ala Asp Glu Gly Gly Pro Arg Gly Arg Leu Arg Ser Arg Asp Ser65 70 75 80Ser Cys Gly Arg Pro Gly Thr Pro Gly Ala Ala Ser Thr Ala Lys Gly 85 90 95Ser Pro Asn Gly Glu Cys Gly Arg Gly Glu Pro Gln Cys Ser Pro Ala 100 105 110Gly Pro Glu Gly Pro Ala Arg Gly Pro Lys Val Ser Phe Ser Cys Arg 115 120 125Gly Ala Ala Ser Gly Pro Ala Pro Gly Pro Gly Pro Ala Glu Glu Ala 130 135 140Gly Ser Glu Glu Ala Gly Pro Ala Gly Glu Pro Arg Gly Ser Gln Ala145 150 155 160Ser Phe Met Gln Arg Gln Phe Gly Ala Leu Leu Gln Pro Gly Val Asn 165 170 175Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val Glu Arg Glu 180 185 190Gln Glu Arg Val Lys Ser Ala Gly Ala Trp Ile Ile His Pro Tyr Ser 195 200 205Asp Phe Arg Phe Tyr Trp Asp Leu Ile Met Leu Ile Met Met Val Gly 210 215 220Asn Leu Val Ile Ile Pro Val Gly Ile Thr Phe Phe Thr Glu Gln Thr225 230 235 240Thr Thr Pro Trp Ile Ile Phe Asn Val Ala Ser Asp Thr Val Phe Leu 245 250 255Leu Asp Leu Ile Met Asn Phe Arg Thr Gly Thr Val Asn Glu Asp Ser 260 265 270Ser Glu Ile Ile Leu Asp Pro Lys Val Ile Lys Met Asn Tyr Leu Lys 275 280 285Ser Trp Ser Val Val Asp Phe Ile Ser Ser Ile Pro Val Asp Tyr Ile 290 295 300Phe Leu Ile Val Glu Lys Gly Met Asp Ser Glu Val Tyr Lys Thr Ala305 310 315 320Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg 325 330 335Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu 340 345 350Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe 355 360 365Asn Leu Ile Gly Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu 370 375 380Gln Phe Leu Val Pro Leu Leu Gln Asp Phe Pro Pro Asp Cys Trp Val385 390 395 400Ser Leu Asn Glu Met Val Asn Asp Ser Trp Gly Lys Gln Tyr Ser Tyr 405 410 415Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Ala 420 425 430Gln Ala Pro Val Ser Met Ser Asp Leu Trp Ile Thr Met Leu Ser Met 435 440 445Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Val Gly His Ala Thr Ala 450 455 460Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr465 470 475 480Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Phe 485 490 495Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met 500 505 510Phe Asp Glu Asp Ser Ile Leu Gly Glu Leu Asn Gly Pro Leu Arg Glu 515 520 525Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val Ala Ser Met Pro Leu 530 535 540Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala Met Leu Thr Lys Leu545 550 555 560Lys Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Thr 565 570 575Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val Ser Val Leu 580 585 590Thr Lys Gly Asn Lys Glu Met Lys Leu Ser Asp Gly Ser Tyr Phe Gly 595 600 605Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala 610 615 620Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu625 630 635 640Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala 645 650 655Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu His 660 665 670Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Asn Gln Glu Asn 675 680 685Ala Ile Ile Gln Glu Ile Val Lys Tyr Asp Arg Glu Met Val Gln Gln 690 695 700Ala Glu Leu Gly Gln Arg Val Gly Leu Phe Pro Pro Pro Pro Pro Pro705 710 715 720Pro Gln Val Thr Ser Ala Ile Ala Thr Leu Gln Gln Ala Ala Ala Met 725 730 735Ser Phe Cys Pro Gln Val Ala Arg Pro Leu Val Gly Pro Leu Ala Leu 740 745 750Gly Ser Pro Arg Leu Val Arg Arg Pro Pro Pro Gly Pro Ala Pro Ala 755 760 765Ala Ala Ser Pro Gly Pro Pro Pro Pro Ala Ser Pro Pro Gly Ala Pro 770 775 780Ala Ser Pro Arg Ala Pro Arg Thr Ser Pro Tyr Gly Gly Leu Pro Ala785 790 795 800Ala Pro Leu Ala Gly Pro Ala Leu Pro Ala Arg Arg Leu Ser Arg Ala 805 810 815Ser Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro His Gly Ala Pro 820 825 830Gly Pro Ala Ala Ser Thr Arg Pro Ala Ser Ser Ser Thr Pro Arg Leu 835 840 845Gly Pro Thr Pro Ala Ala Arg Ala Ala Ala Pro Ser Pro Asp Arg Arg 850 855 860Asp Ser Ala Ser Pro Gly Ala Ala Gly Gly Leu Asp Pro Gln Asp Ser865 870 875 880Ala Arg Ser Arg Leu Ser Ser Asn Leu 88532325DNAHomo sapiens 3atggaggcag agcagcggcc ggcggcgggg gccagcgaag gggcgacccc tggactggag 60gcggtgcctc ccgttgctcc cccgcctgcg accgcggcct caggtccgat ccccaaatct 120gggcctgagc ctaagaggag gcaccttggg acgctgctcc agcctacggt caacaagttc 180tcccttcggg tgttcggcag ccacaaagca gtggaaatcg agcaggagcg ggtgaagtca 240gcgggggcct ggatcatcca cccctacagc gacttccggt tttactggga cctgatcatg 300ctgctgctga tggtggggaa cctcatcgtc ctgcctgtgg gcatcacctt cttcaaggag 360gagaactccc cgccttggat cgtcttcaac gtattgtctg atactttctt cctactggat 420ctggtgctca acttccgaac gggcatcgtg gtggaggagg gtgctgagat cctgctggca 480ccgcgggcca tccgcacgcg ctacctgcgc acctggttcc tggttgacct catctcttct 540atccctgtgg attacatctt cctagtggtg gagctggagc cacggttgga cgctgaggtc 600tacaaaacgg cacgggccct acgcatcgtt cgcttcacca agatcctaag cctgctgagg 660ctgctccgcc tctcccgcct catccgctac atacaccagt gggaggagat ctttcacatg 720acctatgacc tggccagtgc tgtggttcgc atcttcaacc tcattgggat gatgctgctg 780ctatgtcact gggatggctg tctgcagttc ctggtgccca tgctgcagga cttccctccc 840gactgctggg tctccatcaa ccacatggtg aaccactcgt ggggccgcca gtattcccat 900gccctgttca aggccatgag ccacatgctg tgcattggct atgggcagca ggcacctgta 960ggcatgcccg acgtctggct caccatgctc agcatgatcg taggtgccac atgctacgcc 1020atgttcatcg gccatgccac ggcactcatc cagtccctgg actcttcccg gcgtcagtac 1080caggagaagt acaagcaggt ggagcagtac atgtccttcc acaagctgcc agcagacacg 1140cggcagcgca tccacgagta ctatgagcac cgctaccagg gcaagatgtt cgatgaggaa 1200agcatcctgg gcgagctgag cgagccgctt cgcgaggaga tcattaactt cacctgtcgg 1260ggcctggtgg cccacatgcc gctgtttgcc catgccgacc ccagcttcgt cactgcagtt 1320ctcaccaagc tgcgctttga ggtcttccag ccgggggatc tcgtggtgcg tgagggctcc 1380gtggggagga agatgtactt catccagcat gggctgctca gtgtgctggc ccgcggcgcc 1440cgggacacac gcctcaccga tggatcctac tttggggaga tctgcctgct aactaggggc 1500cggcgcacag ccagtgttcg ggctgacacc tactgccgcc tttactcact cagcgtggac 1560catttcaatg ctgtgcttga ggagttcccc atgatgcgcc gggcctttga gactgtggcc 1620atggatcggc tgctccgcat cggcaagaag aattccatac tgcagcggaa gcgctccgag 1680ccaagtccag gcagcagtgg tggcatcatg gagcagcact tggtgcaaca tgacagagac 1740atggctcggg gtgttcgggg tcgggccccg agcacaggag ctcagcttag tggaaagcca 1800gtactgtggg agccactggt acatgcgccc cttcaggcag ctgctgtgac ctccaatgtg 1860gccattgccc tgactcatca gcggggccct ctgcccctct cccctgactc tccagccacc 1920ctccttgctc gctctgcttg gcgctcagca ggctctccag cttccccgct ggtgcccgtc 1980cgagctggcc catgggcatc cacctcccgc ctgcccgccc cacctgcccg aaccctgcac 2040gccagcctat cccgggcagg gcgctcccag gtctccctgc tgggtccccc tccaggagga 2100ggtggacggc ggctaggacc tcggggccgc ccactctcag cctcccaacc ctctctgcct 2160cagcgggcaa caggcgatgg ctctcctggg cgtaagggat caggaagtga gcggctgcct 2220ccctcagggc tcctggccaa acctccaagg acagcccagc cccccaggcc accagtgcct 2280gagccagcca caccccgggg tctccagctt tctgccaaca tgtaa 23254774PRTHomo sapiens 4Met Glu Ala Glu Gln Arg Pro Ala Ala Gly Ala Ser Glu Gly Ala Thr1 5 10 15Pro Gly Leu Glu Ala Val Pro Pro Val Ala Pro Pro Pro Ala Thr Ala 20 25 30Ala Ser Gly Pro Ile Pro Lys Ser Gly Pro Glu Pro Lys Arg Arg His 35 40 45Leu Gly Thr Leu Leu Gln Pro Thr Val Asn Lys Phe Ser Leu Arg Val 50 55 60Phe Gly Ser His Lys Ala Val Glu Ile Glu Gln Glu Arg Val Lys Ser65 70 75 80Ala Gly Ala Trp Ile Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr Trp 85 90 95Asp Leu Ile Met Leu Leu Leu Met Val Gly Asn Leu Ile Val Leu Pro 100 105 110Val Gly Ile Thr Phe Phe Lys Glu Glu Asn Ser Pro Pro Trp Ile Val 115 120 125Phe Asn Val Leu Ser Asp Thr Phe Phe Leu Leu Asp Leu Val Leu Asn 130 135 140Phe Arg Thr Gly Ile Val Val Glu Glu Gly Ala Glu Ile Leu Leu Ala145 150 155 160Pro Arg Ala Ile Arg Thr Arg Tyr Leu Arg Thr Trp Phe Leu Val Asp 165 170 175Leu Ile Ser Ser Ile Pro Val Asp Tyr Ile Phe Leu Val Val Glu Leu 180 185 190Glu Pro Arg Leu Asp Ala Glu Val Tyr Lys Thr Ala Arg Ala Leu Arg 195 200 205Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg Leu Leu Arg Leu 210 215 220Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu Ile Phe His Met225 230 235 240Thr Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe Asn Leu Ile Gly 245 250 255Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu Gln Phe Leu Val 260 265 270Pro Met Leu Gln Asp Phe Pro Pro Asp Cys Trp Val Ser Ile Asn His 275 280 285Met Val Asn His Ser Trp Gly Arg Gln Tyr Ser His Ala Leu Phe Lys 290 295 300Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Gln Gln Ala Pro Val305 310 315 320Gly Met Pro Asp Val Trp Leu Thr Met Leu Ser Met Ile Val Gly Ala 325 330 335Thr Cys Tyr Ala Met Phe Ile Gly His Ala Thr Ala Leu Ile Gln Ser 340 345 350Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr Lys Gln Val Glu 355 360 365Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Thr Arg Gln Arg Ile 370 375 380His Glu Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met Phe Asp Glu Glu385 390 395 400Ser Ile Leu Gly Glu Leu Ser Glu Pro Leu Arg Glu Glu Ile Ile Asn 405 410 415Phe Thr Cys Arg Gly Leu Val Ala His Met Pro Leu Phe Ala His Ala 420 425 430Asp Pro Ser Phe Val Thr Ala Val Leu Thr Lys Leu Arg Phe Glu Val 435 440 445Phe Gln Pro Gly Asp Leu Val Val Arg Glu Gly Ser Val Gly Arg Lys 450 455 460Met Tyr Phe Ile Gln His Gly Leu Leu Ser Val Leu Ala Arg Gly Ala465 470 475 480Arg Asp Thr Arg Leu Thr Asp Gly Ser Tyr Phe Gly Glu Ile Cys Leu 485 490 495Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala Asp Thr Tyr Cys 500 505 510Arg Leu Tyr Ser Leu Ser Val Asp His Phe Asn Ala Val Leu Glu Glu 515 520 525Phe Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala Met Asp Arg Leu 530 535 540Leu Arg Ile Gly Lys Lys Asn Ser Ile Leu Gln Arg Lys Arg Ser Glu545 550 555 560Pro Ser Pro Gly Ser Ser Gly Gly Ile Met Glu Gln His Leu Val Gln 565 570 575His Asp Arg Asp Met Ala Arg Gly Val Arg Gly Arg Ala Pro Ser Thr 580 585 590Gly Ala Gln Leu Ser Gly Lys Pro Val Leu Trp Glu Pro Leu Val His 595 600 605Ala Pro Leu Gln Ala Ala Ala Val Thr Ser Asn Val Ala Ile Ala Leu 610 615 620Thr His Gln Arg Gly Pro Leu Pro Leu Ser Pro Asp Ser Pro Ala Thr625 630 635 640Leu Leu Ala Arg Ser Ala Trp Arg Ser Ala Gly Ser Pro Ala Ser Pro 645 650 655Leu Val Pro Val Arg Ala Gly Pro Trp Ala Ser Thr Ser Arg Leu Pro 660 665 670Ala Pro Pro Ala Arg Thr Leu His Ala Ser Leu Ser Arg Ala Gly Arg 675 680 685Ser Gln Val Ser Leu Leu Gly Pro Pro Pro Gly Gly Gly Gly Arg Arg 690 695 700Leu Gly Pro Arg Gly Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro705 710 715 720Gln Arg Ala Thr Gly Asp Gly Ser Pro Gly Arg Lys Gly Ser Gly Ser 725 730 735Glu Arg Leu Pro Pro Ser Gly Leu Leu Ala Lys Pro Pro Arg Thr Ala

740 745 750Gln Pro Pro Arg Pro Pro Val Pro Glu Pro Ala Thr Pro Arg Gly Leu 755 760 765Gln Leu Ser Ala Asn Met 77052592DNAMus musculus 5atggatgcgc gcgggggcgg cgggcggccg ggcgatagtc cgggcacgac ccctgcgccg 60gggccgccgc caccgccgcc gccgcccgcg ccccctcagc ctcagccacc acccgcgcca 120cccccgaacc ccacgacccc ctcgcacccg gagtcggcgg acgagcccgg cccgcgcgcc 180cggctctgca gccgcgacag cgcctgcacc cctggcgcgg ccaagggcgg cgcgaatggc 240gagtgcgggc gcggggagcc gcagtgcagc cccgagggcc ccgcgcgcgg ccccaaggtt 300tcgttctcat gccgcggggc ggcctccggg ccctcggcgg ccgaggaggc gggcagcgag 360gaggcgggcc cggcgggtga gccgcgcggc agccaggcta gcttcctgca gcgccaattc 420ggggcgcttc tgcagcccgg cgtcaacaag ttctccctgc ggatgttcgg cagccagaag 480gccgtggagc gcgagcagga acgcgtgaag tcggcggggg cctggatcat ccacccctac 540agcgacttca ggttttattg gggattaatc atgcttataa tgatggttgg aaatttggtc 600atcataccag ttggaatcac gttcttcaca gagcagacga caacaccgtg gattattttc 660aacgtggcat ccgatactgt tttcctgttg gacttaatca tgaattttag gactgggact 720gtcaatgaag acagctcgga aatcatcctg gaccctaaag tgatcaagat gaattattta 780aaaagctggt ttgtggtgga cttcatctca tcgatcccgg tggattatat ctttctcatt 840gtagagaaag ggatggactc agaagtttac aagacagcca gagcacttcg tatcgtgagg 900tttacaaaaa ttctcagtct cttgcggtta ttacgccttt caaggttaat cagatacata 960caccagtggg aagagatatt ccacatgacc tatgacctcg ccagtgctgt ggtgaggatc 1020ttcaacctca ttggcatgat gctgcttctg tgccactggg atggctgtct tcagttcctg 1080gttcccctgc tgcaggactt cccaccagat tgctgggttt ctctgaatga aatggttaat 1140gattcctggg gaaaacaata ttcctacgca ctcttcaaag ctatgagtca catgctgtgc 1200attggttatg gcgcccaagc ccctgtcagc atgtctgacc tctggattac catgctgagc 1260atgattgtgg gcgccacctg ctacgcaatg tttgttggcc atgccacagc tttgatccag 1320tctttggatt cgtcacggcg ccaataccag gagaagtaca agcaagtaga gcaatacatg 1380tccttccaca aactgcccgc tgacttccgc cagaagatcc acgattacta tgaacaccgg 1440taccaaggga agatgtttga tgaggacagc atccttgggg aactcaacgg gccactgcgt 1500gaggagattg tgaacttcaa ctgccggaag ctggtggctt ccatgccgct gtttgccaat 1560gcagacccca acttcgtcac agccatgctg acaaagctca aatttgaggt cttccagcct 1620ggagattaca tcatccgaga ggggaccatc gggaagaaga tgtacttcat ccagcatggg 1680gtggtgagcg tgctcaccaa gggcaacaag gagatgaagc tgtcggatgg ctcctatttc 1740ggggagatct gcttgctcac gaggggccgg cgtacggcca gcgtgcgagc tgacacctac 1800tgtcgcctct actcactgag tgtggacaat ttcaacgaag tactggagga ataccccatg 1860atgcggcgtg cctttgagac tgtggctatt gaccggctag atcgcatagg caagaagaac 1920tccatcttgc tgcacaaggt tcagcatgat ctcagctcag gtgtgttcaa caaccaggag 1980aatgccatca tccaggagat tgtcaaatat gaccgtgaga tggtgcagca ggcagagctt 2040ggacagcgtg tggggctctt cccaccaccg ccaccaccgc aggtcacatc ggccattgcc 2100accctacagc aggctgtggc catgagcttc tgcccgcagg tggcccgccc gctcgtgggg 2160cccctggcgc taggctcccc acgcctagtg cgccgcgcgc ccccagggcc tctgcctcct 2220gcagcctcgc cagggccacc cgcagcaagc cccccggctg caccctcgag ccctcgggca 2280ccgcggacct caccctacgg tgtgcctggc tctccggcaa cgcgtgtggg gcccgcattg 2340cccgcacgtc gcctgagccg cgcctcgcgc ccactgtccg cctcgcagcc ctcgctgccc 2400catggcgtgc ccgcgcccag ccccgcggcc tctgcgcgcc cggccagcag ctccacgccg 2460cgcctgggac ccgcacccac cgcccggacc gccgcgccca gtccggaccg cagggactca 2520gcctcgccgg gcgctgccag tggcctcgac ccactggact ctgcgcgctc gcgcctctct 2580tccaacttgt ga 25926863PRTMus musculus 6Met Asp Ala Arg Gly Gly Gly Gly Arg Pro Gly Asp Ser Pro Gly Thr1 5 10 15Thr Pro Ala Pro Gly Pro Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro 20 25 30Gln Pro Gln Pro Pro Pro Ala Pro Pro Pro Asn Pro Thr Thr Pro Ser 35 40 45His Pro Glu Ser Ala Asp Glu Pro Gly Pro Arg Ala Arg Leu Cys Ser 50 55 60Arg Asp Ser Ala Cys Thr Pro Gly Ala Ala Lys Gly Gly Ala Asn Gly65 70 75 80Glu Cys Gly Arg Gly Glu Pro Gln Cys Ser Pro Glu Gly Pro Ala Arg 85 90 95Gly Pro Lys Val Ser Phe Ser Cys Arg Gly Ala Ala Ser Gly Pro Ser 100 105 110Ala Ala Glu Glu Ala Gly Ser Glu Glu Ala Gly Pro Ala Gly Glu Pro 115 120 125Arg Gly Ser Gln Ala Ser Phe Leu Gln Arg Gln Phe Gly Ala Leu Leu 130 135 140Gln Pro Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys145 150 155 160Ala Val Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Ala Trp Ile 165 170 175Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Gly Leu Ile Met Leu 180 185 190Ile Met Met Val Gly Asn Leu Val Ile Ile Pro Val Gly Ile Thr Phe 195 200 205Phe Thr Glu Gln Thr Thr Thr Pro Trp Ile Ile Phe Asn Val Ala Ser 210 215 220Asp Thr Val Phe Leu Leu Asp Leu Ile Met Asn Phe Arg Thr Gly Thr225 230 235 240Val Asn Glu Asp Ser Ser Glu Ile Ile Leu Asp Pro Lys Val Ile Lys 245 250 255Met Asn Tyr Leu Lys Ser Trp Phe Val Val Asp Phe Ile Ser Ser Ile 260 265 270Pro Val Asp Tyr Ile Phe Leu Ile Val Glu Lys Gly Met Asp Ser Glu 275 280 285Val Tyr Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile 290 295 300Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile305 310 315 320His Gln Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala 325 330 335Val Val Arg Ile Phe Asn Leu Ile Gly Met Met Leu Leu Leu Cys His 340 345 350Trp Asp Gly Cys Leu Gln Phe Leu Val Pro Leu Leu Gln Asp Phe Pro 355 360 365Pro Asp Cys Trp Val Ser Leu Asn Glu Met Val Asn Asp Ser Trp Gly 370 375 380Lys Gln Tyr Ser Tyr Ala Leu Phe Lys Ala Met Ser His Met Leu Cys385 390 395 400Ile Gly Tyr Gly Ala Gln Ala Pro Val Ser Met Ser Asp Leu Trp Ile 405 410 415Thr Met Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Val 420 425 430Gly His Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln 435 440 445Tyr Gln Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys 450 455 460Leu Pro Ala Asp Phe Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg465 470 475 480Tyr Gln Gly Lys Met Phe Asp Glu Asp Ser Ile Leu Gly Glu Leu Asn 485 490 495Gly Pro Leu Arg Glu Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val 500 505 510Ala Ser Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala 515 520 525Met Leu Thr Lys Leu Lys Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile 530 535 540Ile Arg Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly545 550 555 560Val Val Ser Val Leu Thr Lys Gly Asn Lys Glu Met Lys Leu Ser Asp 565 570 575Gly Ser Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr 580 585 590Ala Ser Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val 595 600 605Asp Asn Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala 610 615 620Phe Glu Thr Val Ala Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn625 630 635 640Ser Ile Leu Leu His Lys Val Gln His Asp Leu Ser Ser Gly Val Phe 645 650 655Asn Asn Gln Glu Asn Ala Ile Ile Gln Glu Ile Val Lys Tyr Asp Arg 660 665 670Glu Met Val Gln Gln Ala Glu Leu Gly Gln Arg Val Gly Leu Phe Pro 675 680 685Pro Pro Pro Pro Pro Gln Val Thr Ser Ala Ile Ala Thr Leu Gln Gln 690 695 700Ala Val Ala Met Ser Phe Cys Pro Gln Val Ala Arg Pro Leu Val Gly705 710 715 720Pro Leu Ala Leu Gly Ser Pro Arg Leu Val Arg Arg Ala Pro Pro Gly 725 730 735Pro Leu Pro Pro Ala Ala Ser Pro Gly Pro Pro Ala Ala Ser Pro Pro 740 745 750Ala Ala Pro Ser Ser Pro Arg Ala Pro Arg Thr Ser Pro Tyr Gly Val 755 760 765Pro Gly Ser Pro Ala Thr Arg Val Gly Pro Ala Leu Pro Ala Arg Arg 770 775 780Leu Ser Arg Ala Ser Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro785 790 795 800His Gly Val Pro Ala Pro Ser Pro Ala Ala Ser Ala Arg Pro Ala Ser 805 810 815Ser Ser Thr Pro Arg Leu Gly Pro Ala Pro Thr Ala Arg Thr Ala Ala 820 825 830Pro Ser Pro Asp Arg Arg Asp Ser Ala Ser Pro Gly Ala Ala Ser Gly 835 840 845Leu Asp Pro Leu Asp Ser Ala Arg Ser Arg Leu Ser Ser Asn Leu 850 855 86072340DNAMus musculus 7atggaggagg aggcgcggcc ggcggcgggg gccggcgaag cggcgacccc tgcacgcgag 60acgcctcctg cggctccggc ccaggcccgc gcggcctcag gtggggtgcc ggagtctgcg 120cccgagccga agaggcggca gctcgggacg ctgctgcagc cgacggtcaa caagttctct 180ctccgggtct tcggcagcca caaagcagta gaaatcgagc aggagagggt gaagtccgcc 240ggggcctgga tcatccaccc ctacagcgac ttccggtttt actgggatct catcatgctg 300ctgctgatgg tggggaacct catagttctg cctgtgggta tcactttctt caaggaggag 360aactctccac cctggatcgt cttcaatgtc ctctctgaca ctttcttcct gctggatctg 420gtgctcaact tccgaactgg catcgtggtg gaggaaggtg ccgagatcct gctggcgcca 480agggccatcc gaacgcgtta cctgcgcacc tggttcctgg ttgatctgat ctcctccatc 540cctgtggatt atatcttcct agtggtggag ctggagccac gactagatgc tgaggtctac 600aaaacggcac gggccctgcg catcgttaga ttcaccaaga tccttagcct gctgcggctg 660ctccgcctct cccgcctcat ccgctacata caccagtggg aggagatctt tcacatgacc 720tacgacctgg ccagtgcagt ggttcgcatc ttcaacctca ttggaatgat gttgctgctg 780tgtcactggg acggctgtct gcagtttctg gtccctatgc tgcaggactt cccgtccgac 840tgctgggtct ccatgaaccg catggtgaac cactcgtggg gccgccagta ttcccacgcc 900ctgttcaagg ccatgagtca catgctatgc attggctatg ggcagcaggc accggtaggc 960atgcctgacg tctggctcac catgctcagt atgattgtgg gcgccacgtg ttatgccatg 1020ttcatcggtc acgccaccgc cctcatccag tccctggact cttcccggcg acagtaccag 1080gagaagtaca agcaggtgga gcagtacatg tccttccaca agctgcccgc tgacacccgg 1140cagcgcatcc acgagtacta cgagcatcgc taccagggca agatgtttga tgaagagagc 1200atcctggggg agctgagcga gccacttcgg gaggagatta ttaacttcac ctgccggggc 1260ctggtggccc acatgccgct gtttgctcat gctgacccca gcttcgtcac cgcagtgctc 1320accaagctcc gttttgaggt cttccaacca ggggacctgg tggtgcgtga gggctccgtg 1380ggcaggaaga tgtacttcat ccagcacggg ctgctgagtg tgctggcacg tggcgcccgc 1440gacacccgcc tcactgatgg atcctacttt ggggagatct gcctgctgac tcgaggtcgg 1500agaacagcca gtgtaagggc tgacacctat tgtcgcctct actcgctcag cgtggaccac 1560ttcaatgcgg tgcttgagga gttcccaatg atgcgcaggg cttttgagac ggtggccatg 1620gaccggcttc ggcgcatcgg caaaaagaat tcgatactgc agcggaaacg ctctgagccg 1680agtccaggca gcagcggtgg cgtcatggag cagcatttgg tacaacacga cagagacatg 1740gctcgtggtg ttcggggcct ggctcctggt acaggagctc gactcagtgg aaagccagtg 1800ctgtgggaac cactggtgca cgcccctctg caggcagctg ctgtgacctc caacgtggcc 1860atagccttga ctcaccagcg aggccctctg cccctctccc ctgattctcc agccaccctc 1920ctagctcgat ctgctagacg ctcagcaggc tccccagcct ccccactggt gcctgtccga 1980gcaggtcctc tgctggcccg gggaccctgg gcgtccactt ctcgcctgcc tgctccacct 2040gcccgaaccc tccatgccag cctatcccgg acagggcgtt cccaggtatc tctgttgggc 2100cctcccccag gaggaggtgc tcggaggcta ggacctcggg gccgcccact ttctgcctcg 2160caaccctctc tgcctcagcg agcaacaggg gatggctctc ctaggcgtaa aggctctgga 2220agtgagcgcc tgcccccctc tgggctcttg gccaaacctc cagggacagt ccagccaccc 2280aggtcatcag tgcctgagcc agttaccccc agaggtcccc aaatttctgc caacatgtga 23408779PRTMus musculus 8Met Glu Glu Glu Ala Arg Pro Ala Ala Gly Ala Gly Glu Ala Ala Thr1 5 10 15Pro Ala Arg Glu Thr Pro Pro Ala Ala Pro Ala Gln Ala Arg Ala Ala 20 25 30Ser Gly Gly Val Pro Glu Ser Ala Pro Glu Pro Lys Arg Arg Gln Leu 35 40 45Gly Thr Leu Leu Gln Pro Thr Val Asn Lys Phe Ser Leu Arg Val Phe 50 55 60Gly Ser His Lys Ala Val Glu Ile Glu Gln Glu Arg Val Lys Ser Ala65 70 75 80Gly Ala Trp Ile Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp 85 90 95Leu Ile Met Leu Leu Leu Met Val Gly Asn Leu Ile Val Leu Pro Val 100 105 110Gly Ile Thr Phe Phe Lys Glu Glu Asn Ser Pro Pro Trp Ile Val Phe 115 120 125Asn Val Leu Ser Asp Thr Phe Phe Leu Leu Asp Leu Val Leu Asn Phe 130 135 140Arg Thr Gly Ile Val Val Glu Glu Gly Ala Glu Ile Leu Leu Ala Pro145 150 155 160Arg Ala Ile Arg Thr Arg Tyr Leu Arg Thr Trp Phe Leu Val Asp Leu 165 170 175Ile Ser Ser Ile Pro Val Asp Tyr Ile Phe Leu Val Val Glu Leu Glu 180 185 190Pro Arg Leu Asp Ala Glu Val Tyr Lys Thr Ala Arg Ala Leu Arg Ile 195 200 205Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser 210 215 220Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu Ile Phe His Met Thr225 230 235 240Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe Asn Leu Ile Gly Met 245 250 255Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu Gln Phe Leu Val Pro 260 265 270Met Leu Gln Asp Phe Pro Ser Asp Cys Trp Val Ser Met Asn Arg Met 275 280 285Val Asn His Ser Trp Gly Arg Gln Tyr Ser His Ala Leu Phe Lys Ala 290 295 300Met Ser His Met Leu Cys Ile Gly Tyr Gly Gln Gln Ala Pro Val Gly305 310 315 320Met Pro Asp Val Trp Leu Thr Met Leu Ser Met Ile Val Gly Ala Thr 325 330 335Cys Tyr Ala Met Phe Ile Gly His Ala Thr Ala Leu Ile Gln Ser Leu 340 345 350Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr Lys Gln Val Glu Gln 355 360 365Tyr Met Ser Phe His Lys Leu Pro Ala Asp Thr Arg Gln Arg Ile His 370 375 380Glu Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met Phe Asp Glu Glu Ser385 390 395 400Ile Leu Gly Glu Leu Ser Glu Pro Leu Arg Glu Glu Ile Ile Asn Phe 405 410 415Thr Cys Arg Gly Leu Val Ala His Met Pro Leu Phe Ala His Ala Asp 420 425 430Pro Ser Phe Val Thr Ala Val Leu Thr Lys Leu Arg Phe Glu Val Phe 435 440 445Gln Pro Gly Asp Leu Val Val Arg Glu Gly Ser Val Gly Arg Lys Met 450 455 460Tyr Phe Ile Gln His Gly Leu Leu Ser Val Leu Ala Arg Gly Ala Arg465 470 475 480Asp Thr Arg Leu Thr Asp Gly Ser Tyr Phe Gly Glu Ile Cys Leu Leu 485 490 495Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala Asp Thr Tyr Cys Arg 500 505 510Leu Tyr Ser Leu Ser Val Asp His Phe Asn Ala Val Leu Glu Glu Phe 515 520 525Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala Met Asp Arg Leu Arg 530 535 540Arg Ile Gly Lys Lys Asn Ser Ile Leu Gln Arg Lys Arg Ser Glu Pro545 550 555 560Ser Pro Gly Ser Ser Gly Gly Val Met Glu Gln His Leu Val Gln His 565 570 575Asp Arg Asp Met Ala Arg Gly Val Arg Gly Leu Ala Pro Gly Thr Gly 580 585 590Ala Arg Leu Ser Gly Lys Pro Val Leu Trp Glu Pro Leu Val His Ala 595 600 605Pro Leu Gln Ala Ala Ala Val Thr Ser Asn Val Ala Ile Ala Leu Thr 610 615 620His Gln Arg Gly Pro Leu Pro Leu Ser Pro Asp Ser Pro Ala Thr Leu625 630 635 640Leu Ala Arg Ser Ala Arg Arg Ser Ala Gly Ser Pro Ala Ser Pro Leu 645 650 655Val Pro Val Arg Ala Gly Pro Leu Leu Ala Arg Gly Pro Trp Ala Ser 660 665 670Thr Ser Arg Leu Pro Ala Pro Pro Ala Arg Thr Leu His Ala Ser Leu 675 680 685Ser Arg Thr Gly Arg Ser Gln Val Ser Leu Leu Gly Pro Pro Pro Gly 690 695 700Gly Gly Ala Arg Arg Leu Gly Pro Arg Gly Arg Pro Leu Ser Ala Ser705 710 715 720Gln Pro Ser Leu Pro Gln Arg Ala Thr Gly Asp Gly Ser Pro Arg Arg 725 730 735Lys Gly Ser Gly Ser Glu Arg Leu Pro Pro Ser Gly Leu Leu Ala Lys 740 745 750Pro Pro Gly Thr Val

Gln Pro Pro Arg Ser Ser Val Pro Glu Pro Val 755 760 765Thr Pro Arg Gly Pro Gln Ile Ser Ala Asn Met 770 7759910PRTMus musculus 9Met Glu Gly Gly Gly Lys Pro Asn Ser Ala Ser Asn Ser Arg Asp Asp1 5 10 15Gly Asn Ser Val Phe Pro Ser Lys Ala Pro Ala Thr Gly Pro Val Ala 20 25 30Ala Asp Lys Arg Leu Gly Thr Pro Pro Arg Gly Gly Ala Ala Gly Lys 35 40 45Glu His Gly Asn Ser Val Cys Phe Lys Val Asp Gly Gly Gly Gly Glu 50 55 60Glu Pro Ala Gly Ser Phe Glu Asp Ala Glu Gly Pro Arg Arg Gln Tyr65 70 75 80Gly Phe Met Gln Arg Gln Phe Thr Ser Met Leu Gln Pro Gly Val Asn 85 90 95Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val Glu Lys Glu 100 105 110Gln Glu Arg Val Lys Thr Ala Gly Phe Trp Ile Ile His Pro Tyr Ser 115 120 125Asp Phe Arg Phe Tyr Trp Asp Leu Ile Met Leu Ile Met Met Val Gly 130 135 140Asn Leu Val Ile Ile Pro Val Gly Ile Thr Phe Phe Thr Glu Gln Thr145 150 155 160Thr Thr Pro Trp Ile Ile Phe Asn Val Ala Ser Asp Thr Val Phe Leu 165 170 175Leu Asp Leu Ile Met Asn Phe Arg Thr Gly Thr Val Asn Glu Asp Ser 180 185 190Ser Glu Ile Ile Leu Asp Pro Lys Val Ile Lys Met Asn Tyr Leu Lys 195 200 205Ser Trp Phe Val Val Asp Phe Ile Ser Ser Ile Pro Val Asp Tyr Ile 210 215 220Phe Leu Ile Val Glu Lys Gly Met Asp Ser Glu Val Tyr Lys Thr Ala225 230 235 240Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg 245 250 255Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu 260 265 270Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe 275 280 285Asn Leu Ile Gly Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu 290 295 300Gln Phe Leu Val Pro Leu Leu Gln Asp Phe Pro Pro Asp Cys Trp Val305 310 315 320Ser Leu Asn Glu Met Val Asn Asp Ser Trp Gly Lys Gln Tyr Ser Tyr 325 330 335Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Ala 340 345 350Gln Ala Pro Val Ser Met Ser Asp Leu Trp Ile Thr Met Leu Ser Met 355 360 365Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Val Gly His Ala Thr Ala 370 375 380Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr385 390 395 400Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Met 405 410 415Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Ile 420 425 430Phe Asp Glu Glu Asn Ile Leu Ser Glu Leu Asn Asp Pro Leu Arg Glu 435 440 445Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val Ala Thr Met Pro Leu 450 455 460Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala Met Leu Ser Lys Leu465 470 475 480Arg Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Ala 485 490 495Val Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Ala Gly Val Ile 500 505 510Thr Lys Ser Ser Lys Glu Met Lys Leu Thr Asp Gly Ser Tyr Phe Gly 515 520 525Glu Ile Cys Leu Leu Thr Lys Gly Arg Arg Thr Ala Ser Val Arg Ala 530 535 540Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu545 550 555 560Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala 565 570 575Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu Gln 580 585 590Lys Phe Gln Lys Asp Leu Asn Thr Gly Val Phe Asn Asn Gln Glu Asn 595 600 605Glu Ile Leu Lys Gln Ile Val Lys His Asp Arg Glu Met Val Gln Ala 610 615 620Ile Pro Pro Ile Asn Tyr Pro Gln Met Thr Ala Leu Asn Cys Thr Ser625 630 635 640Ser Thr Thr Thr Pro Thr Ser Arg Met Arg Thr Gln Ser Pro Pro Val 645 650 655Tyr Thr Ala Thr Ser Leu Ser His Ser Asn Leu His Ser Pro Ser Pro 660 665 670Ser Thr Gln Thr Pro Gln Pro Ser Ala Ile Leu Ser Pro Cys Ser Tyr 675 680 685Thr Thr Ala Val Cys Ser Pro Pro Ile Gln Ser Pro Leu Ala Thr Arg 690 695 700Thr Phe His Tyr Ala Ser Pro Thr Ala Ser Gln Leu Ser Leu Met Gln705 710 715 720Gln Pro Gln Gln Gln Leu Pro Gln Ser Gln Val Gln Gln Thr Gln Thr 725 730 735Gln Thr Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 740 745 750Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 755 760 765Gln Gln Gln Gln Gln Gln Gln Pro Gln Thr Pro Gly Ser Ser Thr Pro 770 775 780Lys Asn Glu Val His Lys Ser Thr Gln Ala Leu His Asn Thr Asn Leu785 790 795 800Thr Lys Glu Val Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro His 805 810 815Glu Val Ser Thr Leu Ile Ser Arg Pro His Pro Thr Val Gly Glu Ser 820 825 830Leu Ala Ser Ile Pro Gln Pro Val Ala Ala Val His Ser Thr Gly Leu 835 840 845Gln Ala Gly Ser Arg Ser Thr Val Pro Gln Arg Val Thr Leu Phe Arg 850 855 860Gln Met Ser Ser Gly Ala Ile Pro Pro Asn Arg Gly Val Pro Pro Ala865 870 875 880Pro Pro Pro Pro Ala Ala Val Gln Arg Glu Ser Pro Ser Val Leu Asn 885 890 895Thr Asp Pro Asp Ala Glu Lys Pro Arg Phe Ala Ser Asn Leu 900 905 91010910PRTRattus norvegicus 10Met Glu Gly Gly Gly Lys Pro Asn Ser Ala Ser Asn Ser Arg Asp Asp1 5 10 15Gly Asn Ser Val Tyr Pro Ser Lys Ala Pro Ala Thr Gly Pro Ala Ala 20 25 30Ala Asp Lys Arg Leu Gly Thr Pro Pro Gly Gly Gly Ala Ala Gly Lys 35 40 45Glu His Gly Asn Ser Val Cys Phe Lys Val Asp Gly Gly Gly Gly Glu 50 55 60Glu Pro Ala Gly Ser Phe Glu Asp Ala Glu Gly Pro Arg Arg Gln Tyr65 70 75 80Gly Phe Met Gln Arg Gln Phe Thr Ser Met Leu Gln Pro Gly Val Asn 85 90 95Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val Glu Lys Glu 100 105 110Gln Glu Arg Val Lys Thr Ala Gly Phe Trp Ile Ile His Pro Tyr Ser 115 120 125Asp Phe Arg Phe Tyr Trp Asp Leu Ile Met Leu Ile Met Met Val Gly 130 135 140Asn Leu Val Ile Ile Pro Val Gly Ile Thr Phe Phe Thr Glu Gln Thr145 150 155 160Thr Thr Pro Trp Ile Ile Phe Asn Val Ala Ser Asp Thr Val Phe Leu 165 170 175Leu Asp Leu Ile Met Asn Phe Arg Thr Gly Thr Val Asn Glu Asp Ser 180 185 190Ser Glu Ile Ile Leu Asp Pro Lys Val Ile Lys Met Asn Tyr Leu Lys 195 200 205Ser Trp Phe Val Val Asp Phe Ile Ser Ser Ile Pro Val Asp Tyr Ile 210 215 220Phe Leu Ile Val Glu Lys Gly Met Asp Ser Glu Val Tyr Lys Thr Ala225 230 235 240Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg 245 250 255Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu 260 265 270Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe 275 280 285Asn Leu Ile Gly Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu 290 295 300Gln Phe Leu Val Pro Leu Leu Gln Asp Phe Pro Pro Asp Cys Trp Val305 310 315 320Ser Leu Asn Glu Met Val Asn Asp Ser Trp Gly Lys Gln Tyr Ser Tyr 325 330 335Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Ala 340 345 350Gln Ala Pro Val Ser Met Ser Asp Leu Trp Ile Thr Met Leu Ser Met 355 360 365Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Val Gly His Ala Thr Ala 370 375 380Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr385 390 395 400Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Met 405 410 415Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Ile 420 425 430Phe Asp Glu Glu Asn Ile Leu Ser Glu Leu Asn Asp Pro Leu Arg Glu 435 440 445Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val Ala Thr Met Pro Leu 450 455 460Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala Met Leu Ser Lys Leu465 470 475 480Arg Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Ala 485 490 495Val Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Ala Gly Val Ile 500 505 510Thr Lys Ser Ser Lys Glu Met Lys Leu Thr Asp Gly Ser Tyr Phe Gly 515 520 525Glu Ile Cys Leu Leu Thr Lys Gly Arg Arg Thr Ala Ser Val Arg Ala 530 535 540Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu545 550 555 560Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala 565 570 575Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu Gln 580 585 590Lys Phe Gln Lys Asp Leu Asn Thr Gly Val Phe Asn Asn Gln Glu Asn 595 600 605Glu Ile Leu Lys Gln Ile Val Lys His Asp Arg Glu Met Val Gln Ala 610 615 620Ile Pro Pro Ile Asn Tyr Pro Gln Met Thr Ala Leu Asn Cys Thr Ser625 630 635 640Ser Thr Thr Thr Pro Thr Ser Arg Met Arg Thr Gln Ser Pro Pro Val 645 650 655Tyr Thr Ala Thr Ser Leu Ser His Ser Asn Leu His Ser Pro Ser Pro 660 665 670Ser Thr Gln Thr Pro Gln Pro Ser Ala Ile Leu Ser Pro Cys Ser Tyr 675 680 685Thr Thr Ala Val Cys Ser Pro Pro Ile Gln Ser Pro Leu Ala Thr Arg 690 695 700Thr Phe His Tyr Ala Ser Pro Thr Ala Ser Gln Leu Ser Leu Met Gln705 710 715 720Gln Pro Gln Pro Gln Leu Gln Gln Ser Gln Val Gln Gln Thr Gln Thr 725 730 735Gln Thr Gln Gln Gln Gln Gln Gln Gln Gln Pro Gln Pro Gln Pro Gln 740 745 750Gln Pro Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln 755 760 765Gln Gln Gln Gln Gln Gln Gln Pro Gln Thr Pro Gly Ser Ser Thr Pro 770 775 780Lys Asn Glu Val His Lys Ser Thr Gln Ala Leu His Asn Thr His Leu785 790 795 800Thr Arg Glu Val Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro His 805 810 815Glu Val Ser Thr Met Ile Ser Arg Pro His Pro Thr Val Gly Glu Ser 820 825 830Leu Ala Ser Ile Pro Gln Pro Val Ala Thr Val His Ser Thr Gly Leu 835 840 845Gln Ala Gly Ser Arg Ser Thr Val Pro Gln Arg Val Thr Leu Phe Arg 850 855 860Gln Met Ser Ser Gly Ala Ile Pro Pro Asn Arg Gly Val Pro Pro Ala865 870 875 880Pro Pro Pro Pro Ala Ala Val Gln Arg Glu Ser Pro Ser Val Leu Asn 885 890 895Lys Asp Pro Asp Ala Glu Lys Pro Arg Phe Ala Ser Asn Leu 900 905 91011890PRTHomo sapiens 11Met Glu Gly Gly Gly Lys Pro Asn Ser Ser Ser Asn Ser Arg Asp Asp1 5 10 15Gly Asn Ser Val Phe Pro Ala Lys Ala Ser Ala Thr Gly Ala Gly Pro 20 25 30Ala Ala Ala Glu Lys Arg Leu Gly Thr Pro Pro Gly Gly Gly Gly Ala 35 40 45Gly Ala Lys Glu His Gly Asn Ser Val Cys Phe Lys Val Asp Gly Gly 50 55 60Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Glu Glu Pro Ala Gly Gly65 70 75 80Phe Glu Asp Ala Glu Gly Pro Arg Arg Gln Tyr Gly Phe Met Gln Arg 85 90 95Gln Phe Thr Ser Met Leu Gln Pro Gly Val Asn Lys Phe Ser Leu Arg 100 105 110Met Phe Gly Ser Gln Lys Ala Val Glu Lys Glu Gln Glu Arg Val Lys 115 120 125Thr Ala Gly Phe Trp Ile Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr 130 135 140Trp Asp Leu Ile Met Leu Ile Met Met Val Gly Asn Leu Val Ile Ile145 150 155 160Pro Val Gly Ile Thr Phe Phe Thr Glu Gln Thr Thr Thr Pro Trp Ile 165 170 175Ile Phe Asn Val Ala Ser Asp Thr Val Phe Leu Leu Asp Leu Ile Met 180 185 190Asn Phe Arg Thr Gly Thr Val Asn Glu Asp Ser Ser Glu Ile Ile Leu 195 200 205Asp Pro Lys Val Ile Lys Met Asn Tyr Leu Lys Ser Trp Ser Val Val 210 215 220Asp Phe Ile Ser Ser Ile Pro Val Asp Tyr Ile Phe Leu Ile Val Glu225 230 235 240Lys Gly Met Asp Ser Glu Val Tyr Lys Thr Ala Arg Ala Leu Arg Ile 245 250 255Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser 260 265 270Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu Ile Phe His Met Thr 275 280 285Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe Asn Leu Ile Gly Met 290 295 300Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu Gln Phe Leu Val Pro305 310 315 320Leu Leu Gln Asp Phe Pro Pro Asp Cys Trp Val Ser Leu Asn Glu Met 325 330 335Val Asn Asp Ser Trp Gly Lys Gln Tyr Ser Tyr Ala Leu Phe Lys Ala 340 345 350Met Ser His Met Leu Cys Ile Gly Tyr Gly Ala Gln Ala Pro Val Ser 355 360 365Met Ser Asp Leu Trp Ile Thr Met Leu Ser Met Ile Val Gly Ala Thr 370 375 380Cys Tyr Ala Met Phe Val Gly His Ala Thr Ala Leu Ile Gln Ser Leu385 390 395 400Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr Lys Gln Val Glu Gln 405 410 415Tyr Met Ser Phe His Lys Leu Pro Ala Asp Met Arg Gln Lys Ile His 420 425 430Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Ile Phe Asp Glu Glu Asn 435 440 445Ile Leu Asn Glu Leu Asn Asp Pro Leu Arg Gly Glu Ile Val Asn Phe 450 455 460Asn Cys Arg Lys Leu Val Ala Thr Met Pro Leu Phe Ala Asn Ala Asp465 470 475 480Pro Asn Phe Val Thr Ala Met Leu Ser Lys Leu Arg Phe Glu Val Phe 485 490 495Gln Pro Gly Asp Tyr Ile Val Arg Glu Gly Ala Val Gly Lys Lys Met 500 505 510Tyr Phe Ile Gln His Gly Val Ala Gly Val Ile Thr Lys Ser Ser Lys 515 520 525Glu Met Lys Leu Thr Asp Gly Ser Tyr Phe Gly Glu Ile Cys Leu Leu 530 535 540Thr Lys Gly Arg Arg Thr Ala Ser Val Arg Ala Asp Thr Tyr Cys Arg545 550 555 560Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu Val Pro Glu Glu Tyr 565 570 575Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala Ile Asp Arg Leu Asp 580 585 590Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu Gln Lys Phe Gln Lys Asp 595 600 605Leu Asn Thr Gly Val Phe Asn Asn Gln Glu Asn Glu Ile Leu Lys Gln 610

615 620Ile Val Lys His Asp Arg Glu Met Val Gln Ala Ile Ala Pro Ile Asn625 630 635 640Tyr Pro Gln Met Thr Thr Leu Asn Ser Ala Ser Ser Thr Thr Thr Pro 645 650 655Thr Ser Arg Met Arg Thr Gln Ser Pro Pro Val Tyr Thr Ala Thr Ser 660 665 670Leu Ser His Ser Asn Leu His Ser Pro Ser Pro Ser Thr Gln Thr Pro 675 680 685Gln Pro Ser Ala Ile Leu Ser Pro Cys Ser Tyr Thr Thr Ala Val Cys 690 695 700Ser Pro Pro Val Gln Ser Pro Leu Ala Ala Arg Thr Phe His Tyr Ala705 710 715 720Ser Pro Thr Ala Ser Gln Leu Ser Leu Met Gln Gln Gln Pro Gln Gln 725 730 735Gln Val Gln Gln Ser Gln Pro Pro Gln Thr Gln Pro Gln Gln Pro Ser 740 745 750Pro Gln Pro Gln Thr Pro Gly Ser Ser Thr Pro Lys Asn Glu Val His 755 760 765Lys Ser Thr Gln Ala Leu His Asn Thr Asn Leu Thr Arg Glu Val Arg 770 775 780Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro His Glu Val Pro Thr Leu785 790 795 800Ile Ser Arg Pro His Pro Thr Val Gly Glu Ser Leu Ala Ser Ile Pro 805 810 815Gln Pro Val Thr Ala Val Pro Gly Thr Gly Leu Gln Ala Gly Gly Arg 820 825 830Ser Thr Val Pro Gln Arg Val Thr Leu Phe Arg Gln Met Ser Ser Gly 835 840 845Ala Ile Pro Pro Asn Arg Gly Val Pro Pro Ala Pro Pro Pro Pro Ala 850 855 860Ala Ala Leu Pro Arg Glu Ser Ser Ser Val Leu Asn Thr Asp Pro Asp865 870 875 880Ala Glu Lys Pro Arg Phe Ala Ser Asn Leu 885 89012822PRTOryctolagus cuniculus 12Met Ala Thr Ala Ser Ser Pro Pro Arg Arg Pro Arg Arg Ala Arg Gly1 5 10 15Leu Glu Asp Ala Glu Gly Pro Arg Arg Gln Tyr Gly Phe Met Gln Arg 20 25 30Gln Phe Thr Ser Met Leu Gln Pro Gly Val Asn Lys Phe Ser Leu Arg 35 40 45Met Phe Gly Ser Gln Lys Ala Val Glu Lys Glu Gln Glu Arg Val Lys 50 55 60Thr Ala Gly Phe Trp Ile Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr65 70 75 80Trp Asp Leu Ile Met Leu Ile Met Met Val Gly Asn Leu Val Ile Ile 85 90 95Pro Val Gly Ile Thr Phe Phe Thr Glu Gln Thr Thr Thr Pro Trp Ile 100 105 110Ile Phe Asn Val Ala Ser Asp Thr Val Phe Leu Leu Asp Leu Ile Met 115 120 125Asn Phe Arg Thr Gly Thr Val Asn Glu Asp Ser Ser Glu Ile Ile Leu 130 135 140Asp Pro Lys Val Ile Lys Met Asn Tyr Leu Lys Ser Trp Phe Val Val145 150 155 160Asp Phe Ile Ser Ser Ile Pro Val Asp Tyr Ile Phe Leu Ile Val Glu 165 170 175Lys Gly Met Asp Ser Glu Val Tyr Lys Thr Ala Arg Ala Leu Arg Ile 180 185 190Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser 195 200 205Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu Ile Phe His Met Thr 210 215 220Tyr Asp Leu Ala Ser Ala Val Val Arg Ile Phe Asn Leu Ile Gly Met225 230 235 240Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu Gln Phe Leu Val Pro 245 250 255Leu Leu Gln Asp Phe Pro Pro Asp Cys Trp Val Ser Leu Asn Glu Met 260 265 270Val Asn Asp Ser Trp Gly Lys Gln Tyr Ser Tyr Ala Leu Phe Lys Ala 275 280 285Met Ser His Met Leu Cys Ile Gly Tyr Gly Ala Gln Ala Pro Val Ser 290 295 300Met Ser Asp Leu Trp Ile Thr Met Leu Ser Met Ile Val Gly Ala Thr305 310 315 320Cys Tyr Ala Met Phe Val Gly His Ala Thr Ala Leu Ile Gln Ser Leu 325 330 335Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr Lys Gln Val Glu Gln 340 345 350Tyr Met Ser Phe His Lys Leu Pro Ala Asp Met Arg Gln Lys Ile His 355 360 365Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Ile Phe Asp Glu Glu Asn 370 375 380Ile Leu Asn Glu Leu Asn Asp Pro Leu Arg Glu Glu Ile Val Asn Phe385 390 395 400Asn Cys Arg Lys Leu Val Ala Thr Met Pro Leu Phe Ala Asn Ala Asp 405 410 415Pro Asn Phe Val Thr Ala Met Leu Ser Lys Leu Arg Phe Glu Val Phe 420 425 430Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Ala Val Gly Lys Lys Met 435 440 445Tyr Phe Ile Gln His Gly Val Ala Gly Val Ile Thr Lys Ser Ser Lys 450 455 460Glu Met Lys Leu Thr Asp Gly Ser Tyr Phe Gly Glu Ile Cys Leu Leu465 470 475 480Thr Lys Gly Arg Arg Thr Ala Ser Val Arg Ala Asp Thr Tyr Cys Arg 485 490 495Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu Val Leu Glu Glu Tyr 500 505 510Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala Ile Asp Arg Leu Asp 515 520 525Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu Gln Lys Phe Gln Lys Asp 530 535 540Leu Asn Thr Gly Val Phe Asn Asn Gln Glu Asn Glu Ile Leu Lys Gln545 550 555 560Ile Val Lys His Asp Arg Glu Met Val Gln Ala Ile Ala Pro Ile Ser 565 570 575Tyr Pro Gln Met Thr Ala Leu Asn Ser Thr Ser Ser Thr Ala Thr Pro 580 585 590Thr Ser Arg Met Arg Thr Gln Ser Pro Pro Val Tyr Thr Ala Thr Ser 595 600 605Leu Ser His Ser Asn Leu His Ser Pro Ser Pro Ser Thr Gln Thr Pro 610 615 620Gln Pro Ser Ala Ile Leu Ser Pro Cys Ser Tyr Thr Thr Ala Val Cys625 630 635 640Ser Pro Pro Val Gln Ser Pro Leu Ala Thr Arg Thr Phe His Tyr Ala 645 650 655Ser Pro Thr Ala Ser Gln Leu Ser Leu Met Pro Gln Gln Gln Gln Gln 660 665 670Pro Gln Ala Pro Gln Thr Gln Pro Gln Gln Pro Pro Gln Gln Pro Gln 675 680 685Thr Pro Gly Ser Ala Thr Pro Lys Asn Glu Val His Arg Ser Thr Gln 690 695 700Ala Leu Pro Asn Thr Ser Leu Thr Arg Glu Val Arg Pro Leu Ser Ala705 710 715 720Ser Gln Pro Ser Leu Pro His Glu Val Ser Thr Leu Ile Ser Arg Pro 725 730 735His Pro Thr Val Gly Glu Ser Leu Ala Ser Ile Pro Gln Pro Val Ala 740 745 750Ala Val His Ser Ala Gly Leu Gln Ala Ala Gly Arg Ser Thr Val Pro 755 760 765Gln Arg Val Thr Leu Phe Arg Gln Met Ser Ser Gly Ala Ile Pro Pro 770 775 780Asn Arg Gly Val Pro Pro Ala Pro Pro Pro Pro Ala Ala Pro Leu Gln785 790 795 800Arg Glu Ala Ser Ser Val Leu Asn Thr Asp Pro Glu Ala Glu Lys Pro 805 810 815Arg Phe Ala Ser Asn Leu 82013202PRTCavia porcellus 13Ile Met Met Val Gly Asn Leu Val Ile Ile Pro Val Gly Ile Thr Phe1 5 10 15Phe Thr Glu Gln Thr Thr Thr Pro Trp Ile Ile Phe Asn Val Ala Ser 20 25 30Asp Thr Val Phe Leu Leu Asp Leu Ile Met Asn Phe Arg Thr Gly Thr 35 40 45Val Asn Glu Asp Ser Ser Glu Ile Ile Leu Asp Pro Lys Val Ile Lys 50 55 60Met Asn Tyr Leu Lys Ser Trp Phe Val Val Asp Phe Ile Ser Ser Ile65 70 75 80Pro Val Asp Tyr Ile Phe Leu Ile Val Glu Lys Gly Met Asp Ser Glu 85 90 95Val Tyr Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile 100 105 110Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile 115 120 125His Gln Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala 130 135 140Val Val Arg Ile Phe Asn Leu Ile Gly Met Met Leu Leu Leu Cys His145 150 155 160Trp Asp Gly Cys Leu Gln Phe Leu Val Pro Leu Leu Gln Asp Phe Pro 165 170 175Pro Asp Cys Trp Val Ser Leu Asn Lys Met Val Asn Val Ser Trp Gly 180 185 190Gln Gln Tyr Ser Tyr Ala Leu Phe Lys Ala 195 20014863PRTMus musculus 14Met Asp Ala Arg Gly Gly Gly Gly Arg Pro Gly Asp Ser Pro Gly Thr1 5 10 15Thr Pro Ala Pro Gly Pro Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro 20 25 30Gln Pro Gln Pro Pro Pro Ala Pro Pro Pro Asn Pro Thr Thr Pro Ser 35 40 45His Pro Glu Ser Ala Asp Glu Pro Gly Pro Arg Ala Arg Leu Cys Ser 50 55 60Arg Asp Ser Ala Cys Thr Pro Gly Ala Ala Lys Gly Gly Ala Asn Gly65 70 75 80Glu Cys Gly Arg Gly Glu Pro Gln Cys Ser Pro Glu Gly Pro Ala Arg 85 90 95Gly Pro Lys Val Ser Phe Ser Cys Arg Gly Ala Ala Ser Gly Pro Ser 100 105 110Ala Ala Glu Glu Ala Gly Ser Glu Glu Ala Gly Pro Ala Gly Glu Pro 115 120 125Arg Gly Ser Gln Ala Ser Phe Leu Gln Arg Gln Phe Gly Ala Leu Leu 130 135 140Gln Pro Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys145 150 155 160Ala Val Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Ala Trp Ile 165 170 175Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp Phe Thr Met Leu 180 185 190Leu Phe Met Val Gly Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe 195 200 205Phe Lys Asp Glu Thr Thr Ala Pro Trp Ile Val Phe Asn Val Val Ser 210 215 220Asp Thr Phe Phe Leu Met Asp Leu Val Leu Asn Phe Arg Thr Gly Ile225 230 235 240Val Ile Glu Asp Asn Thr Glu Ile Ile Leu Asp Pro Glu Lys Ile Lys 245 250 255Lys Lys Tyr Leu Arg Thr Trp Phe Val Val Asp Phe Val Ser Ser Ile 260 265 270Pro Val Asp Tyr Ile Phe Leu Ile Val Glu Lys Gly Ile Asp Ser Glu 275 280 285Val Tyr Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile 290 295 300Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile305 310 315 320His Gln Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala 325 330 335Val Met Arg Ile Cys Asn Leu Ile Ser Met Met Leu Leu Leu Cys His 340 345 350Trp Asp Gly Cys Leu Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro 355 360 365Ser Asp Cys Trp Val Ser Ile Asn Asn Met Val Asn His Ser Trp Ser 370 375 380Glu Leu Tyr Ser Phe Ala Leu Phe Lys Ala Met Ser His Met Leu Cys385 390 395 400Ile Gly Tyr Gly Arg Gln Ala Pro Glu Ser Met Thr Asp Ile Trp Leu 405 410 415Thr Met Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile 420 425 430Gly His Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln 435 440 445Tyr Gln Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys 450 455 460Leu Pro Ala Asp Phe Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg465 470 475 480Tyr Gln Gly Lys Met Phe Asp Glu Asp Ser Ile Leu Gly Glu Leu Asn 485 490 495Gly Pro Leu Arg Glu Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val 500 505 510Ala Ser Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala 515 520 525Met Leu Thr Lys Leu Lys Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile 530 535 540Ile Arg Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly545 550 555 560Val Val Ser Val Leu Thr Lys Gly Asn Lys Glu Met Lys Leu Ser Asp 565 570 575Gly Ser Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr 580 585 590Ala Ser Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val 595 600 605Asp Asn Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala 610 615 620Phe Glu Thr Val Ala Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn625 630 635 640Ser Ile Leu Leu His Lys Val Gln His Asp Leu Ser Ser Gly Val Phe 645 650 655Asn Asn Gln Glu Asn Ala Ile Ile Gln Glu Ile Val Lys Tyr Asp Arg 660 665 670Glu Met Val Gln Gln Ala Glu Leu Gly Gln Arg Val Gly Leu Phe Pro 675 680 685Pro Pro Pro Pro Pro Gln Val Thr Ser Ala Ile Ala Thr Leu Gln Gln 690 695 700Ala Val Ala Met Ser Phe Cys Pro Gln Val Ala Arg Pro Leu Val Gly705 710 715 720Pro Leu Ala Leu Gly Ser Pro Arg Leu Val Arg Arg Ala Pro Pro Gly 725 730 735Pro Leu Pro Pro Ala Ala Ser Pro Gly Pro Pro Ala Ala Ser Pro Pro 740 745 750Ala Ala Pro Ser Ser Pro Arg Ala Pro Arg Thr Ser Pro Tyr Gly Val 755 760 765Pro Gly Ser Pro Ala Thr Arg Val Gly Pro Ala Leu Pro Ala Arg Arg 770 775 780Leu Ser Arg Ala Ser Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro785 790 795 800His Gly Val Pro Ala Pro Ser Pro Ala Ala Ser Ala Arg Pro Ala Ser 805 810 815Ser Ser Thr Pro Arg Leu Gly Pro Ala Pro Thr Ala Arg Thr Ala Ala 820 825 830Pro Ser Pro Asp Arg Arg Asp Ser Ala Ser Pro Gly Ala Ala Ser Gly 835 840 845Leu Asp Pro Leu Asp Ser Ala Arg Ser Arg Leu Ser Ser Asn Leu 850 855 86015863PRTRattus norvegicus 15Met Asp Ala Arg Gly Gly Gly Gly Arg Pro Gly Asp Ser Pro Gly Ala1 5 10 15Thr Pro Ala Pro Gly Pro Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro 20 25 30Gln Pro Gln Pro Pro Pro Ala Pro Pro Pro Asn Pro Thr Thr Pro Ser 35 40 45His Pro Glu Ser Ala Asp Glu Pro Gly Pro Arg Ser Arg Leu Cys Ser 50 55 60Arg Asp Ser Ser Cys Thr Pro Gly Ala Ala Lys Gly Gly Ala Asn Gly65 70 75 80Glu Cys Gly Arg Gly Glu Pro Gln Cys Ser Pro Glu Gly Pro Ala Arg 85 90 95Gly Pro Lys Val Ser Phe Ser Cys Arg Gly Ala Ala Ser Gly Pro Ala 100 105 110Ala Ala Glu Glu Ala Gly Ser Glu Glu Ala Gly Pro Ala Gly Glu Pro 115 120 125Arg Gly Ser Gln Ala Ser Phe Leu Gln Arg Gln Phe Gly Ala Leu Leu 130 135 140Gln Pro Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys145 150 155 160Ala Val Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Ala Trp Ile 165 170 175Ile His Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp Phe Thr Met Leu 180 185 190Leu Phe Met Val Gly Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe 195 200 205Phe Lys Asp Glu Thr Thr Ala Pro Trp Ile Val Phe Asn Val Val Ser 210 215 220Asp Thr Phe Phe Leu Met Asp Leu Val Leu Asn Phe Arg Thr Gly Ile225 230 235 240Val Ile Glu Asp Asn Thr Glu Ile Ile Leu Asp Pro Glu Lys Ile Lys 245 250 255Lys Lys Tyr Leu Arg Thr Trp Phe Val Val Asp Phe Val Ser Ser Ile 260 265 270Pro Val Asp Tyr Ile Phe Leu Ile Val Glu Lys Gly Ile Asp Ser Glu 275 280 285Val Tyr Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile 290

295 300Leu Ser Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile305 310 315 320His Gln Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala 325 330 335Val Met Arg Ile Cys Asn Leu Ile Ser Met Met Leu Leu Leu Cys His 340 345 350Trp Asp Gly Cys Leu Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro 355 360 365Ser Asp Cys Trp Val Ser Ile Asn Asn Met Val Asn His Ser Trp Ser 370 375 380Glu Leu Tyr Ser Phe Ala Leu Phe Lys Ala Met Ser His Met Leu Cys385 390 395 400Ile Gly Tyr Gly Arg Gln Ala Pro Glu Ser Met Thr Asp Ile Trp Leu 405 410 415Thr Met Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile 420 425 430Gly His Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln 435 440 445Tyr Gln Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys 450 455 460Leu Pro Ala Asp Phe Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg465 470 475 480Tyr Gln Gly Lys Met Phe Asp Glu Asp Ser Ile Leu Gly Glu Leu Asn 485 490 495Gly Pro Leu Arg Glu Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val 500 505 510Ala Ser Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala 515 520 525Met Leu Thr Lys Leu Lys Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile 530 535 540Ile Arg Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly545 550 555 560Val Val Ser Val Leu Thr Lys Gly Asn Lys Glu Met Lys Leu Ser Asp 565 570 575Gly Ser Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr 580 585 590Ala Ser Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val 595 600 605Asp Asn Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala 610 615 620Phe Glu Thr Val Ala Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn625 630 635 640Ser Ile Leu Leu His Lys Val Gln His Asp Leu Ser Ser Gly Val Phe 645 650 655Asn Asn Gln Glu Asn Ala Ile Ile Gln Glu Ile Val Lys Tyr Asp Arg 660 665 670Glu Met Val Gln Gln Ala Glu Leu Gly Gln Arg Val Gly Leu Phe Pro 675 680 685Pro Pro Pro Pro Pro Gln Val Thr Ser Ala Ile Ala Thr Leu Gln Gln 690 695 700Ala Val Ala Met Ser Phe Cys Pro Gln Val Ala Arg Pro Leu Val Gly705 710 715 720Pro Leu Ala Leu Gly Ser Pro Arg Leu Val Arg Arg Ala Pro Pro Gly 725 730 735Pro Leu Pro Pro Ala Ala Ser Pro Gly Pro Pro Ala Ala Ser Pro Pro 740 745 750Ala Ala Pro Ser Ser Pro Arg Ala Pro Arg Thr Ser Pro Tyr Gly Val 755 760 765Pro Gly Ser Pro Ala Thr Arg Val Gly Pro Ala Leu Pro Ala Arg Arg 770 775 780Leu Ser Arg Ala Ser Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro785 790 795 800His Gly Ala Pro Ala Pro Ser Pro Ala Ala Ser Ala Arg Pro Ala Ser 805 810 815Ser Ser Thr Pro Arg Leu Gly Pro Ala Pro Thr Thr Arg Thr Ala Ala 820 825 830Pro Ser Pro Asp Arg Arg Asp Ser Ala Ser Pro Gly Ala Ala Ser Gly 835 840 845Leu Asp Pro Leu Asp Ser Ala Arg Ser Arg Leu Ser Ser Asn Leu 850 855 86016889PRTHomo sapiens 16Met Asp Ala Arg Gly Gly Gly Gly Arg Pro Gly Glu Ser Pro Gly Ala1 5 10 15Thr Pro Ala Pro Gly Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro Gln 20 25 30Gln Gln Pro Pro Pro Pro Pro Pro Pro Ala Pro Pro Pro Gly Pro Gly 35 40 45Pro Ala Pro Pro Gln His Pro Pro Arg Ala Glu Ala Leu Pro Pro Glu 50 55 60Ala Ala Asp Glu Gly Gly Pro Arg Gly Arg Leu Arg Ser Arg Asp Ser65 70 75 80Ser Cys Gly Arg Pro Gly Thr Pro Gly Ala Ala Ser Thr Ala Lys Gly 85 90 95Ser Pro Asn Gly Glu Cys Gly Arg Gly Glu Pro Gln Cys Ser Pro Ala 100 105 110Gly Pro Glu Gly Pro Ala Arg Gly Pro Lys Val Ser Phe Ser Cys Arg 115 120 125Gly Ala Ala Ser Gly Pro Ala Pro Gly Pro Gly Pro Ala Glu Glu Ala 130 135 140Gly Ser Glu Glu Ala Gly Pro Ala Gly Glu Pro Arg Gly Ser Gln Ala145 150 155 160Ser Phe Met Gln Arg Gln Phe Gly Ala Leu Leu Gln Pro Gly Val Asn 165 170 175Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val Glu Arg Glu 180 185 190Gln Glu Arg Val Lys Ser Ala Gly Ala Trp Ile Ile His Pro Tyr Ser 195 200 205Asp Phe Arg Phe Tyr Trp Asp Phe Thr Met Leu Leu Phe Met Val Gly 210 215 220Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys Asp Glu Thr225 230 235 240Thr Ala Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr Phe Phe Leu 245 250 255Met Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Ile Glu Asp Asn 260 265 270Thr Glu Ile Ile Leu Asp Pro Glu Lys Ile Lys Lys Lys Tyr Leu Arg 275 280 285Thr Trp Phe Val Val Asp Phe Val Ser Ser Ile Pro Val Asp Tyr Ile 290 295 300Phe Leu Ile Val Glu Lys Gly Ile Asp Ser Glu Val Tyr Lys Thr Ala305 310 315 320Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser Leu Leu Arg 325 330 335Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln Trp Glu Glu 340 345 350Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Met Arg Ile Cys 355 360 365Asn Leu Ile Ser Met Met Leu Leu Leu Cys His Trp Asp Gly Cys Leu 370 375 380Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro Arg Asn Cys Trp Val385 390 395 400Ser Ile Asn Gly Met Val Asn His Ser Trp Ser Glu Leu Tyr Ser Phe 405 410 415Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Arg 420 425 430Gln Ala Pro Glu Ser Met Thr Asp Ile Trp Leu Thr Met Leu Ser Met 435 440 445Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His Ala Thr Ala 450 455 460Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr465 470 475 480Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Phe 485 490 495Arg Gln Lys Ile His Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met 500 505 510Phe Asp Glu Asp Ser Ile Leu Gly Glu Leu Asn Gly Pro Leu Arg Glu 515 520 525Glu Ile Val Asn Phe Asn Cys Arg Lys Leu Val Ala Ser Met Pro Leu 530 535 540Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ala Met Leu Thr Lys Leu545 550 555 560Lys Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Thr 565 570 575Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val Ser Val Leu 580 585 590Thr Lys Gly Asn Lys Glu Met Lys Leu Ser Asp Gly Ser Tyr Phe Gly 595 600 605Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala 610 615 620Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn Phe Asn Glu625 630 635 640Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu Thr Val Ala 645 650 655Ile Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile Leu Leu His 660 665 670Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Asn Gln Glu Asn 675 680 685Ala Ile Ile Gln Glu Ile Val Lys Tyr Asp Arg Glu Met Val Gln Gln 690 695 700Ala Glu Leu Gly Gln Arg Val Gly Leu Phe Pro Pro Pro Pro Pro Pro705 710 715 720Pro Gln Val Thr Ser Ala Ile Ala Thr Leu Gln Gln Ala Ala Ala Met 725 730 735Ser Phe Cys Pro Gln Val Ala Arg Pro Leu Val Gly Pro Leu Ala Leu 740 745 750Gly Ser Pro Arg Leu Val Arg Arg Pro Pro Pro Gly Pro Ala Pro Ala 755 760 765Ala Ala Ser Pro Gly Pro Pro Pro Pro Ala Ser Pro Pro Gly Ala Pro 770 775 780Ala Ser Pro Arg Ala Pro Arg Thr Ser Pro Tyr Gly Gly Leu Pro Ala785 790 795 800Ala Pro Leu Ala Gly Pro Ala Leu Pro Ala Arg Arg Leu Ser Arg Ala 805 810 815Ser Arg Pro Leu Ser Ala Ser Gln Pro Ser Leu Pro His Gly Ala Pro 820 825 830Gly Pro Ala Ala Ser Thr Arg Pro Ala Ser Ser Ser Thr Pro Arg Leu 835 840 845Arg Pro Thr Pro Ala Ala Arg Ala Ala Ala Pro Ser Pro Asp Arg Arg 850 855 860Asp Ser Ala Ser Pro Gly Ala Ala Gly Gly Leu Asp Pro Gln Asp Ser865 870 875 880Ala Arg Ser Arg Leu Ser Ser Asn Leu 8851797PRTCanis familiaris 17Ala Met Ser His Met Leu Cys Ile Gly Tyr Gly Arg Gln Ala Pro Glu1 5 10 15Ser Met Thr Asp Ile Trp Leu Thr Met Leu Ser Met Ile Val Gly Ala 20 25 30Thr Cys Tyr Ala Met Phe Ile Gly His Ala Thr Ala Leu Ile Gln Ser 35 40 45Leu Asp Ser Ser Arg Arg Gln Tyr Gln Glu Lys Tyr Lys Gln Val Glu 50 55 60Gln Tyr Met Ser Phe His Lys Leu Pro Ala Asp Phe Arg Gln Lys Ile65 70 75 80His Asp Tyr Tyr Glu His Arg Tyr Gln Gly Lys Met Phe Asp Glu Glu 85 90 95Ser181186PRTMus musculus 18Met Asp Lys Leu Pro Pro Ser Met Arg Lys Arg Leu Tyr Ser Leu Pro1 5 10 15Gln Gln Val Gly Ala Lys Ala Trp Ile Met Asp Glu Glu Glu Asp Gly 20 25 30Glu Glu Glu Gly Ala Gly Gly Arg Gln Asp Pro Ser Arg Arg Ser Ile 35 40 45Arg Leu Arg Pro Leu Pro Ser Pro Ser Pro Ser Val Ala Ala Gly Cys 50 55 60Ser Glu Ser Arg Gly Ala Ala Leu Gly Ala Thr Glu Ser Glu Gly Pro65 70 75 80Gly Arg Ser Ala Gly Lys Ser Ser Thr Asn Gly Asp Cys Arg Arg Phe 85 90 95Arg Gly Ser Leu Ala Ser Leu Gly Ser Arg Gly Gly Gly Ser Gly Gly 100 105 110Ala Gly Gly Gly Ser Ser Leu Gly His Leu His Asp Ser Ala Glu Glu 115 120 125Arg Arg Leu Ile Ala Ala Glu Gly Asp Ala Ser Pro Gly Glu Asp Arg 130 135 140Thr Pro Pro Gly Leu Ala Thr Glu Pro Glu Arg Pro Ala Thr Ala Ala145 150 155 160Gln Pro Ala Ala Ser Pro Pro Pro Gln Gln Pro Pro Gln Pro Ala Ser 165 170 175Ala Ser Cys Glu Gln Pro Ser Ala Asp Thr Ala Ile Lys Val Glu Gly 180 185 190Gly Ala Ala Ala Ile Asp His Ile Leu Pro Glu Ala Glu Val Arg Leu 195 200 205Gly Gln Ser Gly Phe Met Gln Arg Gln Phe Gly Ala Met Leu Gln Pro 210 215 220Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val225 230 235 240Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Phe Trp Ile Ile His 245 250 255Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp Leu Thr Met Leu Leu Leu 260 265 270Met Val Gly Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys 275 280 285Asp Glu Asn Thr Thr Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr 290 295 300Phe Phe Leu Ile Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Val305 310 315 320Glu Asp Asn Thr Glu Ile Ile Leu Asp Pro Gln Arg Ile Lys Met Lys 325 330 335Tyr Leu Lys Ser Trp Phe Val Val Asp Phe Ile Ser Ser Ile Pro Val 340 345 350Glu Tyr Ile Phe Leu Ile Val Glu Thr Arg Ile Asp Ser Glu Val Tyr 355 360 365Lys Thr Ala Arg Ala Val Arg Ile Val Arg Phe Thr Lys Ile Leu Ser 370 375 380Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln385 390 395 400Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Val 405 410 415Arg Ile Val Asn Leu Ile Gly Met Met Leu Leu Leu Cys His Trp Asp 420 425 430Gly Cys Leu Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro His Asp 435 440 445Cys Trp Val Ser Ile Asn Gly Met Val Asn Asn Ser Trp Gly Lys Gln 450 455 460Tyr Ser Tyr Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly465 470 475 480Tyr Gly Arg Gln Ala Pro Val Gly Met Ser Asp Val Trp Leu Thr Met 485 490 495Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His 500 505 510Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln 515 520 525Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro 530 535 540Pro Asp Thr Arg Gln Arg Ile His Asp Tyr Tyr Glu His Arg Tyr Gln545 550 555 560Gly Lys Met Phe Asp Glu Glu Ser Ile Leu Gly Glu Leu Ser Glu Pro 565 570 575Leu Arg Glu Glu Ile Ile Asn Phe Asn Cys Arg Lys Leu Val Ala Ser 580 585 590Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ser Met Leu 595 600 605Thr Lys Leu Arg Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg 610 615 620Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val625 630 635 640Ser Val Leu Thr Lys Gly Asn Lys Glu Thr Arg Leu Ala Asp Gly Ser 645 650 655Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser 660 665 670Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn 675 680 685Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Lys Lys Asn Ser 690 695 700Ile Leu Leu His Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn705 710 715 720Tyr Gln Glu Asn Glu Ile Ile Gln Gln Ile Val Arg His Asp Arg Glu 725 730 735Met Ala His Cys Ala His Arg Val Gln Ala Ala Ala Ser Ala Thr Pro 740 745 750Thr Pro Thr Pro Val Ile Trp Thr Pro Leu Ile Gln Ala Pro Leu Gln 755 760 765Ala Ala Ala Ala Thr Thr Ser Val Ala Ile Ala Leu Thr His His Pro 770 775 780Arg Leu Pro Ala Ala Ile Phe Arg Pro Pro Pro Gly Pro Gly Leu Gly785 790 795 800Asn Leu Gly Ala Gly Gln Thr Pro Arg His Pro Arg Arg Leu Gln Ser 805 810 815Leu Ile Pro Ser Ala Leu Gly Ser Ala Ser Pro Ala Ser Ser Pro Ser 820 825 830Gln Val Asp Thr Pro Ser Ser Ser Ser Phe His Ile Gln Gln Leu Ala 835 840 845Gly Phe Ser Ala Pro Pro Gly Leu Ser Pro Leu Leu Pro Ser Ser Ser 850 855 860Ser Ser Pro Pro Pro Gly Ala Cys Gly Ser Pro Pro Ala Pro Thr Pro865 870 875 880Ser Thr Ser Thr Ala Ala Ala Ala Ser Thr Thr Gly Phe Gly His Phe 885 890 895His Lys Ala Leu Gly Gly Ser Leu Ser Ser Ser Asp Ser Pro Leu Leu 900 905 910Thr Pro Leu Gln Pro Gly Ala Arg Ser Pro Gln Ala

Ala Gln Pro Pro 915 920 925Pro Pro Leu Pro Gly Ala Arg Gly Gly Leu Gly Leu Leu Glu His Phe 930 935 940Leu Pro Pro Pro Pro Ser Ser Arg Ser Pro Ser Ser Ser Pro Gly Gln945 950 955 960Leu Gly Gln Pro Pro Gly Glu Leu Ser Leu Gly Leu Ala Ala Gly Pro 965 970 975Ser Ser Thr Pro Glu Thr Pro Pro Arg Pro Glu Arg Pro Ser Phe Met 980 985 990Ala Gly Ala Ser Gly Gly Ala Ser Pro Val Ala Phe Thr Pro Arg Gly 995 1000 1005Gly Leu Ser Pro Pro Gly His Ser Pro Gly Pro Pro Arg Thr Phe Pro 1010 1015 1020Ser Ala Pro Pro Arg Ala Ser Gly Ser His Gly Ser Leu Leu Leu Pro1025 1030 1035 1040Pro Ala Ser Ser Pro Pro Pro Pro Gln Val Pro Gln Arg Arg Gly Thr 1045 1050 1055Pro Pro Leu Thr Pro Gly Arg Leu Thr Gln Asp Leu Lys Leu Ile Ser 1060 1065 1070Ala Ser Gln Pro Ala Leu Pro Gln Asp Gly Ala Gln Thr Leu Arg Arg 1075 1080 1085Ala Ser Pro His Ser Ser Gly Glu Ser Val Ala Ala Phe Ser Leu Tyr 1090 1095 1100Pro Arg Ala Gly Gly Gly Ser Gly Ser Ser Gly Gly Leu Gly Pro Pro1105 1110 1115 1120Gly Arg Pro Tyr Gly Ala Ile Pro Gly Gln His Val Thr Leu Pro Arg 1125 1130 1135Lys Thr Ser Ser Gly Ser Leu Pro Pro Pro Leu Ser Leu Phe Gly Ala 1140 1145 1150Arg Ala Ala Ser Ser Gly Gly Pro Pro Leu Thr Thr Ala Ala Pro Gln 1155 1160 1165Arg Glu Pro Gly Ala Arg Ser Glu Pro Val Arg Ser Lys Leu Pro Ser 1170 1175 1180Asn Leu1185191198PRTRattus norvegicus 19Met Asp Lys Leu Pro Pro Ser Met Arg Lys Arg Leu Tyr Ser Leu Pro1 5 10 15Gln Gln Val Gly Ala Lys Ala Trp Ile Met Asp Glu Glu Glu Asp Gly 20 25 30Glu Glu Glu Gly Ala Gly Gly Leu Gln Asp Pro Ser Arg Arg Ser Ile 35 40 45Arg Leu Arg Pro Leu Pro Ser Pro Ser Pro Ser Val Ala Ala Gly Cys 50 55 60Ser Glu Ser Arg Gly Ala Ala Leu Gly Ala Ala Asp Ser Glu Gly Pro65 70 75 80Gly Arg Ser Ala Gly Lys Ser Ser Thr Asn Gly Asp Cys Arg Arg Phe 85 90 95Arg Gly Ser Leu Ala Ser Leu Gly Ser Arg Gly Gly Gly Ser Gly Gly 100 105 110Ala Gly Gly Gly Ser Ser Leu Gly His Leu His Asp Ser Ala Glu Glu 115 120 125Arg Arg Leu Ile Ala Ala Glu Gly Asp Ala Ser Pro Gly Glu Asp Arg 130 135 140Thr Pro Pro Gly Leu Ala Thr Glu Pro Glu Arg Pro Gly Ala Ala Ala145 150 155 160Gln Pro Ala Ala Ser Pro Pro Pro Gln Gln Pro Pro Gln Pro Ala Ser 165 170 175Ala Ser Cys Glu Gln Pro Ser Ala Asp Thr Ala Ile Lys Val Glu Gly 180 185 190Gly Ala Ala Ala Ser Asp Gln Ile Leu Pro Glu Ala Glu Val Arg Leu 195 200 205Gly Gln Ser Gly Phe Met Gln Arg Gln Phe Gly Ala Met Leu Gln Pro 210 215 220Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val225 230 235 240Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Phe Trp Ile Ile His 245 250 255Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp Leu Thr Met Leu Leu Leu 260 265 270Met Val Gly Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys 275 280 285Asp Glu Asn Thr Thr Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr 290 295 300Phe Phe Leu Ile Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Val305 310 315 320Glu Asp Asn Thr Glu Ile Ile Leu Asp Pro Gln Arg Ile Lys Met Lys 325 330 335Tyr Leu Lys Ser Trp Phe Val Val Asp Phe Ile Ser Ser Ile Pro Val 340 345 350Asp Tyr Ile Phe Leu Ile Val Glu Thr Arg Ile Asp Ser Glu Val Tyr 355 360 365Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser 370 375 380Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln385 390 395 400Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Val 405 410 415Arg Ile Val Asn Leu Ile Gly Met Met Leu Leu Leu Cys His Trp Asp 420 425 430Gly Cys Leu Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro His Asp 435 440 445Cys Trp Val Ser Ile Asn Gly Met Val Asn Asn Ser Trp Gly Lys Gln 450 455 460Tyr Ser Tyr Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly465 470 475 480Tyr Gly Arg Gln Ala Pro Val Gly Met Ser Asp Val Trp Leu Thr Met 485 490 495Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His 500 505 510Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln 515 520 525Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro 530 535 540Pro Asp Thr Arg Gln Arg Ile His Asp Tyr Tyr Glu His Arg Tyr Gln545 550 555 560Gly Lys Met Phe Asp Glu Glu Ser Ile Leu Gly Glu Leu Ser Glu Pro 565 570 575Leu Arg Glu Glu Ile Ile Asn Phe Asn Cys Arg Lys Leu Val Ala Ser 580 585 590Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ser Met Leu 595 600 605Thr Lys Leu Arg Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg 610 615 620Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val625 630 635 640Ser Val Leu Thr Lys Gly Asn Lys Glu Thr Lys Leu Ala Asp Gly Ser 645 650 655Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser 660 665 670Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn 675 680 685Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu 690 695 700Thr Val Ala Leu Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile705 710 715 720Leu Leu His Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Tyr 725 730 735Gln Glu Asn Glu Ile Ile Gln Gln Ile Val Arg His Asp Arg Glu Met 740 745 750Ala His Cys Ala His Arg Val Gln Ala Ala Ala Ser Ala Thr Pro Thr 755 760 765Pro Thr Pro Val Ile Trp Thr Pro Leu Ile Gln Ala Pro Leu Gln Ala 770 775 780Ala Ala Ala Thr Thr Ser Val Ala Ile Ala Leu Thr His His Pro Arg785 790 795 800Leu Pro Ala Ala Ile Phe Arg Pro Pro Pro Gly Pro Gly Leu Gly Asn 805 810 815Leu Gly Ala Gly Gln Thr Pro Arg His Pro Arg Arg Leu Gln Ser Leu 820 825 830Ile Pro Ser Ala Leu Gly Ser Ala Ser Pro Ala Ser Ser Pro Ser Gln 835 840 845Val Asp Thr Pro Ser Ser Ser Ser Phe His Ile Gln Gln Leu Ala Gly 850 855 860Phe Ser Ala Pro Pro Gly Leu Ser Pro Leu Leu Pro Ser Ser Ser Ser865 870 875 880Ser Pro Pro Pro Gly Ala Cys Ser Ser Pro Pro Ala Pro Thr Pro Ser 885 890 895Thr Ser Thr Ala Ala Thr Thr Thr Gly Phe Gly His Phe His Lys Ala 900 905 910Leu Gly Gly Ser Leu Ser Ser Ser Asp Ser Pro Leu Leu Thr Pro Leu 915 920 925Gln Pro Gly Ala Arg Ser Pro Gln Ala Ala Gln Pro Pro Pro Pro Leu 930 935 940Pro Gly Ala Arg Gly Gly Leu Gly Leu Leu Glu His Phe Leu Pro Pro945 950 955 960Pro Pro Ser Ser Arg Ser Pro Ser Ser Ser Pro Gly Gln Leu Gly Gln 965 970 975Pro Pro Gly Glu Leu Ser Pro Gly Leu Ala Ala Gly Pro Pro Ser Thr 980 985 990Pro Glu Thr Pro Pro Arg Pro Glu Arg Pro Ser Phe Met Ala Gly Ala 995 1000 1005Ser Gly Gly Ala Ser Pro Val Ala Phe Thr Pro Arg Gly Gly Leu Ser 1010 1015 1020Pro Pro Gly His Ser Pro Gly Pro Pro Arg Thr Phe Pro Ser Ala Pro1025 1030 1035 1040Pro Arg Ala Ser Gly Ser His Gly Ser Leu Leu Leu Pro Pro Ala Ser 1045 1050 1055Ser Pro Pro Pro Pro Gln Val Pro Gln Arg Arg Gly Thr Pro Pro Leu 1060 1065 1070Thr Pro Gly Arg Leu Thr Gln Asp Leu Lys Leu Ile Ser Ala Ser Gln 1075 1080 1085Pro Ala Leu Pro Gln Asp Gly Ala Gln Thr Leu Arg Arg Ala Ser Pro 1090 1095 1100His Ser Ser Gly Glu Ser Met Ala Ala Phe Ser Leu Tyr Pro Arg Ala1105 1110 1115 1120Gly Gly Gly Ser Gly Ser Ser Gly Gly Leu Gly Pro Pro Gly Arg Pro 1125 1130 1135Tyr Gly Ala Ile Pro Gly Gln His Val Thr Leu Pro Arg Lys Thr Ser 1140 1145 1150Ser Gly Ser Leu Pro Pro Pro Leu Ser Leu Phe Gly Ala Arg Ala Ala 1155 1160 1165Ser Ser Gly Gly Pro Pro Leu Thr Ala Ala Pro Gln Arg Glu Pro Gly 1170 1175 1180Ala Arg Ser Glu Pro Val Arg Ser Lys Leu Pro Ser Asn Leu1185 1190 1195201203PRTHomo sapiens 20Met Asp Lys Leu Pro Pro Ser Met Arg Lys Arg Leu Tyr Ser Leu Pro1 5 10 15Gln Gln Val Gly Ala Lys Ala Trp Ile Met Asp Glu Glu Glu Asp Ala 20 25 30Glu Glu Glu Gly Ala Gly Gly Arg Gln Asp Pro Ser Arg Arg Ser Ile 35 40 45Arg Leu Arg Pro Leu Pro Ser Pro Ser Pro Ser Ala Ala Ala Gly Gly 50 55 60Thr Glu Ser Arg Ser Ser Ala Leu Gly Ala Ala Asp Ser Glu Gly Pro65 70 75 80Ala Arg Gly Ala Gly Lys Ser Ser Thr Asn Gly Asp Cys Arg Arg Phe 85 90 95Arg Gly Ser Leu Ala Ser Leu Gly Ser Arg Gly Gly Gly Ser Gly Gly 100 105 110Thr Gly Ser Gly Ser Ser His Gly His Leu His Asp Ser Ala Glu Glu 115 120 125Arg Arg Leu Ile Ala Glu Gly Asp Ala Ser Pro Gly Glu Asp Arg Thr 130 135 140Pro Pro Gly Leu Ala Ala Glu Pro Glu Arg Pro Gly Ala Ser Ala Gln145 150 155 160Pro Ala Ala Ser Pro Pro Pro Pro Gln Gln Pro Pro Gln Pro Ala Ser 165 170 175Ala Ser Cys Glu Gln Pro Ser Val Asp Thr Ala Ile Lys Val Glu Gly 180 185 190Gly Ala Ala Ala Gly Asp Gln Ile Leu Pro Glu Ala Glu Val Arg Leu 195 200 205Gly Gln Ala Gly Phe Met Gln Arg Gln Phe Gly Ala Met Leu Gln Pro 210 215 220Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala Val225 230 235 240Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Phe Trp Ile Ile His 245 250 255Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp Leu Thr Met Leu Leu Leu 260 265 270Met Val Gly Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe Lys 275 280 285Asp Glu Asn Thr Thr Pro Trp Ile Val Phe Asn Val Val Ser Asp Thr 290 295 300Phe Phe Leu Ile Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val Val305 310 315 320Glu Asp Asn Thr Glu Ile Ile Leu Asp Pro Gln Arg Ile Lys Met Lys 325 330 335Tyr Leu Lys Ser Trp Phe Met Val Asp Phe Ile Ser Ser Ile Pro Val 340 345 350Asp Tyr Ile Phe Leu Ile Val Glu Thr Arg Ile Asp Ser Glu Val Tyr 355 360 365Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu Ser 370 375 380Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His Gln385 390 395 400Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val Val 405 410 415Arg Ile Val Asn Leu Ile Gly Met Met Leu Leu Leu Cys His Trp Asp 420 425 430Gly Cys Leu Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro Asp Asp 435 440 445Cys Trp Val Ser Ile Asn Asn Met Val Asn Asn Ser Trp Gly Lys Gln 450 455 460Tyr Ser Tyr Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile Gly465 470 475 480Tyr Gly Arg Gln Ala Pro Val Gly Met Ser Asp Val Trp Leu Thr Met 485 490 495Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly His 500 505 510Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr Gln 515 520 525Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu Pro 530 535 540Pro Asp Thr Arg Gln Arg Ile His Asp Tyr Tyr Glu His Arg Tyr Gln545 550 555 560Gly Lys Met Phe Asp Glu Glu Ser Ile Leu Gly Glu Leu Ser Glu Pro 565 570 575Leu Arg Glu Glu Ile Ile Asn Phe Asn Cys Arg Lys Leu Val Ala Ser 580 585 590Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ser Met Leu 595 600 605Thr Lys Leu Arg Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg 610 615 620Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val625 630 635 640Ser Val Leu Thr Lys Gly Asn Lys Glu Thr Lys Leu Ala Asp Gly Ser 645 650 655Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser 660 665 670Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn 675 680 685Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe Glu 690 695 700Thr Val Ala Leu Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser Ile705 710 715 720Leu Leu His Lys Val Gln His Asp Leu Asn Ser Gly Val Phe Asn Tyr 725 730 735Gln Glu Asn Glu Ile Ile Gln Gln Ile Val Gln His Asp Arg Glu Met 740 745 750Ala His Cys Ala His Arg Val Gln Ala Ala Ala Ser Ala Thr Pro Thr 755 760 765Pro Thr Pro Val Ile Trp Thr Pro Leu Ile Gln Ala Pro Leu Gln Ala 770 775 780Ala Ala Ala Thr Thr Ser Val Ala Ile Ala Leu Thr His His Pro Arg785 790 795 800Leu Pro Ala Ala Ile Phe Arg Pro Pro Pro Gly Ser Gly Leu Gly Asn 805 810 815Leu Gly Ala Gly Gln Thr Pro Arg His Leu Lys Arg Leu Gln Ser Leu 820 825 830Ile Pro Ser Ala Leu Gly Ser Ala Ser Pro Ala Ser Ser Pro Ser Gln 835 840 845Val Asp Thr Pro Ser Ser Ser Ser Phe His Ile Gln Gln Leu Ala Gly 850 855 860Phe Ser Ala Pro Ala Gly Leu Ser Pro Leu Leu Pro Ser Ser Ser Ser865 870 875 880Ser Pro Pro Pro Gly Ala Cys Gly Ser Pro Ser Ala Pro Thr Pro Ser 885 890 895Ala Gly Val Ala Ala Thr Thr Ile Ala Gly Phe Gly His Phe His Lys 900 905 910Ala Leu Gly Gly Ser Leu Ser Ser Ser Asp Ser Pro Leu Leu Thr Pro 915 920 925Leu Gln Pro Gly Ala Arg Ser Pro Gln Ala Ala Gln Pro Ser Pro Ala 930 935 940Pro Pro Gly Ala Arg Gly Gly Leu Gly Leu Pro Glu His Phe Leu Pro945 950 955 960Pro Pro Pro Ser Ser Arg Ser Pro Ser Ser Ser Pro Gly Gln Leu Gly 965 970 975Gln Pro Pro Gly Glu Leu Ser Leu Gly Leu Ala Thr Gly Pro Leu Ser 980 985 990Thr Pro Glu Thr Pro Pro Arg Gln Pro Glu Pro Pro Ser Leu Val Ala

995 1000 1005Gly Ala Ser Gly Gly Ala Ser Pro Val Gly Phe Thr Pro Arg Gly Gly 1010 1015 1020Leu Ser Pro Pro Gly His Ser Pro Gly Pro Pro Arg Thr Phe Pro Ser1025 1030 1035 1040Ala Pro Pro Arg Ala Ser Gly Ser His Gly Ser Leu Leu Leu Pro Pro 1045 1050 1055Ala Ser Ser Pro Pro Pro Pro Gln Val Pro Gln Arg Arg Gly Thr Pro 1060 1065 1070Pro Leu Thr Pro Gly Arg Leu Thr Gln Asp Leu Lys Leu Ile Ser Ala 1075 1080 1085Ser Gln Pro Ala Leu Pro Gln Asp Gly Ala Gln Thr Leu Arg Arg Ala 1090 1095 1100Ser Pro His Ser Ser Gly Glu Ser Met Ala Ala Phe Pro Leu Phe Pro1105 1110 1115 1120Arg Ala Gly Gly Gly Ser Gly Gly Ser Gly Ser Ser Gly Gly Leu Gly 1125 1130 1135Pro Pro Gly Arg Pro Tyr Gly Ala Ile Pro Gly Gln His Val Thr Leu 1140 1145 1150Pro Arg Lys Thr Ser Ser Gly Ser Leu Pro Pro Pro Leu Ser Leu Phe 1155 1160 1165Gly Ala Arg Ala Thr Ser Ser Gly Gly Pro Pro Leu Thr Ala Gly Pro 1170 1175 1180Gln Arg Glu Pro Gly Ala Arg Pro Glu Pro Val Arg Ser Lys Leu Pro1185 1190 1195 1200Ser Asn Leu211175PRTOryctolagus cuniculus 21Met Asp Lys Leu Pro Pro Ser Met Arg Lys Arg Leu Tyr Ser Leu Pro1 5 10 15Gln Gln Val Gly Ala Lys Ala Trp Ile Met Asp Glu Glu Glu Asp Ala 20 25 30Glu Glu Glu Gly Ala Gly Gly Arg Gln Asp Pro Arg Arg Arg Ser Ile 35 40 45Arg Leu Arg Pro Leu Pro Ser Pro Ser Pro Ser Pro Ser Ala Ala Ala 50 55 60Ala Ala Ala Gly Gly Ala Glu Ser Arg Gly Ala Ala Leu Gly Gly Ala65 70 75 80Ala Asp Gly Glu Gly Pro Ala Arg Gly Ala Ala Lys Ser Ser Thr Asn 85 90 95Gly Asp Cys Arg Arg Phe Arg Gly Ser Leu Ala Ser Leu Gly Ser Arg 100 105 110Gly Gly Gly Gly Gly Gly Gly Ser Thr Gly Gly Gly Ser His Gly His 115 120 125Leu His Asp Ser Ala Glu Glu Arg Arg Leu Ile Ala Glu Gly Asp Ala 130 135 140Ser Pro Gly Glu Asp Arg Thr Pro Pro Gly Leu Ala Ala Glu Pro Glu145 150 155 160Arg Pro Gly Ala Pro Ala Pro Pro Ala Ala Ser Pro Pro Gln Val Pro 165 170 175Ser Ser Cys Gly Glu Gln Arg Pro Ala Asp Ala Ala Val Lys Val Glu 180 185 190Gly Gly Ala Ala Ala Gly Asp Gln Ile Leu Pro Glu Ala Glu Ala Arg 195 200 205Leu Gly Gln Ala Gly Phe Met Gln Arg Gln Phe Gly Ala Met Leu Gln 210 215 220Pro Gly Val Asn Lys Phe Ser Leu Arg Met Phe Gly Ser Gln Lys Ala225 230 235 240Val Glu Arg Glu Gln Glu Arg Val Lys Ser Ala Gly Phe Trp Ile Ile 245 250 255His Pro Tyr Ser Asp Phe Arg Phe Tyr Trp Asp Leu Thr Met Leu Leu 260 265 270Leu Met Val Gly Asn Leu Ile Ile Ile Pro Val Gly Ile Thr Phe Phe 275 280 285Lys Asp Glu Asn Thr Thr Pro Trp Ile Val Phe Asn Val Val Ser Asp 290 295 300Thr Phe Phe Leu Ile Asp Leu Val Leu Asn Phe Arg Thr Gly Ile Val305 310 315 320Val Glu Asp Asn Thr Asp Ile Ile Leu Asp Pro Arg Arg Ile Lys Met 325 330 335Lys Tyr Leu Lys Ser Trp Phe Val Val Asp Phe Val Ser Ser Ile Pro 340 345 350Val Asp Tyr Ile Phe Leu Ile Val Glu Thr Arg Ile Asp Ser Glu Val 355 360 365Tyr Lys Thr Ala Arg Ala Leu Arg Ile Val Arg Phe Thr Lys Ile Leu 370 375 380Ser Leu Leu Arg Leu Leu Arg Leu Ser Arg Leu Ile Arg Tyr Ile His385 390 395 400Gln Trp Glu Glu Ile Phe His Met Thr Tyr Asp Leu Ala Ser Ala Val 405 410 415Val Arg Ile Val Asn Leu Ile Gly Met Met Leu Leu Leu Cys His Trp 420 425 430Asp Gly Cys Leu Gln Phe Leu Val Pro Met Leu Gln Asp Phe Pro Asp 435 440 445Asp Cys Trp Val Ser Leu Asn Asn Met Val Asn Asn Ser Trp Gly Lys 450 455 460Gln Tyr Ser Tyr Ala Leu Phe Lys Ala Met Ser His Met Leu Cys Ile465 470 475 480Gly Tyr Gly Arg Gln Ala Pro Met Gly Met Ser Asp Val Trp Leu Thr 485 490 495Met Leu Ser Met Ile Val Gly Ala Thr Cys Tyr Ala Met Phe Ile Gly 500 505 510His Ala Thr Ala Leu Ile Gln Ser Leu Asp Ser Ser Arg Arg Gln Tyr 515 520 525Gln Glu Lys Tyr Lys Gln Val Glu Gln Tyr Met Ser Phe His Lys Leu 530 535 540Pro Pro Asp Thr Arg Gln Arg Ile His Asp Tyr Tyr Glu His Arg Tyr545 550 555 560Gln Gly Lys Met Phe Asp Glu Glu Ser Ile Leu Gly Glu Leu Ser Glu 565 570 575Pro Leu Arg Glu Glu Ile Ile Asn Phe Asn Cys Arg Lys Leu Val Ala 580 585 590Ser Met Pro Leu Phe Ala Asn Ala Asp Pro Asn Phe Val Thr Ser Met 595 600 605Leu Thr Lys Leu Arg Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile 610 615 620Arg Glu Gly Thr Ile Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val625 630 635 640Val Ser Val Leu Thr Lys Gly Asn Lys Glu Thr Lys Leu Ala Asp Gly 645 650 655Ser Tyr Phe Gly Glu Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala 660 665 670Ser Val Arg Ala Asp Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp 675 680 685Asn Phe Asn Glu Val Leu Glu Glu Tyr Pro Met Met Arg Arg Ala Phe 690 695 700Glu Thr Val Ala Leu Asp Arg Leu Asp Arg Ile Gly Lys Lys Asn Ser705 710 715 720Ile Leu Leu His Lys Val Gln His Asp Leu Ser Ser Gly Val Ser Asn 725 730 735Tyr Gln Glu Asn Ala Ile Val Gln Arg Ile Val Gln His Asp Arg Glu 740 745 750Met Ala His Cys Ala Arg Arg Ala Gln Ala Thr Thr Pro Val Ala Pro 755 760 765Ala Ile Trp Thr Pro Leu Ile Gln Ala Pro Leu Gln Ala Ala Ala Ala 770 775 780Thr Thr Ser Val Ala Ile Ala Leu Thr His His Pro Arg Leu Pro Ala785 790 795 800Ala Ile Phe Arg Pro Pro Pro Gly Pro Thr Thr Leu Gly Ser Leu Gly 805 810 815Ala Gly Gln Thr Pro Arg His Leu Arg Arg Leu Gln Ser Leu Ala Pro 820 825 830Ser Ala Pro Ser Pro Ala Ser Pro Ala Ser Ser Pro Ser Gln Pro Asp 835 840 845Thr Pro Ser Ser Ala Ser Leu His Val Gln Pro Leu Pro Gly Cys Ser 850 855 860Thr Pro Ala Gly Leu Gly Ser Leu Leu Pro Thr Ala Gly Ser Pro Pro865 870 875 880Ala Pro Thr Pro Pro Thr Thr Ala Gly Ala Ala Gly Phe Ser His Phe 885 890 895His Arg Ala Leu Gly Gly Ser Leu Ser Ser Ser Asp Ser Pro Leu Leu 900 905 910Thr Pro Met Gln Ser Ala Ala Arg Ser Pro Gln Gln Pro Pro Pro Pro 915 920 925Pro Gly Ala Pro Ala Gly Leu Gly Leu Leu Glu His Phe Leu Pro Pro 930 935 940Pro Ala Arg Ser Pro Thr Ser Ser Pro Gly Gln Leu Gly Gln Pro Pro945 950 955 960Gly Glu Leu Ser Pro Gly Leu Gly Ser Gly Pro Pro Gly Thr Pro Glu 965 970 975Thr Pro Pro Arg Gln Pro Glu Arg Leu Pro Phe Ala Ala Gly Ala Ser 980 985 990Ala Gly Ala Ser Pro Val Ala Phe Ser Pro Arg Gly Gly Pro Ser Pro 995 1000 1005Pro Gly His Ser Pro Gly Thr Pro Arg Thr Phe Pro Ser Ala Pro Pro 1010 1015 1020Arg Ala Ser Gly Ser His Gly Ser Leu Leu Leu Pro Pro Ala Ser Ser1025 1030 1035 1040Pro Pro Pro Pro Pro Pro Pro Pro Ala Pro Gln Arg Arg Ala Thr Pro 1045 1050 1055Pro Leu Ala Pro Gly Arg Leu Ser Gln Asp Leu Lys Leu Ile Ser Ala 1060 1065 1070Ser Gln Pro Ala Leu Pro Gln Asp Gly Ala Gln Thr Leu Arg Arg Ala 1075 1080 1085Ser Pro His Ser Ser Ser Gly Glu Ser Val Ala Ala Leu Pro Pro Phe 1090 1095 1100Pro Arg Ala Pro Gly Arg Pro Pro Gly Ala Gly Pro Gly Gln His Val1105 1110 1115 1120Thr Leu Thr Leu Pro Arg Lys Ala Ser Ser Gly Ser Leu Pro Pro Pro 1125 1130 1135Leu Ser Leu Phe Gly Pro Arg Ala Ala Pro Ala Gly Gly Pro Arg Leu 1140 1145 1150Thr Ala Ala Pro Gln Arg Glu Pro Gly Ala Lys Ser Glu Pro Val Arg 1155 1160 1165Ser Lys Leu Pro Ser Asn Leu 1170 117522124PRTCanis familiaris 22Asp Glu Asp Ser Ile Leu Gly Glu Leu Ser Glu Pro Leu Arg Glu Glu1 5 10 15Ile Ile Asn Phe Asn Cys Arg Lys Leu Val Ala Ser Met Pro Leu Phe 20 25 30Ala Asn Ala Asp Pro Asn Phe Val Thr Ser Met Leu Thr Lys Leu Arg 35 40 45Phe Glu Val Phe Gln Pro Gly Asp Tyr Ile Ile Arg Glu Gly Thr Ile 50 55 60Gly Lys Lys Met Tyr Phe Ile Gln His Gly Val Val Ser Val Leu Thr65 70 75 80Lys Gly Asn Lys Glu Thr Lys Leu Ala Asp Gly Ser Tyr Phe Gly Glu 85 90 95Ile Cys Leu Leu Thr Arg Gly Arg Arg Thr Ala Ser Val Arg Ala Asp 100 105 110Thr Tyr Cys Arg Leu Tyr Ser Leu Ser Val Asp Asn 115 120231528DNAHomo sapiens 23gaggcagttc acctccatgc tgcagcccgg ggtcaacaaa ttctccctcc gcatgtttgg 60gagccagaag gcggtggaaa aggagcagga aagggttaaa actgcaggct tctggattat 120ccacccttac agtgatttca ggttttactg ggatttaata atgcttataa tgatggttgg 180aaatctagtc atcataccag ttggaatcac attctttaca gagcaaacaa caacaccatg 240gattattttc aatgtggcat cagatacagt tttcctattg gacctgatca tgaattttag 300gactgggact gtcaatgaag acagttctga aatcatcctg gaccccaaag tgatcaagat 360gaattattta aaaagctggt ctgtggttga cttcatctca tccatcccag tggattatat 420ctttcttatt gtagaaaaag gaatggattc tgaagtttac aagacagcca gggcacttcg 480cattgtgagg tttacaaaaa ttctcagtct cttgcgttta ttacgacttt caaggttaat 540tagatacata catcaatggg aagagatatt ccacatgaca tatgatctcg ccagtgcagt 600ggtgagaatt tttaatctca tcggcatgat gctgctcctg tgccactggg atggttgtct 660tcagttctta gtaccactac tgcaggactt cccaccagat tgctgggtgt ctttaaatga 720aatggttaat gattcttggg gaaagcagta ttcatacgca ctcttcaaag ctatgagtca 780catgctgtgc attgggtatg gagcccaagc cccagtcagc atgtctgacc tctggattac 840catgctgagc atgatcgtcg gggccacctg ctatgccatg tttgtcggcc atgccaccgc 900tttaatccag tctctggatt cttcgaggcg gcagtatcaa gagaagtata agcaagtgga 960acaatacatg tcattccata agttaccagc tgatatgcgt cagaagatac atgattacta 1020tgaacacaga taccaaggca aaatctttga tgaggaaaat attctcaatg aactcaatga 1080tcctctgaga ggggagatag tcaacttcaa ctgtcggaaa ctggtggcta caatgccttt 1140atttgctaat gcggatccta attttgtgac tgccatgctg agcaagttga gatttgaggt 1200gtttcaacct ggagattata tcgtacgaga aggagccgtg ggtaaaaaaa tgtatttcat 1260tcaacacggt gttgctggtg tcattacaaa atccagtaaa gaaatgaagc tgacagatgg 1320ctcttacttt ggagagattt gcctgctgac caaaggacgt cgtactgcca gtgttcgagc 1380tgatacatat tgtcgtcttt actcactttc cgtggacaat ttcaacgagg tcccggagga 1440atatccaatg atgaggagag cctttgagac agttgccatt gaccgactag atcgaatagg 1500aaagaaaaat tcaattcttc tgcaaaag 1528241528DNAHomo sapiens 24gcgccagttc ggcgcgctcc tgcagccggg cgtcaacaag ttctcgctgc ggatgttcgg 60cagccagaag gccgtggagc gcgagcagga gcgcgtcaag tcggcggggg cctggatcat 120ccacccgtac agcgacttca ggttctactg ggacttcacc atgctgctgt tcatggtggg 180aaacctcatc atcatcccag tgggcatcac cttcttcaag gatgagacca ctgccccgtg 240gatcgtgttc aacgtggtct cggacacctt cttcctcatg gacctggtgt tgaacttccg 300caccggcatt gtgatcgagg acaacacgga gatcatcctg gaccccgaga agatcaagaa 360gaagtatctg cgcacgtggt tcgtggtgga cttcgtgtcc tccatccccg tggactacat 420cttccttatt gtggagaagg gcattgactc cgaggtctac aagacggcac gcgccctgcg 480catcgtgcgc ttcaccaaga tcctcagcct cctgcggctg ctgcgcctct cacgcctgat 540ccgctacatc catcagtggg aggagatctt ccacatgacc tatgacctgg ccagcgcggt 600gatgaggatc tgcaatctca tcagcatgat gctgctgctc tgccactggg acggctgcct 660gcagttcctg gtgcctatgc tgcaggactt cccgcgcaac tgctgggtgt ccatcaatgg 720catggtgaac cactcgtgga gtgaactgta ctccttcgca ctcttcaagg ccatgagcca 780catgctgtgc atcgggtacg gccggcaggc gcccgagagc atgacggaca tctggctgac 840catgctcagc atgattgtgg gtgccacctg ctacgccatg ttcatcggcc acgccactgc 900cctcatccag tcgctggact cctcgcggcg ccagtaccag gagaagtaca agcaggtgga 960gcagtacatg tccttccaca agctgccagc tgacttccgc cagaagatcc acgactacta 1020tgagcaccgt taccagggca agatgtttga cgaggacagc atcctgggcg agctcaacgg 1080gcccctgcgg gaggagatcg tcaacttcaa ctgccggaag ctggtggcct ccatgccgct 1140gttcgccaac gccgacccca acttcgtcac ggccatgctg accaagctca agttcgaggt 1200cttccagccg ggtgactaca tcatccgcga aggcaccatc gggaagaaga tgtacttcat 1260ccagcacggc gtggtcagcg tgctcactaa gggcaacaag gagatgaagc tgtccgatgg 1320ctcctacttc ggggagatct gcctgctcac ccggggccgc cgcacggcga gcgtgcgggc 1380cgacacctac tgccgcctct attcgctgag cgtggacaac ttcaacgagg tgctggagga 1440gtaccccatg atgcggcgcg ccttcgagac ggtggccatc gaccgcctgg accgcatcgg 1500caagaagaat tccatcctcc tgcacaag 1528251520DNAHomo sapiens 25gcgccagttc ggggccatgc tccaacccgg ggtcaacaaa ttctccctaa ggatgttcgg 60cagccagaaa gccgtggagc gcgaacagga gagggtcaag tcggccggat tttggattat 120ccacccctac agtgacttca gattttactg ggacctgacc atgctgctgc tgatggtggg 180aaacctgatt atcattcctg tgggcatcac cttcttcaag gatgagaaca ccacaccctg 240gattgtcttc aatgtggtgt cagacacatt cttcctcatc gacttggtcc tcaacttccg 300cacagggatc gtggtggagg acaacacaga gatcatcctg gacccgcagc ggattaaaat 360gaagtacctg aaaagctggt tcatggtaga tttcatttcc tccatccccg tggactacat 420cttcctcatt gtggagacac gcatcgactc ggaggtctac aagactgccc gggccctgcg 480cattgtccgc ttcacgaaga tcctcagcct cttacgcctg ttacgcctct cccgcctcat 540tcgatatatt caccagtggg aagagatctt ccacatgacc tacgacctgg ccagcgccgt 600ggtgcgcatc gtgaacctca tcggcatgat gctcctgctc tgccactggg acggctgcct 660gcagttcctg gtacccatgc tacaggactt ccctgacgac tgctgggtgt ccatcaacaa 720catggtgaac aactcctggg ggaagcagta ctcctacgcg ctcttcaagg ccatgagcca 780catgctgtgc atcggctacg ggcggcaggc gcccgtgggc atgtccgacg tctggctcac 840catgctcagc atgatcgtgg gtgccacctg ctacgccatg ttcattggcc acgccactgc 900cctcatccag tccctggact cctcccggcg ccagtaccag gaaaagtaca agcaggtgga 960gcagtacatg tcctttcaca agctcccgcc cgacacccgg cagcgcatcc acgactacta 1020cgagcaccgc taccagggca agatgttcga cgaggagagc atcctgggcg agctaagcga 1080gcccctgcgg gaggagatca tcaactttaa ctgtcggaag ctggtggcct ccatgccact 1140gtttgccaat gcggacccca acttcgtgac gtccatgctg accaagctgc gtttcgaggt 1200cttccagcct ggggactaca tcatccggga aggcaccatt ggcaagaaga tgtacttcat 1260ccagcatggc gtggtcagcg tgctcaccaa gggcaacaag gagaccaagc tggccgacgg 1320ctcctacttt ggagagatct gcctgctgac ccggggccgg cgcacagcca gcgtgagggc 1380cgacacctac tgccgcctct actcgctgag cgtggacaac ttcaatgagg tgctggagga 1440gtaccccatg atgcgaaggg ccttcgagac cgtggcgctg gaccgcctgg accgcattgg 1500caagaagaac tccatcctcc 1520261527DNAMus musculus 26gaggcagttc acctccatgc tgcagcctgg ggtcaacaaa ttctccctcc gcatgtttgg 60gagccagaag gcggtggaga aggagcagga aagggttaaa actgcaggct tctggattat 120ccatccgtac agtgacttca ggttttattg ggatttaatc atgcttataa tgatggttgg 180aaatttggtc atcataccag ttggaatcac gttcttcaca gagcagacga caacaccgtg 240gattattttc aacgtggcat ccgatactgt tttcctgttg gacttaatca tgaattttag 300gactgggact gtcaatgaag acagctcgga aatcatcctg gaccctaaag tgatcaagat 360gaattattta aaaagctggt ttgtggtgga cttcatctca tcgatcccgg tggattatat 420ctttctcatt gtagagaaag ggatggactc agaagtttac aagacagcca gagcacttcg 480tatcgtgagg tttacaaaaa ttctcagtct cttgcggtta ttacgccttt caaggttaat 540cagatacata caccagtggg aagagatatt ccacatgacc tatgacctcg ccagtgctgt 600ggtgaggatc ttcaacctca ttggcatgat gctgcttctg tgccactggg atggctgtct 660tcagttcctg gttcccctgc tgcaggactt cccaccagat tgctgggttt ctctgaatga 720aatggttaat gattcctggg gaaaacaata ttcctacgca ctcttcaaag ctatgagtca 780catgctgtgc attggttatg gcgcccaagc ccctgtcagc atgtctgacc tctggattac 840catgctgagc atgattgtgg gcgccacctg ctacgcaatg tttgttggcc atgccacagc 900tttgatccag tctttggact cttcaaggag gcagtatcaa gagaagtata agcaagtaga 960gcaatacatg tcattccaca agttaccagc tgacatgcgc cagaagatac atgattacta 1020tgagcaccga taccaaggca agatcttcga tgaagaaaat attctcagtg agcttaatga 1080tcctctgaga gaggaaatag tcaacttcaa ctgccggaaa ctggtggcta

ctatgcctct 1140ttttgctaac gccgatccca atttcgtgac ggccatgctg agcaagctga gatttgaggt 1200gttccagccc ggagactata tcattcgaga aggagctgtg gggaagaaaa tgtatttcat 1260ccagcacggt gttgctggcg ttatcaccaa gtccagtaaa gaaatgaagc tgacagatgg 1320ctcttacttc ggagagatat gcctgctgac caagggccgg cgcactgcca gtgtccgagc 1380tgatacctac tgtcgtcttt actccctttc ggtggacaat ttcaatgagg tcttggagga 1440atatccaatg atgagaagag cctttgagac agttgctatt gaccgactcg atcggatagg 1500caagaaaaac tctattctcc tgcagaa 1527271527DNAMus musculus 27gcgccaattc ggggcgcttc tgcagcccgg cgtcaacaag ttctccctgc ggatgttcgg 60cagccagaag gccgtggagc gcgagcagga acgcgtgaag tcggcggggg cctggatcat 120ccacccctac agcgacttca ggttctactg ggacttcacc atgctgttgt tcatggtggg 180aaatctcatt atcattcccg tgggcatcac tttcttcaag gacgagacca ccgcgccctg 240gatcgtcttc aacgtggtct cggacacttt cttcctcatg gacttggtgt tgaacttccg 300caccggcatt gttattgagg acaacacgga gatcatcctg gaccccgaga agataaagaa 360gaagtacttg cgtacgtggt tcgtggtgga cttcgtgtca tccatcccgg tggactacat 420cttcctcata gtggagaagg gaatcgactc cgaggtctac aagacagcgc gtgctctgcg 480catcgtgcgc ttcaccaaga tcctcagtct gctgcggctg ctgcggctat cacggctcat 540ccgatatatc caccagtggg aagagatttt ccacatgacc tacgacctgg caagtgcagt 600gatgcgcatc tgtaacctga tcagcatgat gctactgctc tgccactggg acggttgcct 660gcagttcctg gtgcccatgc tgcaagactt ccccagcgac tgctgggtgt ccatcaacaa 720catggtgaac cactcgtgga gcgagctcta ctcgttcgcg ctcttcaagg ccatgagcca 780catgctgtgc atcggctacg ggcggcaggc gcccgagagc atgacagaca tctggctgac 840catgctcagc atgatcgtag gcgccacctg ctatgccatg ttcattgggc acgccactgc 900gctcatccag tccctggatt cgtcacggcg ccaataccag gagaagtaca agcaagtaga 960gcaatacatg tccttccaca aactgcccgc tgacttccgc cagaagatcc acgattacta 1020tgaacaccgg taccaaggga agatgtttga tgaggacagc atccttgggg aactcaacgg 1080gccactgcgt gaggagattg tgaacttcaa ctgccggaag ctggtggctt ccatgccgct 1140gtttgccaat gcagacccca acttcgtcac agccatgctg acaaagctca aatttgaggt 1200cttccagcct ggagattaca tcatccgaga ggggaccatc gggaagaaga tgtacttcat 1260ccagcatggg gtggtgagcg tgctcaccaa gggcaacaag gagatgaagc tgtcggatgg 1320ctcctatttc ggggagatct gcttgctcac gaggggccgg cgtacggcca gcgtgcgagc 1380tgacacctac tgtcgcctct actcactgag tgtggacaat ttcaacgagg tgctggagga 1440ataccccatg atgcggcgtg cctttgagac tgtggctatt gaccggctag atcgcatagg 1500caagaagaac tccatcttgc tgcacaa 1527281547DNAMus musculus 28gcgcctgggc cagagcggct tcatgcagcg ccagttcggt gccatgctgc aacctggggt 60caacaaattc tccctaagga tgttcggcag ccagaaagcg gtggagcgcg agcaggagag 120ggttaagtca gcagggtttt ggattatcca cccctacagt gacttcagat tttactggga 180cctgacgatg ctgttgctga tggtggggaa tctgatcatc atacccgtgg gcatcacctt 240cttcaaggat gagaacacca caccctggat cgtcttcaat gtggtgtcag acacattctt 300cctcattgac ttggtcctca acttccgcac ggggatcgtg gtggaggaca acacagaaat 360catccttgac ccgcagagga tcaagatgaa gtacctgaaa agctggtttg tggtagattt 420catctcctcc atccctgtcg actacatctt ccttatagtg gagactcgca ttgactcgga 480ggtctacaaa accgctaggg ctctgcgcat tgtccgtttc actaagatcc tcagcctcct 540gcgcctcttg aggctttccc gcctcattcg atacattcat cagtgggaag agatcttcca 600catgacctat gacctggcca gcgccgtggt acgcatcgtg aacctcattg gcatgatgct 660tctgctgtgt cactgggatg gctgcctgca gttcctagtg cccatgctgc aggacttccc 720ccatgactgc tgggtgtcca tcaatggcat ggtgaataac tcctggggga agcagtattc 780ctacgccctc ttcaaggcca tgagccacat gctgtgcatt gggtatggac ggcaggcacc 840cgtaggcatg tctgacgtct ggctcaccat gctcagcatg atcgtggggg ccacctgcta 900tgccatgttc atcggccacg ccactgccct catccagtcg ctagactcct cccggcgcca 960gtaccaggag aagtataaac aggtggagca gtacatgtcc ttccacaagc tcccgcctga 1020cacccgacag cgcatccatg actactatga acaccgctac caaggcaaga tgtttgatga 1080ggaaagcatc ctgggtgagc tgagtgagcc acttcgagag gagatcatca actttaactg 1140ccgaaagctg gtggcatcca tgccactgtt tgccaacgca gatcccaact ttgtgacatc 1200catgctgacc aagttgcgtt tcgaggtctt ccagcctggg gattacatca tccgcgaagg 1260caccatcggc aagaagatgt actttatcca gcacggcgtg gtcagcgtgc tcactaaggg 1320caacaaagag accaagctgg ctgatggctc ctattttgga gagatctgct tgctgacccg 1380gggtcggcgc acagccagcg tcagagcgga tacttattgc cgcctctact cactgagcgt 1440ggacaacttc aatgaggtgc tggaggagta tcccatgatg cggagggcct tcgagacggt 1500tgcgctggac cgcctggacc gcataggcaa gaagaactcc atcctcc 1547

Claims

1. An atrioventricular bypass bridge comprising a tract of gap junction-coupled adult human mesenchymal stem cells obtained from blood, wherein the tract i. is formed in vitro, ii. is at least about 0.5 mm in length, iii. has a first end that is capable of being attached to a first site in the atrium of the heart and a second end that is capable of being attached to a second site in the ventricle of the heart so as to allow the conduction of at least one of a pacemaker signal, pacemaker current, electrical signal, and electrical current across the tract between the two sites, and iv. wherein the adult mesenchymal stem cells functionally express both a sodium channel and a potassium channel which channels are each encoded by a respective nucleic acid that has been introduced into the cells.

2. The bypass bridge of claim 1, wherein the mesenchymal stem cells further functionally express a pacemaker ion channel encoded by a nucleic acid that has been introduced into the cells which channel induces a pacemaker in the cells.

3-6. (canceled)

7. The bypass bridge of claim 1 or 2, wherein the sodium channel is a SKM-1 channel.

8. The bypass bridge of claim 7, wherein the SKM-1 channel comprises an alpha subunit.

9. The bypass bridge of claim 8, wherein the SKM-1 channel further comprises an accessory subunit.

10. The bypass bridge of claim 1, wherein the cells in the tract further functionally express a calcium channel encoded by a nucleic acid that has been introduced into the cells.

11. The bypass bridge of claim 1, wherein the potassium channel comprises the potassium inwardly-rectifying channel 12 (Kir2.1) alpha subunit or potassium inwardly-rectifying channel, subfamily 1, member 12 (Kir2.2) alpha subunit.

12. The bypass bridge of claim 11, wherein the potassium channel further comprises an accessory subunit.

13. The bypass bridge of claim 10, wherein the calcium channel is an L-type calcium channel.

14. The bypass bridge of claim 13, wherein the L-type calcium channel comprises an alpha subunit and accessory subunits.

15. The bypass bridge of claim 1, wherein cells in the tract further functionally express one or more of at least one cardiac connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel.

16. The bypass bridge of claim 15, wherein the at least one connexin is Cx43, Cx40, or Cx45.

17. The bypass bridge of claim 2, wherein the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or chimera thereof, and (b) a MiRP1 beta subunit.

18. The bypass bridge of claim 17, wherein the pacemaker ion channel is expressed in cells in the first end of the tract.

19. The bypass bridge of claim 18, wherein the cells expressing the pacemaker ion channel are located in a region extending 0.5 mm from the first end.

20. The bypass bridge of claim 17, wherein the chimeric HCN channel provides an improved characteristic, as compared to a wild-type HCN channel, selected from the group consisting of faster kinetics, more positive activation, increased expression, increased stability, preserved or enhanced cAMP responsiveness, and preserved or enhanced neurohumoral response.

21. The bypass bridge of claim 17, wherein the HCN chimera comprises portions derived from more than one HCN channel isoform.

22. The bypass bridge of claim 21, wherein the portions are an amino terminal portion, an intramembranous portion, and a carboxy terminal portion.

23. The bypass bridge of claim 21, wherein at least one portion of the HCN chimera is derived from an animal species which is different from the animal species from which at least one of the other two portions is derived.

24. The bypass bridge of claim 21, wherein the HCN chimera is mHCN112, mHCN212, mHCN312, mHCN412, mHCN114, mHCN214, mHCN314, mHCN414, hHCN112, hHCN212, hHCN312, hHCN412, hHCN114, hHCN214, hHCN314, or hHCN414.

25. The bypass bridge of claim 24, wherein the HCN chimera is hHCN212 having the sequence set forth in SEQ ID NO: 2.

26. The bypass bridge of claim 24, wherein the HCN chimera is mHCN212 having the sequence set forth in SEQ ID NO: 6.

27. The bypass bridge of claim 21, wherein at least one portion of the chimera is derived from a HCN channel containing a mutation which provides an improved characteristic, as compared to a wild-type HCN channel, selected from the group consisting of faster kinetics, more positive activation, increased expression, increased stability, preserved or enhanced cAMP responsiveness, and preserved or enhanced neurohumoral response.

28. The bypass bridge of claim 17, wherein the HCN channel is a mutant channel derived from mHCN2 having the sequence set forth in SEQ ID NO: 14 and comprises E324A-mHCN2, Y331A-mHCN2, R339A-mHCN2, or Y331A, E324A-mHCN2.

29. The bypass bridge of claim 28, wherein the mutant HCN channel is E324A-HCN2.

30. The bypass bridge of claim 19, wherein the pacemaker current is conducted by electrotonic conduction.

31. The bypass bridge of claim 17, wherein the pacemaker current is actively propagated by an action potential.

32. The bypass bridge of claim 31, wherein the action potential is a sodium-dependent action potential.

33. The bypass bridge of claim 31, wherein cells in the tract further functionally express an L-type calcium channel and the action potential is a calcium-dependent action potential.

34. A method of making a bypass bridge for implantation in a heart comprising: (a) transfecting a cell with, and functionally expressing therein, a nucleic acid encoding a sodium channel; and (b) growing the transfected cell into a tract of cells having a first and a second end capable of being attached to two selected sites in the heart, wherein the cells are physically interconnected via electrically conductive gap junctions.

35. The method of claim 34 for making a bypass bridge, further comprising transfecting cells in the tract with a nucleic acid encoding a pacemaker ion channel, wherein the nucleic acid is functionally expressed so as to induce a pacemaker current in the cells.

36. The method of claim 34 or 35, wherein the cells are human adult mesenchymal stem cells.

37. The method of claim 34 or 35, further comprising transfecting the cell with, and expressing therein, at least one nucleic acid encoding one or more of at least one cardiac connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel, such that implantation of a bypass bridge in a heart changes the voltage-time course of repolarization and/or refractoriness of the heart.

38. The method of claim 41, wherein the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or a mutant or chimera thereof, and (b) a MiRP1 beta subunit.

39. A method of implanting a bypass bridge in a heart comprising: (a) making a bypass bridge by the method of claim 34; (b) selecting a first and a second site in the heart; and (c) attaching the first end of the tract to the first site and the second end of the tract to the second site; so as to thereby implant a bypass bridge in the heart that allows the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites.

40. The method of claim 39, wherein the a pacemaker and/or electrical signal/current is generated in the atrium by the sinus node or an electronic pacemaker.

41. The method of claim 39, further comprising transfecting cells in the tract with a nucleic acid encoding a pacemaker ion channel, wherein the nucleic acid is functionally expressed so as to induce a pacemaker current in the cells.

42. The method of claim 41, wherein the pacemaker ion channel is expressed in cells in the first end of the tract.

43. The method of claim 42, wherein the cells expressing the pacemaker ion channel are located in a region extending 0.5 mm from the first end.

44. The method of claim 39 or 41, wherein the first site is in an atrium and the second site is in a ventricle, so as to allow propagation of a pacemaker and/or electrical signal/current across the tract from the atrium to the ventricle.

45. The method of claim 39 or 41, wherein the cells are stem cells, cardiomyocytes, fibroblasts or skeletal muscle cells engineered to express connexins, or endothelial cells.

46. The method of claim 44, wherein the stem cells are adult mesenchymal stem cells or embryonic stem cells, wherein said cells are substantially incapable of differentiation.

47. The method of claim 46, wherein the stem cells are human adult mesenchymal stem cells or human embryonic stem cells.

48. The method of claim 39 or 41, further comprising transfecting the cells in the tract with, and expressing therein, at least one nucleic acid encoding one or more of at least one cardiac connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel, so as to change the voltage-time course of repolarization and/or refractoriness of the heart.

49. The method of claim 48, wherein the at least one cardiac connexin is Cx43, Cx40, or Cx45.

50. The method of claim 41, wherein the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or a mutant or chimera thereof, and (b) a MiRP1 beta subunit.

51. A method of treating a disorder associated with an impaired conduction in a subject's heart comprising: (a) transfecting a cell with a nucleic acid encoding a sodium channel, wherein the cell functionally expresses the sodium channel; (b) growing the transfected cell into a tract of cells having a first end and a second end, wherein the cells are physically interconnected via electrically conductive gap junctions; (c) selecting a first site and a second site in the heart between which sites conduction is impaired; and (d) so as to allow the conduction of a pacemaker and/or electrical signal/current across the tract between the two sites and thereby treat the subject.

52. A method of treating a disorder associated with an impaired conduction and impaired sinus node activity in a subject's heart comprising: (a) transfecting a cell with at least one nucleic acid encoding a sodium channel and a pacemaker ion channel, wherein the cell functionally expresses the sodium channel and the pacemaker ion channel; (b) growing the transfected cell into a tract of cells having a first end and a second end, wherein the cells are physically interconnected via electrically conductive gap junctions; (c) selecting a first site in the left atrium of the heart and a second site, between which sites conduction is impaired; and (d) attaching the first end of the tract to the first site and the second end of the tract to the second site; so as to allow the propagation of a pacemaker and/or electrical signal/current generated by the sinus node and/or tract of cells between the two sites and thereby treat the subject.

53. The method of claim 51 or 52, wherein the cells are human adult mesenchymal stem cells or human embryonic stem cells, wherein said cells are substantially incapable of differentiation.

54. The method of claim 51 or 52, further comprising transfecting the cells with, and expressing therein, at least one nucleic acid encoding one or more of at least one connexin, an alpha subunit with accessory subunits of an L-type calcium channel, or an alpha subunit with or without accessory subunits of the potassium channel, so as to change the voltage-time course of repolarization and/or refractoriness of the heart.

55. The method of claim 52, wherein the pacemaker ion channel is at least one of (a) a hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channel or a mutant or chimera thereof, and (b) a MiRP1 beta subunit.

56. An atrioventricular bypass tract made by growing mesenchymal stem cells into a strip with two ends and transfecting the mesenchymal stems cells with a nucleic acid encoding as a sodium channel so that the mesenchymal stem cells functionally express the sodium channel, wherein the strip can be attached to a heart so as to create a tract between an atrium of the heart and a ventricle of the heart, which tract is capable of propagating electrical signals.