Abstract:
The present invention discloses the cDNA sequence and the expressed amino acid sequence of a sodium channel α subunit, termed hH1b. A specific mutation in hH1b has been shown to display a different phenotype in relation to a human heart disease than other known human sodium channel α subunits with corresponding mutations. The present inventions provides new tools to design or identify new diagnostic and treatment strategies or agents for sodium channel related diseases or conditions.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    Not applicable. 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
       [0002] This invention was made with United States government support awarded by the following agency: NIH, Grant No. HL-56441. The United States has certain rights in this invention. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0003]    Sodium channel proteins embedded in cellular membranes of muscle cells, neurons and other excitable cells help produce and propagate electrical impulses and are implicated in many human diseases and conditions. Sodium channels are often composed of a pore-forming α subunit, having four homologous domains DI-DIV and six transmembrane regions S1-S6 per domain, and two auxiliary subunits β1 and β2. The α subunit is sufficient to form a functional channel for generating sodium current flow across cellular membranes.  
           [0004]    Human cardiac sodium channels play a critical role in cardiac excitation. hNa v 1.5, a human cardiac sodium channel α subunit encoded by the SCN5A gene forms a functioning monomeric sodium channel that carries the inward Na current (I Na ) in the heart. The I Na  current is vital for excitation and conduction in working myocardium and in specialized conduction tissue such as Purkinje fibers.  
           [0005]    Two distinct full-length polymorphic SCN5A clones (designated SCN5A hH1 and SCN5A hH1a, or simply hH1 and hH1a, respectively) that encode the hNaV1.5 human cardiac sodium channel have been isolated from human cardiac cDNA libraries (Gellens, M. E. et al., “Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel,”  Proc.Natl.Acad.Sci. U.S.A . 89, 554-558 (1992); Hartmann, H. A. et al., Effects of III-IV linker mutations on human heart Na +  channel inactivation gating.  Circ.Res . 75, 114-122 (1994)). The significance of polymorphisms in the sodium channel is still unknown. For example, it is not known how such polymorphisms affect the mutation phenotype of SCN5A. Nonetheless, identified polymorphisms can help identify disease-associated mutations in SCN5A. For example, various SCN5A mutations are associated with congenital Long QT syndrome, idiopathic ventricular fibrillation and the Brugada syndrome (Keating and Sanguinetti 2001).  
           [0006]    The reported sequences for hH1 and hH1a differ by nine amino acids with hH1 containing one additional amino acid (Q1077). However, the applicants independently sequenced both SCN5A forms again, thereby confirming the sequence of hH1a, while noting that the re-sequenced hH1 differed from the published hH1 sequence at seven amino acid positions, where each difference is, in fact, identical to the hH1a sequence. This change in the published sequence reduces the difference between hH1 and hH1a to just 3 amino acids (T559 vs. A559, Q1027 vs. R1027, and Q1077 vs. Q1077del (hH1 vs. hH1a, respectively) over a total length of 2016/2015 amino acids, respectively. All of the differences are confined to the cytoplasmic linkers between DI-II and between DII-III. Both hH1 and hH1a contain a histidine residue at amino acid 558 (H558), the site of a histidine-to-arginine polymorphism (H558R) (Iwasa, et al., “Twenty single nucleotide polymorphisms (SNPs) and their allelic frequencies in four genes that are responsible for familial long QT syndrome in the Japanese population,”  J.Hum.Genet . 45, 182-183). The amino acid numbering follows that of the original hH1 clone which contains 2016 amino acids. In separate studies, the two known polymorphic forms showed only minor kinetic differences that can be attributed to different expression systems and study techniques including solutions, temperature, and protocols. (Gellens, M. E. et al., supra; Hartmann et. al., supra; and Wattanasirichaigoon et. al. 1999). Subtle differences in kinetics such as decay rates, inactivation midpoints, and late I Na , however, may be important in controlling repolarization.  
           [0007]    Sodium channel α subunits encoded by an SCN5A hH1a clone carrying an arrhythmogenic missense methionine-to-leucine mutation at amino acid 1766 (M1766L) further exhibit a significant inward sodium current level drop, relative to the current level in channels encoded by a wild type hH1a clone. Recently, M1766L in the hH1a background was shown to have a trafficking defect and to cause QT prolongation and ventricular arrhythmia (Valdivia et. al. 2001). These conditions can be rescued by low temperature, antiarrhythmic drug and β1 subunit (Valdivia et. al. 2001).  
           [0008]    In another aspect, drugs that can alter sodium channel activities can relieve or prevent symptoms of certain conditions such as cardiac arrhythmias. Cardiac arrhythmias are abnormalities in the rate, regularity, or site of origin of the cardiac impulse, or a disturbance in conduction of the impulse that alters the normal sequence of atrial or ventricular activation. One known way to treat cardiac arrhythmias is to block the activity of a cardiac sodium channel. Sodium channel blockers used to treat cardiac arrhythmias include: Quinidine, Lidocaine, Procainamide, Mexiletine, Flecainide, Moricizine, and Disopyramide. Identifying other polymorphic forms of human cardiac sodium channel will advance our understanding of sodium channel-related heart problems and provide new tools for developing diagnostic, prophylactic and therapeutic strategies.  
         BRIEF SUMMARY OF THE INVENTION  
         [0009]    The present invention discloses a novel polymorphism in the hNaV1.5 sodium channel α subunit polypeptide, designated SCN5A hH1b or simply hH1b, as well as a nucleic acid molecule encoding same. A specific mutation in hH1b displays a different phenotype in relation to a human heart disease than other known human sodium channel α subunits carrying a corresponding mutation.  
           [0010]    The present invention also includes various related nucleic acid molecules and polypeptides that are useful in various applications such as detecting the subunit and generating antibodies to the subunit. The present invention also relates to cloning and expression vectors and host cells containing same. In addition, the present invention includes methods for screening for an agent for altering (increasing or reducing) sodium channel activities. Furthermore, methods of using the nucleic acids and polypeptides to detect hH1b and generate antibodies to detect and purify hH1b are also included in the present invention. New diagnostic and treatment strategies for various sodium channel-related diseases and conditions are also enabled by the present invention.  
           [0011]    It is an object of the present invention to identify a new polymorphic form of a human cardiac sodium channel.  
           [0012]    It is another object of the present invention to provide new tools for designing diagnostic and treatment strategies for sodium channel related diseases and conditions.  
           [0013]    Other objects, advantages and features of the present invention will become apparent from the following specifications and claims. 
       
    
    
     BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS  
       [0014]    [0014]FIG. 1 depicts the structure of the hNaV1.5 sodium channel α subunit polypeptide including amino acid variations among hH1, hH1a, and hH1b.  
         [0015]    [0015]FIG. 2 shows whole cell I Na  for hH1, hH1a, and hH1b.  
         [0016]    [0016]FIG. 3 shows current magnitude for wild type and hH1, hH1a, and hH1b clones and for each clone containing the M1766L arrhythmia mutation.  
         [0017]    [0017]FIG. 4 summarizes the data of FIG. 3 as relative I Na  density. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0018]    The present invention relates to a new variant form of the hNa v 1.5 sodium channel α subunit, designated SCN5A hH1b. A polynucleotide sequence that encodes SCN5A hH1b is attached as SEQ ID NO: 1. The amino acid sequence of the polypeptide encoded by SCN5A hH1b is attached as SEQ ID NO:2. The new clone differs from SCN5A hH1 by just four amino acids in the cytoplasmic linkers between DI-DII and DII-DIII (R558 vs. H558, 1618 vs. L618, R1027 vs. Q1027, and Q1077del vs. Q1077 (hH1b vs. hH1, respectively)). SCN5A hH1b differs from SCN5A hH1a by just three amino acids in the cytoplasmic linker between DI-DII (R558 vs. H558, T559 vs. A559, and I618 vs. L618 (hH1b vs. hH1a respectively). The only two amino acid positions at which hH1b differs from both hH1 and hH1a are amino acids 558 and 618. Table 1 summarizes the differences among the three hNa v 1.5 clones and from a sequence found in the Celera® human genome database. BLAST searching of the Celera human genome sequence showed only 2 differences between hH1b and the Celera sequence (R558 vs. H558 and I618 vs. L618), both of which are in the cyotplasmic linker between DI-DII. FIG. 1 also depicts the set of relevant mutations in the various clones, as well as the M1766L mutation.  
                                                           TABLE 1                           Deduced amino acid sequence comparisons for SCN5A clones.            AA#   hH1   HH1a   hH1b   ® Celera                    558   H   H   R   H       559   T   A   T   T       618   L   L   I   L       1027   Q   R   R   R       1077   Q   .   .   .                  
 
         [0019]    The hH1, hH1a, and hH1b sodium channel α subunits exhibits similar activation, inactivation and recovery kinetics when expressed in a cell line and, accordingly, hH1b provides an additional target for diagnosing and treating sodium channel-related diseases or conditions or for determining an agent&#39;s effects on sodium ion channels. More interestingly, hH1b differs from hH1 and hH1a in that the sodium channel current of the hH1b form is not lost in the presence of the M1766L mutation, as it is in the case of the hH1 and hH1a forms of the SCN5A gene. Because M1766L is associated with long QT syndrome in subjects that carry the hH1a form, the present invention further enables a test for determining the form of SCN5A carried by a subject, thereby facilitating assessment of the subject&#39;s risk for developing long QT syndrome or other condition.  
         [0020]    The nucleic acid and amino acid sequences of hH1b disclosed herein enable the skilled artisan to produce diagnostic, prevention and treatment tools for sodium channel-related diseases including probes or hH1b specific antibodies for detecting hH1b expression in a biological sample.  
         [0021]    In one aspect, the present invention relates to an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2. An isolated polypeptide, as used herein, is one either synthetically derived or removed from its natural environment. An isolated polypeptide identical to SEQ ID NO: 2 carrying a substitution at one or more non-critical amino acid positions, where the substitution does not materially affect the function of the polypeptide. A non-critical amino acid position is an amino acid at a position other than those disclosed for hH1b in Table 1. Furthermore, an isolated polypeptide of the invention can also include any of the foregoing polypeptides having one or more amino acids at either or both of the N-terminus and C-terminus, where the additional amino acid(s) do not materially affect the function of the polypeptide. A change does not materially affect the function if the protein retains kinetic parameters within the ranges of those disclosed for hH1b in Table 2 and if the protein retains the ability to rescue the M1766L mutation, as described above. Any additional amino acids can, but need not, have advantageous use in purifying, detecting, or stabilizing the polypeptide. Likewise, small deletions or other rearrangements in the polypeptide that do not affect the function of the polypeptide are also within the scope of the invention. Such deletions are preferably deletions of fewer than 100 amino acids, more preferably of fewer than 50 amino acids, still more preferably of fewer than 10 amino acids.  
                                                                                                                                                 TABLE 2                           Kinetic parameters for 3 SCN5A clones.                hH1   hH1a   hH1b                        Activation                V 1/2  (mV)   −44 ± 5    −40 ± 7    −43 ± 6            Slope factor   5 ± 1   5.5 ± 0.6   5.5 ± 0.7           N   10   7    9            Inactivation                V 1/2  (mV)    −95 ± 4.8*    −86 ± 5.8*   −90 ± 8.8            N    9   7   10            Recovery                τ f  (ms)   8 ± 5   5 ± 3   5 ± 2           τ s  (ms)   215 ± 101   279 ± 312   111 ± 52            As   0.31 ± 0.1    0.25 ± 0.1    0.24 ± 0.1            N    8   6    9            Decay (−30 mV)                τ f  (ms)   1.2 ± 0.4   1.5 ± 0.5   1.3 ± 0.5           τ s  (ms)   6.4 ± 5     6.0 ± 3     4.0 ± 1             A f     0.71 ± 0.1    0.65 ± 0.1    0.56 ± 0.2            N    9   6    9                      
 
         [0022]    In a related aspect, the present invention also includes an immunogenic fragment of SEQ ID NO: 2 that contains at least one of amino acid 558 and amino acid 618 of SEQ ID NO: 2 and an antibody that binds specifically to such an immunogenic fragment. Such immunogenic fragments are used to generate an hH1b-specific antibody that can be used to detect or to isolate hH1b protein, or both. An hH1b-specific antibody has a higher affinity for hH1b protein than for either hH1- or hH1a protein. It is well within the ability of an artisan having ordinary skill in possession of the disclosed SEQ ID NO: 2 to make monoclonal or polyclonal antibodies against some or all of the polypeptide and to assess the specificity of the antibodies.  
         [0023]    In another aspect, the present invention relates to an isolated nucleic acid containing a polynucleotide having an uninterrupted sequence that encodes a polypeptide of the invention as set forth above. An “isolated nucleic acid” has structure not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA that has the sequence of part of a naturally occurring genomic DNA molecule but which is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of (i) DNA molecules, (ii) transfected cells, and (iii) cell clones, e.g., as these occur in a DNA library such as a cDNA or genomic DNA library. An isolated nucleic acid molecule can be modified or unmodified DNA or RNA, whether fully or partially single-stranded or double-stranded or even triple-stranded. A modified nucleic acid molecule can be chemically or enzymatically induced and can include so-called non-standard bases such as inosine.  
         [0024]    In a related aspect, any nucleic acid of the present invention described above can be provided in a vector in a manner known to those skilled in the art. The vector can be a cloning vector or an expression vector. In an expression vector, the polypeptide-encoding polynucleotide is under the transcriptional control of one or more non-native expression control sequences which can include a promoter not natively found adjacent to the polynucleotide such that the encoded polypeptide can be produced when the vector is provided in a compatible host cell or in a cell-free transcription and translation system. Such cell-based and cell-free systems are well known to the skilled artisan. Cells comprising a vector containing a nucleic acid of the invention are themselves within the scope of the present invention. Also within the scope of the present invention is a host cell having the nucleic acid of the present invention integrated into its genome at a non-native site.  
         [0025]    The present invention also includes an isolated nucleic acid molecule that contains a fragment of at least 20 contiguous nucleotides of SEQ ID NO: 1 or its complement wherein the fragment includes codon(s) encoding at least one of amino acids 558 and 618 and wherein the fragment hybridizes under stringent hybridization conditions to SEQ ID NO: 1 or its complement. Such a nucleic acid molecule can be used to detect the expression of hH1b in a cell. A stringent hybridization is, for example, hybridizing at 68° C. in 5×SSC/5×Denhardt&#39;s solution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature. Moderately stringent conditions include washing in 3×SSC at 42° C.  
         [0026]    The present invention also enables a screening method for agents that can either inhibit or enhance sodium channel activities. In such a method, an agent is exposed to a cell line that expresses a functional hH1b and the agent&#39;s effect on hH1b activity is determined. hH1b activity can be measured in many ways, including but not limited to measuring a sodium current across the cell membrane, a sodium current kinetic activity, a membrane potential, or an intracellular sodium level. Also, a phenotype associated with over-expression of a sodium channel or absence of expression (e.g., in a transgenic knockout animal) can be monitored. In vitro, an effect on action potential can be measured after a channel of interest is transfected into suitable cells, such as cardiac cells. An arsenal of agents affecting the sodium channel activity are desired because many diseases and conditions, such as arrhythmias and Brugada syndrome, result from elevated or reduced sodium channel activity. Particularly in view of the understanding that various forms of the sodium channel α subunit differ functionally, it is important to evaluate the effects of every form that may be present in an individual. Indeed, one can tailor a suitable treatment to an individual after evaluating the form of α subunit present in that individual. Sodium channel activity means the open channel activity leading to a peak sodium current. Sodium channel activity is enhanced or inhibited when the open state probability is greater or less, and the peak current is higher or lower, respectively, than in the absence of a modulating agent.  
         [0027]    Batteries of agents for screening are commercially available in the form of various chemical libraries including peptide libraries. Examples of such libraries include those from ASINEX (e.g., the Combined Wisdom Library of 24,000 manually synthesized organic molecules) and from CHEMBRIDGE CORPORATION (e.g., the DIVERSet™ library of 50,000 manually synthesized chemical compounds; the SCREEN-Set™ library of 24,000 manually synthesized chemical compounds; the CNS-Set™ library of 11,000 compounds; the Cherry-Pick™ library of up to 300,000 compounds). Once an agent having desired ability to increase or decrease activity of the sodium channel protein is identified, further iterations of the screen using one or more libraries of derivatives of that agent can be screened to identify agents having superior effects.  
         [0028]    The above screening methods also enable one to determine the likelihood that an agent intended to be administered to a human or non-human subject will induce an undesired and unintended side effect, namely by altering the activity of cellular hH1b in a subject in which such alteration is not indicated. Any product of the invention described herein can be combined with one or more other reagent, buffer or the like in the form of a kit useful, e.g., for diagnostic or therapeutic purposes, in accord with the understanding of a skilled artisan.  
         [0029]    The present invention is not intended to be limited to the foregoing, but rather to encompass all such variations and modifications as come within the scope of the appended claims. The invention will be more fully understood upon consideration of the following Examples which are, likewise, not intended to limit the scope of the invention.  
       EXAMPLES  
     Structural Differences Among Three Complete hNa v 1.5 Clones  
       [0030]    We generated a novel, complete hNa v 1.5-encoding clone (SCN5A form hH1b) from human cardiac mRNA (Clontech®, Palo Alto, Calif.) using RT-PCR. About 3 kb of 5′ Na v 1.5 gene was cloned using 5′-GATGAGAAGATGGCAAACTTC C-3′ (SEQ ID NO: 3) and 5′-GCTCTGGATCCCCGGGGGTGCC-3′(SEQ ID NO: 4) primers. The 3 kb of 3′ Na v 1.5 gene was cloned with 5′-CACCCCCGGGGATCCAGAGC-3′ (SEQ ID NO: 5) and 5′-TTCAGTGTGTCCTGGCCAG-3′ (SEQ ID NO: 6) primers. RETRscript (Ambion®, Austin, Tex.) and pfu DNA polymerase were used to perform RT-PCR according to the protocol recommended by the manufacturer. PCR thermocycling involved one denaturation cycle at 94° C. for 1 min, 35 amplification cycles at 94° C. for 1 min, 50° C. for 1 min and 72° C. for 8 min, and one extension cycle at 72° C. for 20 min. The PCR products were cloned into pCR-BluntII-TOPO vector (Invitrogen®, Carlsbad, Calif.). The sequence of the hH1b gene was determined using thermostable polymerases and fluorescently labeled dideoxy terminators and automated DNA sequence analyzers at the University of Wisconsin Biotechnology Center. The two known hNa v 1.5-encoding clones (SCN5A forms hH1 and hH1a) were also re-sequenced completely.  
       Kinetic Studies of the Three Complete hNa v 1.5 Clones  
       [0031]    We measured macroscopic !Na of the three clones concurrently (on the same day) under identical conditions in the same experimental whole cell patch-clamp set-up. All techniques, protocols, and analysis techniques are standard and have been previously published (Nagatomo, et al., “Temperature dependence of early and late currents in human cardiac wild-type and long QT Δ KPQ Na +  channels,”  Am.J.Physiol . (Heart44) 275, H2016-H2024 (1998), incorporated by reference herein as if set forth in its entirety). The pipette solution contained (in mM) 120 CsF, 15 CsCl, 2 EGTA, 5 HEPES and 5 NaCl (pH7.4 with CsOH). Data were recorded at room temperature. Peak I Na  and late I Na  were obtained after passive leak subtraction as described previously (Nagatomo, supra). Activation and inactivation data were fitted to a standard Boltzmann equation. Recovery and decay data were fitted to a two-exponential equation. Goodness of fit was determined both visually and by a sum of squares errors. One way ANOVA was performed to determine statistical significance among the three groups of mean data. Statistical significance was determined by a P value &lt;0.05.  
         [0032]    The 3 Na v 1.5 clones were introduced by transfection into the HEK293 host cell line (commercially available from ATCC) using Superfect (Qiagen®) according to the protocol recommended by the manufacturer. A Green Fluorescent Protein was co-transfected (at 1:10) as a marker to identify transfected cells. The vectors transfected included SCN5A form hH1 cloned into prcCMV vector (Invitrogen®, Carlsbad, Calif.), and forms hH1a and hH1b separately cloned into pcDNA3 vector (Invitrogen). HEK293 cells were cultured as previously described (Nagatomo et. al. 1998). The HEK 293 host cells were harvested 24 hours later to measure macroscopic I Na  current.  
         [0033]    The three clones generally showed similar current time courses (FIG. 2 a : representative traces were recorded with test potentials of −120 mV to +60 mV from a holding potential of −120 mV). Summary data for activation, steady-state inactivation, and recovery show minor differences (FIG. 2 b  Current-voltage relationship (left), “steady state” inactivation relationship (middle) and recovery from inactivation relationship (right) for I Na : the y-axis represent normalized current; diagrams depicting the voltage clamp protocols are inset into each plot; peak I Na  was measured in response to the last depolarizing step and it was then normalized to the maximal peak I Na  found; solid symbols represent the mean data for between 6 to 10 experiments (see Table 2 for exact n numbers) with hH1 (square), hH1a (circle), and hH1b (triangle), and the bars represent SD). Although not identical, the kinetic parameters determined by the fitted lines (Table 2) were only statistically different for the midpoint of inactivation for hH1 (−95 mV) versus hH1a (−86 mV). Late I Na  measured at 240 ms after the start of the depolarization was no different among the three clones.  
         [0034]    The three SCN5A clones, hH1, hH1a, and hH1b show minor differences in activation, inactivation, and recovery from inactivation kinetics. These parameters were obtained from fitting the individual experiments as in FIG. 2 to the appropriate model equations (Nagatomo, T. et al. (1998)). The fitted kinetic parameters from n experiments were averaged and are reported ±SD. For the Boltzmann fits (activation and inactivation), the parameters are V 1/2  midpoint and slope factor. For the exponential fits (recovery and current decay), the parameters are τ f —the fast time constant, τ s —the slow time constant, A s —the fractional amplitude of slow component, and A f —the fractional amplitude of fast component. All parameters were analyzed by one-way ANOVA across the 3 clones, and statistically significant values are marked with an asterisk.  
       Rescue of Expression for the M1766L Mutant by Using the hH1b Background  
       [0035]    By site-directed mutagenesis, the M1766L arrhythmia mutation was engineered into all three clones, expressed in HEK cells, and studied by voltage clamp. Mutations were generated using the Excite® mutagenesis kit (Stratagene®). The mutagenesis method was based on the protocol recommended by the manufacturer. The M1766L mutation was created with 5′-TTCCTCATCGTGGTTAACCTGTACATTGCCATC-3 ′ (SEQ ID NO: 7) and 5′-GGAGATGATGATGTAGGTGG-3′ (SEQ ID NO: 8) primers. The histamine-to-arginine change at position 558 was generated with 5′-CGAGAGCCACCGCGCATCACTGCTG-3′ (SEQ ID NO: 9) and 5° CTCTCCCCCGCTGTGCTGTTTTC-3′ (SEQ ID NO: 10) primers. DNA was isolated and purified using a Qiagen® (Hilden, Germany) column and protocol.  
         [0036]    Examples Of I Na  traces show that the amount of current for the M1766L mutant channel was greatly reduced compared to wild type when expressed in hH1 and hH1a, but the current level was surprisingly large when expressed in hH1b (FIG. 3: Examples of I Na  traces (protocol inset) for each construct show that M1766L expressed very poorly in hH1 and hH1a, but expressed normally in hH1b; expression levels for the double mutant H558R/M1766L in hH1a were normal). Summary data of FIG. 4 show that for wild type, hH1b and hH1a clones generally expressed more I Na  than the hH1 clone. For wild type, but not mutant channels, the data here represent an underestimate of true current density because larger current densities were discarded to assure better voltage control for kinetic measurement. In general, however, wild type hH1 expressed at lower levels than hH1a and hH1b. Currents for M1766L in hH1b were dramatically greater than those for the mutant in hH1 and hH1a and were not affected by selection. I Na  were normalized to cell capacitance and mean values shown as a column with SD bar and the number of experiments indicated next to the bar. Without intending to be limited as to the theory underlying the invention, the applicants speculate that the H558R polymorphism underlies the restoration of expression in sodium channels carrying the M1766L mutation. Again using site-directed mutagenesis, we engineered the double mutation H558R/M1766L in the hH1a clone. Now, rather than the 97% reduction in current expression observed previously for M1766L in hH1a, the H558R/M1766L-hH1a mutant manifested a fully restored I Na  density (FIGS. 3 and 4).  
     
       
       
         1 
         
           
             10  
           
           
             1  
             6091  
             DNA  
             Homo sapiens  
             
               CDS  
               (10)..(6054)  
             
           
            1 

gatgagaag atg gca aac ttc cta tta cct cgg ggc acc agc agc ttc cgc     51 
          Met Ala Asn Phe Leu Leu Pro Arg Gly Thr Ser Ser Phe Arg 
            1               5                  10 

agg ttc aca cgg gag tcc ctg gca gcc atc gag aag cgc atg gcg gag       99 
Arg Phe Thr Arg Glu Ser Leu Ala Ala Ile Glu Lys Arg Met Ala Glu 
 15                  20                  25                  30 

aag caa gcc cgc ggc tca acc acc ttg cag gag agc cga gag ggg ctg      147 
Lys Gln Ala Arg Gly Ser Thr Thr Leu Gln Glu Ser Arg Glu Gly Leu 
                 35                  40                  45 

ccc gag gag gag gct ccc cgg ccc cag ctg gac ctg cag gcc tcc aaa      195 
Pro Glu Glu Glu Ala Pro Arg Pro Gln Leu Asp Leu Gln Ala Ser Lys 
             50                  55                  60 

aag ctg cca gat ctc tat ggc aat cca ccc caa gag ctc atc gga gag      243 
Lys Leu Pro Asp Leu Tyr Gly Asn Pro Pro Gln Glu Leu Ile Gly Glu 
         65                  70                  75 

ccc ctg gag gac ctg gac ccc ttc tat agc acc caa aag act ttc atc      291 
Pro Leu Glu Asp Leu Asp Pro Phe Tyr Ser Thr Gln Lys Thr Phe Ile 
     80                  85                  90 

gta ctg aat aaa ggc aag acc atc ttc cgg ttc agt gcc acc aac gcc      339 
Val Leu Asn Lys Gly Lys Thr Ile Phe Arg Phe Ser Ala Thr Asn Ala 
 95                 100                 105                 110 

ttg tat gtc ctc agt ccc ttc cac ccc atc cgg aga gcg gct gtg aag      387 
Leu Tyr Val Leu Ser Pro Phe His Pro Ile Arg Arg Ala Ala Val Lys 
                115                 120                 125 

att ctg gtt cac tcg ctc ttc aac atg ctc atc atg tgc acc atc ctc      435 
Ile Leu Val His Ser Leu Phe Asn Met Leu Ile Met Cys Thr Ile Leu 
            130                 135                 140 

acc aac tgc gtg ttc atg gcc cag cac gac cct cca ccc tgg acc aag      483 
Thr Asn Cys Val Phe Met Ala Gln His Asp Pro Pro Pro Trp Thr Lys 
        145                 150                 155 

tat gtc gag tac acc ttc acc gcc att tac acc ttt gag tct ctg gtc      531 
Tyr Val Glu Tyr Thr Phe Thr Ala Ile Tyr Thr Phe Glu Ser Leu Val 
    160                 165                 170 

aag att ctg gct cga ggc ttc tgc ctg cac gcg ttc act ttc ctt cgg      579 
Lys Ile Leu Ala Arg Gly Phe Cys Leu His Ala Phe Thr Phe Leu Arg 
175                 180                 185                 190 

gac cca tgg aac tgg ctg gac ttt agt gtg att atc atg gca tac aca      627 
Asp Pro Trp Asn Trp Leu Asp Phe Ser Val Ile Ile Met Ala Tyr Thr 
                195                 200                 205 

act gaa ttt gtg gac ctg ggc aat gtc tca gcc tta cgc acc ttc cga      675 
Thr Glu Phe Val Asp Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg 
            210                 215                 220 

gtc ctc cgg gcc ctg aaa act ata tca gtc att tca ggg ctg aag acc      723 
Val Leu Arg Ala Leu Lys Thr Ile Ser Val Ile Ser Gly Leu Lys Thr 
        225                 230                 235 

atc gtg ggg gcc ctg atc cag tct gtg aag aag ctg gct gat gtg atg      771 
Ile Val Gly Ala Leu Ile Gln Ser Val Lys Lys Leu Ala Asp Val Met 
    240                 245                 250 

gtc ctc aca gtc ttc tgc ctc agc gtc ttt gcc ctc atc ggc ctg cag      819 
Val Leu Thr Val Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln 
255                 260                 265                 270 

ctc ttc atg ggc aac cta agg cac aag tgc gtg cgc aac ttc aca gcg      867 
Leu Phe Met Gly Asn Leu Arg His Lys Cys Val Arg Asn Phe Thr Ala 
                275                 280                 285 

ctc aac ggc acc aac ggc tcc gtg gag gcc gac ggc ttg gtc tgg gaa      915 
Leu Asn Gly Thr Asn Gly Ser Val Glu Ala Asp Gly Leu Val Trp Glu 
            290                 295                 300 

tcc ctg gac ctt tac ctc agt gat cca gaa aat tac ctg ctc aag aac      963 
Ser Leu Asp Leu Tyr Leu Ser Asp Pro Glu Asn Tyr Leu Leu Lys Asn 
        305                 310                 315 

ggc acc tct gat gtg tta ctg tgt ggg aac agc tct gac gct ggg aca     1011 
Gly Thr Ser Asp Val Leu Leu Cys Gly Asn Ser Ser Asp Ala Gly Thr 
    320                 325                 330 

tgt ccg gag ggc tac cgg tgc cta aag gca ggc gag aac ccc gac cac     1059 
Cys Pro Glu Gly Tyr Arg Cys Leu Lys Ala Gly Glu Asn Pro Asp His 
335                 340                 345                 350 

ggc tac acc agc ttc gat tcc ttt gcc tgg gcc ttt ctt gca ctc ttc     1107 
Gly Tyr Thr Ser Phe Asp Ser Phe Ala Trp Ala Phe Leu Ala Leu Phe 
                355                 360                 365 

cgc ctg atg acg cag gac tgc tgg gag cgc ctc tat cag cag acc ctc     1155 
Arg Leu Met Thr Gln Asp Cys Trp Glu Arg Leu Tyr Gln Gln Thr Leu 
            370                 375                 380 

agg tcc gca ggg aag atc tac atg atc ttc ttc atg ctt gtc atc ttc     1203 
Arg Ser Ala Gly Lys Ile Tyr Met Ile Phe Phe Met Leu Val Ile Phe 
        385                 390                 395 

ctg ggg tcc ttc tac ctg gtg aac ctg atc ctg gcc gtg gtc gca atg     1251 
Leu Gly Ser Phe Tyr Leu Val Asn Leu Ile Leu Ala Val Val Ala Met 
    400                 405                 410 

gcc tat gag gag caa aac caa gcc acc atc gct gag acc gag gag aag     1299 
Ala Tyr Glu Glu Gln Asn Gln Ala Thr Ile Ala Glu Thr Glu Glu Lys 
415                 420                 425                 430 

gaa aag cgc ttc cag gag gcc atg gaa atg ctc aag aaa gaa cac gag     1347 
Glu Lys Arg Phe Gln Glu Ala Met Glu Met Leu Lys Lys Glu His Glu 
                435                 440                 445 

gcc ctc acc atc agg ggt gtg gat acc gtg tcc cgt agc tcc ttg gag     1395 
Ala Leu Thr Ile Arg Gly Val Asp Thr Val Ser Arg Ser Ser Leu Glu 
            450                 455                 460 

atg tcc cct ttg gcc cca gta aac agc cat gag aga aga agc aag agg     1443 
Met Ser Pro Leu Ala Pro Val Asn Ser His Glu Arg Arg Ser Lys Arg 
        465                 470                 475 

aga aaa cgg atg tct tca gga act gag gag tgt ggg gag gac agg ctc     1491 
Arg Lys Arg Met Ser Ser Gly Thr Glu Glu Cys Gly Glu Asp Arg Leu 
    480                 485                 490 

ccc aag tct gac tca gaa gat ggt ccc aga gca atg aat cat ctc agc     1539 
Pro Lys Ser Asp Ser Glu Asp Gly Pro Arg Ala Met Asn His Leu Ser 
495                 500                 505                 510 

ctc acc cgt ggc ctc agc agg act tct atg aag cca cgt tcc agc cgc     1587 
Leu Thr Arg Gly Leu Ser Arg Thr Ser Met Lys Pro Arg Ser Ser Arg 
                515                 520                 525 

ggg agc att ttc acc ttt cgc agg cga gac ctg ggt tct gaa gca gat     1635 
Gly Ser Ile Phe Thr Phe Arg Arg Arg Asp Leu Gly Ser Glu Ala Asp 
            530                 535                 540 

ttt gca gat gat gaa aac agc aca gcg ggg gag agc gag agc cac cgc     1683 
Phe Ala Asp Asp Glu Asn Ser Thr Ala Gly Glu Ser Glu Ser His Arg 
        545                 550                 555 

aca tca ctg ctg gtg ccc tgg ccc ctg cgc cgg acc agt gcc cag gga     1731 
Thr Ser Leu Leu Val Pro Trp Pro Leu Arg Arg Thr Ser Ala Gln Gly 
    560                 565                 570 

cag ccc agt ccc gga acc tcg gct cct ggc cac gcc ctc cat ggc aaa     1779 
Gln Pro Ser Pro Gly Thr Ser Ala Pro Gly His Ala Leu His Gly Lys 
575                 580                 585                 590 

aag aac agc act gtg gac tgc aat ggg gtg gtc tca tta ctg ggg gca     1827 
Lys Asn Ser Thr Val Asp Cys Asn Gly Val Val Ser Leu Leu Gly Ala 
                595                 600                 605 

ggc gac cca gag gcc aca tcc cca gga agc cac atc ctc cgc cct gtg     1875 
Gly Asp Pro Glu Ala Thr Ser Pro Gly Ser His Ile Leu Arg Pro Val 
            610                 615                 620 

atg cta gag cac ccg cca gac acg acc acg cca tcg gag gag cca ggc     1923 
Met Leu Glu His Pro Pro Asp Thr Thr Thr Pro Ser Glu Glu Pro Gly 
        625                 630                 635 

ggg ccc cag atg ctg acc tcc cag gct ccg tgt gta gat ggc ttc gag     1971 
Gly Pro Gln Met Leu Thr Ser Gln Ala Pro Cys Val Asp Gly Phe Glu 
    640                 645                 650 

gag cca gga gca cgg cag cgg gcc ctc agc gca gtc agc gtc ctc acc     2019 
Glu Pro Gly Ala Arg Gln Arg Ala Leu Ser Ala Val Ser Val Leu Thr 
655                 660                 665                 670 

agc gca ctg gaa gag tta gag gag tct cgc cac aag tgt cca cca tgc     2067 
Ser Ala Leu Glu Glu Leu Glu Glu Ser Arg His Lys Cys Pro Pro Cys 
                675                 680                 685 

tgg aac cgt ctc gcc cag cgc tac ctg atc tgg gag tgc tgc ccg ctg     2115 
Trp Asn Arg Leu Ala Gln Arg Tyr Leu Ile Trp Glu Cys Cys Pro Leu 
            690                 695                 700 

tgg atg tcc atc aag cag gga gtg aag ttg gtg gtc atg gac ccg ttt     2163 
Trp Met Ser Ile Lys Gln Gly Val Lys Leu Val Val Met Asp Pro Phe 
        705                 710                 715 

act gac ctc acc atc act atg tgc atc gta ctc aac aca ctc ttc atg     2211 
Thr Asp Leu Thr Ile Thr Met Cys Ile Val Leu Asn Thr Leu Phe Met 
    720                 725                 730 

gcg ctg gag cac tac aac atg aca agt gaa ttc gag gag atg ctg cag     2259 
Ala Leu Glu His Tyr Asn Met Thr Ser Glu Phe Glu Glu Met Leu Gln 
735                 740                 745                 750 

gtc gga aac ctg gtc ttc aca ggg att ttc aca gca gag atg acc ttc     2307 
Val Gly Asn Leu Val Phe Thr Gly Ile Phe Thr Ala Glu Met Thr Phe 
                755                 760                 765 

aag atc att gcc ctc gac ccc tac tac tac ttc caa cag ggc tgg aac     2355 
Lys Ile Ile Ala Leu Asp Pro Tyr Tyr Tyr Phe Gln Gln Gly Trp Asn 
            770                 775                 780 

atc ttc gac agc atc atc gtc atc ctt agc ctc atg gag ctg ggc ctg     2403 
Ile Phe Asp Ser Ile Ile Val Ile Leu Ser Leu Met Glu Leu Gly Leu 
        785                 790                 795 

tcc cgc atg agc aac ttg tcg gtg ctg cgc tcc ttc cgc ctg ctg cgg     2451 
Ser Arg Met Ser Asn Leu Ser Val Leu Arg Ser Phe Arg Leu Leu Arg 
    800                 805                 810 

gtc ttc aag ctg gcc aaa tca tgg ccc acc ctg aac aca ctc atc aag     2499 
Val Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Thr Leu Ile Lys 
815                 820                 825                 830 

atc atc ggg aac tca gtg ggg gca ctg ggg aac ctg aca ctg gtg cta     2547 
Ile Ile Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Leu Val Leu 
                835                 840                 845 

gcc atc atc gtg ttc atc ttt gct gtg gtg ggc atg cag ctc ttt ggc     2595 
Ala Ile Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe Gly 
            850                 855                 860 

aag aac tac tcg gag ctg agg gac agc gac tca ggc ctg ctg cct cgc     2643 
Lys Asn Tyr Ser Glu Leu Arg Asp Ser Asp Ser Gly Leu Leu Pro Arg 
        865                 870                 875 

tgg cac atg atg gac ttc ttt cat gcc ttc ctc atc atc ttc cgc atc     2691 
Trp His Met Met Asp Phe Phe His Ala Phe Leu Ile Ile Phe Arg Ile 
    880                 885                 890 

ctc tgt gga gag tgg atc gag acc atg tgg gac tgc atg gag gtg tcg     2739 
Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met Glu Val Ser 
895                 900                 905                 910 

ggg cag tca tta tgc ctg ctg gtc ttc ttg ctt gtt atg gtc att ggc     2787 
Gly Gln Ser Leu Cys Leu Leu Val Phe Leu Leu Val Met Val Ile Gly 
                915                 920                 925 

aac ctt gtg gtc ctg aat ctc ttc ctg gcc ttg ctg ctc agc tcc ttc     2835 
Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe 
            930                 935                 940 

agt gca gac aac ctc aca gcc cct gat gag gac aga gag atg aac aac     2883 
Ser Ala Asp Asn Leu Thr Ala Pro Asp Glu Asp Arg Glu Met Asn Asn 
        945                 950                 955 

ctc cag ctg gcc ctg gcc cgc atc cag agg ggc ctg cgc ttt gtc aag     2931 
Leu Gln Leu Ala Leu Ala Arg Ile Gln Arg Gly Leu Arg Phe Val Lys 
    960                 965                 970 

cgg acc acc tgg gat ttc tgc tgt ggt ctc ctg cgg cag cgg cct cag     2979 
Arg Thr Thr Trp Asp Phe Cys Cys Gly Leu Leu Arg Gln Arg Pro Gln 
975                 980                 985                 990 

aag ccc gca gcc ctt gcc gcc cag ggc cag ctg ccc agc tgc att gcc     3027 
Lys Pro Ala Ala Leu Ala Ala Gln Gly Gln Leu Pro Ser Cys Ile Ala 
                995                1000                1005 

acc ccc tac tcc ccg cca ccc cca gag acg gag aag gtg cct ccc acc     3075 
Thr Pro Tyr Ser Pro Pro Pro Pro Glu Thr Glu Lys Val Pro Pro Thr 
           1010                1015                1020 

cgc aag gaa aca cgg ttt gag gaa ggc gag caa cca ggc cag ggc acc     3123 
Arg Lys Glu Thr Arg Phe Glu Glu Gly Glu Gln Pro Gly Gln Gly Thr 
       1025                1030                1035 

ccc ggg gat cca gag ccc gtg tgt gtg ccc atc gct gtg gcc gag tca     3171 
Pro Gly Asp Pro Glu Pro Val Cys Val Pro Ile Ala Val Ala Glu Ser 
   1040                1045                1050 

gac aca gat gac caa gaa gaa gat gag gag aac agc ctg ggc acg gag     3219 
Asp Thr Asp Asp Gln Glu Glu Asp Glu Glu Asn Ser Leu Gly Thr Glu 
1055               1060                1065                1070 

gag gag tcc agc aag cag gaa tcc cag cct gtg tcc ggt ggc cca gag     3267 
Glu Glu Ser Ser Lys Gln Glu Ser Gln Pro Val Ser Gly Gly Pro Glu 
               1075                1080                1085 

gcc cct ccg gat tcc agg acc tgg agc cag gtg tca gcg act gcc tcc     3315 
Ala Pro Pro Asp Ser Arg Thr Trp Ser Gln Val Ser Ala Thr Ala Ser 
           1090                1095                1100 

tct gag gcc gag gcc agt gca tct cag gcc gac tgg cgg cag cag tgg     3363 
Ser Glu Ala Glu Ala Ser Ala Ser Gln Ala Asp Trp Arg Gln Gln Trp 
       1105                1110                1115 

aaa gcg gaa ccc cag gcc cca ggg tgc ggt gag acc cca gag gac agt     3411 
Lys Ala Glu Pro Gln Ala Pro Gly Cys Gly Glu Thr Pro Glu Asp Ser 
   1120                1125                1130 

tgc tcc gag ggc agc aca gca gac atg acc aac acc gct gag ctc ctg     3459 
Cys Ser Glu Gly Ser Thr Ala Asp Met Thr Asn Thr Ala Glu Leu Leu 
1135               1140                1145                1150 

gag cag atc cct gac ctc ggc cag gat gtc aag gac cca gag gac tgc     3507 
Glu Gln Ile Pro Asp Leu Gly Gln Asp Val Lys Asp Pro Glu Asp Cys 
               1155                1160                1165 

ttc act gaa ggc tgt gtc cgg cgc tgt ccc tgc tgt gcg gtg gac acc     3555 
Phe Thr Glu Gly Cys Val Arg Arg Cys Pro Cys Cys Ala Val Asp Thr 
           1170                1175                1180 

aca cag gcc cca ggg aag gtc tgg tgg cgg ttg cgc aag acc tgc tac     3603 
Thr Gln Ala Pro Gly Lys Val Trp Trp Arg Leu Arg Lys Thr Cys Tyr 
       1185                1190                1195 

cac atc gtg gag cac agc tgg ttc gag aca ttc atc atc ttc atg atc     3651 
His Ile Val Glu His Ser Trp Phe Glu Thr Phe Ile Ile Phe Met Ile 
   1200                1205                1210 

cta ctc agc agt gga gcg ctg gcc ttc gag gac atc tac cta gag gag     3699 
Leu Leu Ser Ser Gly Ala Leu Ala Phe Glu Asp Ile Tyr Leu Glu Glu 
1215               1220                1225                1230 

cgg aag acc atc aag gtt ctg ctt gag tat gcc gac aag atg ttc aca     3747 
Arg Lys Thr Ile Lys Val Leu Leu Glu Tyr Ala Asp Lys Met Phe Thr 
               1235                1240                1245 

tat gtc ttc gtg ctg gag atg ctg ctc aag tgg gtg gcc tac ggc ttc     3795 
Tyr Val Phe Val Leu Glu Met Leu Leu Lys Trp Val Ala Tyr Gly Phe 
           1250                1255                1260 

aag aag tac ttc acc aat gcc tgg tgc tgg ctc gac ttc ctc atc gta     3843 
Lys Lys Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu Ile Val 
       1265                1270                1275 

gac gtc tct ctg gtc agc ctg gtg gcc aac acc ctg ggc ttt gcc gag     3891 
Asp Val Ser Leu Val Ser Leu Val Ala Asn Thr Leu Gly Phe Ala Glu 
   1280                1285                1290 

atg ggc ccc atc aag tca ctg cgg acg ctg cgt gca ctc cgt cct ctg     3939 
Met Gly Pro Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu 
1295               1300                1305                1310 

aga gct ctg tca cga ttt gag ggc atg agg gtg gtg gtc aat gcc ctg     3987 
Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn Ala Leu 
               1315                1320                1325 

gtg ggc gcc atc ccg tcc atc atg aac gtc ctc ctc gtc tgc ctc atc     4035 
Val Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val Cys Leu Ile 
           1330                1335                1340 

ttc tgg ctc atc ttc agc atc atg ggc gtg aac ctc ttt gcg ggg aag     4083 
Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu Phe Ala Gly Lys 
       1345                1350                1355 

ttt ggg agg tgc atc aac cag aca gag gga gac ttg cct ttg aac tac     4131 
Phe Gly Arg Cys Ile Asn Gln Thr Glu Gly Asp Leu Pro Leu Asn Tyr 
   1360                1365                1370 

acc atc gtg aac aac aag agc cag tgt gag tcc ttg aac ttg acc gga     4179 
Thr Ile Val Asn Asn Lys Ser Gln Cys Glu Ser Leu Asn Leu Thr Gly 
1375               1380                1385                1390 

gaa ttg tac tgg acc aag gtg aaa gtc aac ttt gac aac gtg ggg gcc     4227 
Glu Leu Tyr Trp Thr Lys Val Lys Val Asn Phe Asp Asn Val Gly Ala 
               1395                1400                1405 

ggg tac ctg gcc ctt ctg cag gtg gca aca ttt aaa ggc tgg atg gac     4275 
Gly Tyr Leu Ala Leu Leu Gln Val Ala Thr Phe Lys Gly Trp Met Asp 
           1410                1415                1420 

att atg tat gca gct gtg gac tcc agg ggg tat gaa gag cag cct cag     4323 
Ile Met Tyr Ala Ala Val Asp Ser Arg Gly Tyr Glu Glu Gln Pro Gln 
       1425                1430                1435 

tgg gaa tac aac ctc tac atg tac atc tat ttt gtc att ttc atc atc     4371 
Trp Glu Tyr Asn Leu Tyr Met Tyr Ile Tyr Phe Val Ile Phe Ile Ile 
   1440                1445                1450 

ttt ggg tct ttc ttc acc ctg aac ctc ttt att ggt gtc atc att gac     4419 
Phe Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp 
1455               1460                1465                1470 

aac ttc aac caa cag aag aaa aag tta ggg ggc cag gac atc ttc atg     4467 
Asn Phe Asn Gln Gln Lys Lys Lys Leu Gly Gly Gln Asp Ile Phe Met 
               1475                1480                1485 

aca gag gag cag aag aag tac tac aat gcc atg aag aag ctg ggc tcc     4515 
Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly Ser 
           1490                1495                1500 

aag aag ccc cag aag ccc atc cca cgg ccc ctg aac aag tac cag ggc     4563 
Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Leu Asn Lys Tyr Gln Gly 
       1505                1510                1515 

ttc ata ttc gac att gtg acc aag cag gcc ttt gac gtc acc atc atg     4611 
Phe Ile Phe Asp Ile Val Thr Lys Gln Ala Phe Asp Val Thr Ile Met 
   1520                1525                1530 

ttt ctg atc tgc ttg aat atg gtg acc atg atg gtg gag aca gat gac     4659 
Phe Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Thr Asp Asp 
1535               1540                1545                1550 

caa agt cct gag aaa atc aac atc ttg gcc aag atc aac ctg ctc ttt     4707 
Gln Ser Pro Glu Lys Ile Asn Ile Leu Ala Lys Ile Asn Leu Leu Phe 
               1555                1560                1565 

gtg gcc atc ttc aca ggc gag tgt att gtc aag ctg gct gcc ctg cgc     4755 
Val Ala Ile Phe Thr Gly Glu Cys Ile Val Lys Leu Ala Ala Leu Arg 
           1570                1575                1580 

cac tac tac ttc acc aac agc tgg aat atc ttc gac ttc gtg gtt gtc     4803 
His Tyr Tyr Phe Thr Asn Ser Trp Asn Ile Phe Asp Phe Val Val Val 
       1585                1590                1595 

atc ctc tcc atc gtg ggc act gtg ctc tcg gac atc atc cag aag tac     4851 
Ile Leu Ser Ile Val Gly Thr Val Leu Ser Asp Ile Ile Gln Lys Tyr 
   1600                1605                1610 

ttc ttc tcc ccg acg ctc ttc cga gtc atc cgc ctg gcc cga ata ggc     4899 
Phe Phe Ser Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg Ile Gly 
1615               1620                1625                1630 

cgc atc ctc aga ctg atc cga ggg gcc aag ggg atc cgc acg ctg ctc     4947 
Arg Ile Leu Arg Leu Ile Arg Gly Ala Lys Gly Ile Arg Thr Leu Leu 
               1635                1640                1645 

ttt gcc ctc atg atg tcc ctg cct gcc ctc ttc aac atc ggg ctg ctg     4995 
Phe Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly Leu Leu 
           1650                1655                1660 

ctc ttc ctc gtc atg ttc atc tac tcc atc ttt ggc atg gcc aac ttc     5043 
Leu Phe Leu Val Met Phe Ile Tyr Ser Ile Phe Gly Met Ala Asn Phe 
       1665                1670                1675 

gct tat gtc aag tgg gag gct ggc atc gac gac atg ttc aac ttc cag     5091 
Ala Tyr Val Lys Trp Glu Ala Gly Ile Asp Asp Met Phe Asn Phe Gln 
   1680                1685                1690 

acc ttc gcc aac agc atg ctg tgc ctc ttc cag atc acc acg tcg gcc     5139 
Thr Phe Ala Asn Ser Met Leu Cys Leu Phe Gln Ile Thr Thr Ser Ala 
1695               1700                1705                1710 

ggc tgg gat ggc ctc ctc agc ccc atc ctc aac act ggg ccg ccc tac     5187 
Gly Trp Asp Gly Leu Leu Ser Pro Ile Leu Asn Thr Gly Pro Pro Tyr 
               1715                1720                1725 

tgc gac ccc act ctg ccc aac agc aat ggc tct cgg ggg gac tgc ggg     5235 
Cys Asp Pro Thr Leu Pro Asn Ser Asn Gly Ser Arg Gly Asp Cys Gly 
           1730                1735                1740 

agc cca gcc gtg ggc atc ctc ttc ttc acc acc tac atc atc atc tcc     5283 
Ser Pro Ala Val Gly Ile Leu Phe Phe Thr Thr Tyr Ile Ile Ile Ser 
       1745                1750                1755 

ttc ctc atc gtg gtc aac atg tac att gcc atc atc ctg gag aac ttc     5331 
Phe Leu Ile Val Val Asn Met Tyr Ile Ala Ile Ile Leu Glu Asn Phe 
   1760                1765                1770 

agc gtg gcc acg gag gag agc acc gag ccc ctg agt gag gac gac ttc     5379 
Ser Val Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu Asp Asp Phe 
1775               1780                1785                1790 

gat atg ttc tat gag atc tgg gag aaa ttt gac cca gag gcc act cag     5427 
Asp Met Phe Tyr Glu Ile Trp Glu Lys Phe Asp Pro Glu Ala Thr Gln 
               1795                1800                1805 

ttt att gag tat tcg gtc ctg tct gac ttt gcc gat gcc ctg tct gag     5475 
Phe Ile Glu Tyr Ser Val Leu Ser Asp Phe Ala Asp Ala Leu Ser Glu 
           1810                1815                1820 

cca ctc cgt atc gcc aag ccc aac cag ata agc ctc atc aac atg gac     5523 
Pro Leu Arg Ile Ala Lys Pro Asn Gln Ile Ser Leu Ile Asn Met Asp 
       1825                1830                1835 

ctg ccc atg gtg agt ggg gac cgc atc cat tgc atg gac att ctc ttt     5571 
Leu Pro Met Val Ser Gly Asp Arg Ile His Cys Met Asp Ile Leu Phe 
   1840                1845                1850 

gcc ttc acc aaa agg gtc ctg ggg gag tct ggg gag atg gac gcc ctg     5619 
Ala Phe Thr Lys Arg Val Leu Gly Glu Ser Gly Glu Met Asp Ala Leu 
1855               1860                1865                1870 

aag atc cag atg gag gag aag ttc atg gca gcc aac cca tcc aag atc     5667 
Lys Ile Gln Met Glu Glu Lys Phe Met Ala Ala Asn Pro Ser Lys Ile 
               1875                1880                1885 

tcc tac gag ccc atc acc acc aca ctc cgg cgc aag cac gaa gag gtg     5715 
Ser Tyr Glu Pro Ile Thr Thr Thr Leu Arg Arg Lys His Glu Glu Val 
           1890                1895                1900 

tcg gcc atg gtt atc cag aga gcc ttc cgc agg cac ctg ctg caa cgc     5763 
Ser Ala Met Val Ile Gln Arg Ala Phe Arg Arg His Leu Leu Gln Arg 
       1905                1910                1915 

tct ttg aag cat gcc tcc ttc ctc ttc cgt cag cag gcg ggc agc ggc     5811 
Ser Leu Lys His Ala Ser Phe Leu Phe Arg Gln Gln Ala Gly Ser Gly 
   1920                1925                1930 

ctc tcc gaa gag gat gcc cct gag cga gag ggc ctc atc gcc tac gtg     5859 
Leu Ser Glu Glu Asp Ala Pro Glu Arg Glu Gly Leu Ile Ala Tyr Val 
1935               1940                1945                1950 

atg agt gag aac ttc tcc cga ccc ctt ggc cca ccc tcc agc tcc tcc     5907 
Met Ser Glu Asn Phe Ser Arg Pro Leu Gly Pro Pro Ser Ser Ser Ser 
               1955                1960                1965 

atc tcc tcc act tcc ttc cca ccc tcc tat gac agt gtc act aga gcc     5955 
Ile Ser Ser Thr Ser Phe Pro Pro Ser Tyr Asp Ser Val Thr Arg Ala 
           1970                1975                1980 

acc agc gat aac ctc cag gtg cgg ggg tct gac tac agc cac agt gaa     6003 
Thr Ser Asp Asn Leu Gln Val Arg Gly Ser Asp Tyr Ser His Ser Glu 
       1985                1990                1995 

gat ctc gcc gac ttc ccc cct tct ccg gac agg gac cgt gag tcc atc     6051 
Asp Leu Ala Asp Phe Pro Pro Ser Pro Asp Arg Asp Arg Glu Ser Ile 
   2000                2005                2010 

gtg tgagcctcgg cctggctggc caggacacac tgaaaag                        6091 
Val 
2015 

 
           
             2  
             2015  
             PRT  
             Homo sapiens  
           
            2 

Met Ala Asn Phe Leu Leu Pro Arg Gly Thr Ser Ser Phe Arg Arg Phe 
  1               5                  10                  15 

Thr Arg Glu Ser Leu Ala Ala Ile Glu Lys Arg Met Ala Glu Lys Gln 
             20                  25                  30 

Ala Arg Gly Ser Thr Thr Leu Gln Glu Ser Arg Glu Gly Leu Pro Glu 
         35                  40                  45 

Glu Glu Ala Pro Arg Pro Gln Leu Asp Leu Gln Ala Ser Lys Lys Leu 
     50                  55                  60 

Pro Asp Leu Tyr Gly Asn Pro Pro Gln Glu Leu Ile Gly Glu Pro Leu 
 65                  70                  75                  80 

Glu Asp Leu Asp Pro Phe Tyr Ser Thr Gln Lys Thr Phe Ile Val Leu 
                 85                  90                  95 

Asn Lys Gly Lys Thr Ile Phe Arg Phe Ser Ala Thr Asn Ala Leu Tyr 
            100                 105                 110 

Val Leu Ser Pro Phe His Pro Ile Arg Arg Ala Ala Val Lys Ile Leu 
        115                 120                 125 

Val His Ser Leu Phe Asn Met Leu Ile Met Cys Thr Ile Leu Thr Asn 
    130                 135                 140 

Cys Val Phe Met Ala Gln His Asp Pro Pro Pro Trp Thr Lys Tyr Val 
145                 150                 155                 160 

Glu Tyr Thr Phe Thr Ala Ile Tyr Thr Phe Glu Ser Leu Val Lys Ile 
                165                 170                 175 

Leu Ala Arg Gly Phe Cys Leu His Ala Phe Thr Phe Leu Arg Asp Pro 
            180                 185                 190 

Trp Asn Trp Leu Asp Phe Ser Val Ile Ile Met Ala Tyr Thr Thr Glu 
        195                 200                 205 

Phe Val Asp Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val Leu 
    210                 215                 220 

Arg Ala Leu Lys Thr Ile Ser Val Ile Ser Gly Leu Lys Thr Ile Val 
225                 230                 235                 240 

Gly Ala Leu Ile Gln Ser Val Lys Lys Leu Ala Asp Val Met Val Leu 
                245                 250                 255 

Thr Val Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu Phe 
            260                 265                 270 

Met Gly Asn Leu Arg His Lys Cys Val Arg Asn Phe Thr Ala Leu Asn 
        275                 280                 285 

Gly Thr Asn Gly Ser Val Glu Ala Asp Gly Leu Val Trp Glu Ser Leu 
    290                 295                 300 

Asp Leu Tyr Leu Ser Asp Pro Glu Asn Tyr Leu Leu Lys Asn Gly Thr 
305                 310                 315                 320 

Ser Asp Val Leu Leu Cys Gly Asn Ser Ser Asp Ala Gly Thr Cys Pro 
                325                 330                 335 

Glu Gly Tyr Arg Cys Leu Lys Ala Gly Glu Asn Pro Asp His Gly Tyr 
            340                 345                 350 

Thr Ser Phe Asp Ser Phe Ala Trp Ala Phe Leu Ala Leu Phe Arg Leu 
        355                 360                 365 

Met Thr Gln Asp Cys Trp Glu Arg Leu Tyr Gln Gln Thr Leu Arg Ser 
    370                 375                 380 

Ala Gly Lys Ile Tyr Met Ile Phe Phe Met Leu Val Ile Phe Leu Gly 
385                 390                 395                 400 

Ser Phe Tyr Leu Val Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr 
                405                 410                 415 

Glu Glu Gln Asn Gln Ala Thr Ile Ala Glu Thr Glu Glu Lys Glu Lys 
            420                 425                 430 

Arg Phe Gln Glu Ala Met Glu Met Leu Lys Lys Glu His Glu Ala Leu 
        435                 440                 445 

Thr Ile Arg Gly Val Asp Thr Val Ser Arg Ser Ser Leu Glu Met Ser 
    450                 455                 460 

Pro Leu Ala Pro Val Asn Ser His Glu Arg Arg Ser Lys Arg Arg Lys 
465                 470                 475                 480 

Arg Met Ser Ser Gly Thr Glu Glu Cys Gly Glu Asp Arg Leu Pro Lys 
                485                 490                 495 

Ser Asp Ser Glu Asp Gly Pro Arg Ala Met Asn His Leu Ser Leu Thr 
            500                 505                 510 

Arg Gly Leu Ser Arg Thr Ser Met Lys Pro Arg Ser Ser Arg Gly Ser 
        515                 520                 525 

Ile Phe Thr Phe Arg Arg Arg Asp Leu Gly Ser Glu Ala Asp Phe Ala 
    530                 535                 540 

Asp Asp Glu Asn Ser Thr Ala Gly Glu Ser Glu Ser His Arg Thr Ser 
545                 550                 555                 560 

Leu Leu Val Pro Trp Pro Leu Arg Arg Thr Ser Ala Gln Gly Gln Pro 
                565                 570                 575 

Ser Pro Gly Thr Ser Ala Pro Gly His Ala Leu His Gly Lys Lys Asn 
            580                 585                 590 

Ser Thr Val Asp Cys Asn Gly Val Val Ser Leu Leu Gly Ala Gly Asp 
        595                 600                 605 

Pro Glu Ala Thr Ser Pro Gly Ser His Ile Leu Arg Pro Val Met Leu 
    610                 615                 620 

Glu His Pro Pro Asp Thr Thr Thr Pro Ser Glu Glu Pro Gly Gly Pro 
625                 630                 635                 640 

Gln Met Leu Thr Ser Gln Ala Pro Cys Val Asp Gly Phe Glu Glu Pro 
                645                 650                 655 

Gly Ala Arg Gln Arg Ala Leu Ser Ala Val Ser Val Leu Thr Ser Ala 
            660                 665                 670 

Leu Glu Glu Leu Glu Glu Ser Arg His Lys Cys Pro Pro Cys Trp Asn 
        675                 680                 685 

Arg Leu Ala Gln Arg Tyr Leu Ile Trp Glu Cys Cys Pro Leu Trp Met 
    690                 695                 700 

Ser Ile Lys Gln Gly Val Lys Leu Val Val Met Asp Pro Phe Thr Asp 
705                 710                 715                 720 

Leu Thr Ile Thr Met Cys Ile Val Leu Asn Thr Leu Phe Met Ala Leu 
                725                 730                 735 

Glu His Tyr Asn Met Thr Ser Glu Phe Glu Glu Met Leu Gln Val Gly 
            740                 745                 750 

Asn Leu Val Phe Thr Gly Ile Phe Thr Ala Glu Met Thr Phe Lys Ile 
        755                 760                 765 

Ile Ala Leu Asp Pro Tyr Tyr Tyr Phe Gln Gln Gly Trp Asn Ile Phe 
    770                 775                 780 

Asp Ser Ile Ile Val Ile Leu Ser Leu Met Glu Leu Gly Leu Ser Arg 
785                 790                 795                 800 

Met Ser Asn Leu Ser Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe 
                805                 810                 815 

Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Thr Leu Ile Lys Ile Ile 
            820                 825                 830 

Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile 
        835                 840                 845 

Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe Gly Lys Asn 
    850                 855                 860 

Tyr Ser Glu Leu Arg Asp Ser Asp Ser Gly Leu Leu Pro Arg Trp His 
865                 870                 875                 880 

Met Met Asp Phe Phe His Ala Phe Leu Ile Ile Phe Arg Ile Leu Cys 
                885                 890                 895 

Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met Glu Val Ser Gly Gln 
            900                 905                 910 

Ser Leu Cys Leu Leu Val Phe Leu Leu Val Met Val Ile Gly Asn Leu 
        915                 920                 925 

Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe Ser Ala 
    930                 935                 940 

Asp Asn Leu Thr Ala Pro Asp Glu Asp Arg Glu Met Asn Asn Leu Gln 
945                 950                 955                 960 

Leu Ala Leu Ala Arg Ile Gln Arg Gly Leu Arg Phe Val Lys Arg Thr 
                965                 970                 975 

Thr Trp Asp Phe Cys Cys Gly Leu Leu Arg Gln Arg Pro Gln Lys Pro 
            980                 985                 990 

Ala Ala Leu Ala Ala Gln Gly Gln Leu Pro Ser Cys Ile Ala Thr Pro 
        995                1000                1005 

Tyr Ser Pro Pro Pro Pro Glu Thr Glu Lys Val Pro Pro Thr Arg Lys 
   1010                1015                1020 

Glu Thr Arg Phe Glu Glu Gly Glu Gln Pro Gly Gln Gly Thr Pro Gly 
1025               1030                1035                1040 

Asp Pro Glu Pro Val Cys Val Pro Ile Ala Val Ala Glu Ser Asp Thr 
               1045                1050                1055 

Asp Asp Gln Glu Glu Asp Glu Glu Asn Ser Leu Gly Thr Glu Glu Glu 
           1060                1065                1070 

Ser Ser Lys Gln Glu Ser Gln Pro Val Ser Gly Gly Pro Glu Ala Pro 
       1075                1080                1085 

Pro Asp Ser Arg Thr Trp Ser Gln Val Ser Ala Thr Ala Ser Ser Glu 
   1090                1095                1100 

Ala Glu Ala Ser Ala Ser Gln Ala Asp Trp Arg Gln Gln Trp Lys Ala 
1105               1110                1115                1120 

Glu Pro Gln Ala Pro Gly Cys Gly Glu Thr Pro Glu Asp Ser Cys Ser 
               1125                1130                1135 

Glu Gly Ser Thr Ala Asp Met Thr Asn Thr Ala Glu Leu Leu Glu Gln 
           1140                1145                1150 

Ile Pro Asp Leu Gly Gln Asp Val Lys Asp Pro Glu Asp Cys Phe Thr 
       1155                1160                1165 

Glu Gly Cys Val Arg Arg Cys Pro Cys Cys Ala Val Asp Thr Thr Gln 
   1170                1175                1180 

Ala Pro Gly Lys Val Trp Trp Arg Leu Arg Lys Thr Cys Tyr His Ile 
1185               1190                1195                1200 

Val Glu His Ser Trp Phe Glu Thr Phe Ile Ile Phe Met Ile Leu Leu 
               1205                1210                1215 

Ser Ser Gly Ala Leu Ala Phe Glu Asp Ile Tyr Leu Glu Glu Arg Lys 
           1220                1225                1230 

Thr Ile Lys Val Leu Leu Glu Tyr Ala Asp Lys Met Phe Thr Tyr Val 
       1235                1240                1245 

Phe Val Leu Glu Met Leu Leu Lys Trp Val Ala Tyr Gly Phe Lys Lys 
   1250                1255                1260 

Tyr Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu Ile Val Asp Val 
1265               1270                1275                1280 

Ser Leu Val Ser Leu Val Ala Asn Thr Leu Gly Phe Ala Glu Met Gly 
               1285                1290                1295 

Pro Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala 
           1300                1305                1310 

Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn Ala Leu Val Gly 
       1315                1320                1325 

Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val Cys Leu Ile Phe Trp 
   1330                1335                1340 

Leu Ile Phe Ser Ile Met Gly Val Asn Leu Phe Ala Gly Lys Phe Gly 
1345               1350                1355                1360 

Arg Cys Ile Asn Gln Thr Glu Gly Asp Leu Pro Leu Asn Tyr Thr Ile 
               1365                1370                1375 

Val Asn Asn Lys Ser Gln Cys Glu Ser Leu Asn Leu Thr Gly Glu Leu 
           1380                1385                1390 

Tyr Trp Thr Lys Val Lys Val Asn Phe Asp Asn Val Gly Ala Gly Tyr 
       1395                1400                1405 

Leu Ala Leu Leu Gln Val Ala Thr Phe Lys Gly Trp Met Asp Ile Met 
   1410                1415                1420 

Tyr Ala Ala Val Asp Ser Arg Gly Tyr Glu Glu Gln Pro Gln Trp Glu 
1425               1430                1435                1440 

Tyr Asn Leu Tyr Met Tyr Ile Tyr Phe Val Ile Phe Ile Ile Phe Gly 
               1445                1450                1455 

Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile Asp Asn Phe 
           1460                1465                1470 

Asn Gln Gln Lys Lys Lys Leu Gly Gly Gln Asp Ile Phe Met Thr Glu 
       1475                1480                1485 

Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly Ser Lys Lys 
   1490                1495                1500 

Pro Gln Lys Pro Ile Pro Arg Pro Leu Asn Lys Tyr Gln Gly Phe Ile 
1505               1510                1515                1520 

Phe Asp Ile Val Thr Lys Gln Ala Phe Asp Val Thr Ile Met Phe Leu 
               1525                1530                1535 

Ile Cys Leu Asn Met Val Thr Met Met Val Glu Thr Asp Asp Gln Ser 
           1540                1545                1550 

Pro Glu Lys Ile Asn Ile Leu Ala Lys Ile Asn Leu Leu Phe Val Ala 
       1555                1560                1565 

Ile Phe Thr Gly Glu Cys Ile Val Lys Leu Ala Ala Leu Arg His Tyr 
   1570                1575                1580 

Tyr Phe Thr Asn Ser Trp Asn Ile Phe Asp Phe Val Val Val Ile Leu 
1585               1590                1595                1600 

Ser Ile Val Gly Thr Val Leu Ser Asp Ile Ile Gln Lys Tyr Phe Phe 
               1605                1610                1615 

Ser Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg Ile Gly Arg Ile 
           1620                1625                1630 

Leu Arg Leu Ile Arg Gly Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala 
       1635                1640                1645 

Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly Leu Leu Leu Phe 
   1650                1655                1660 

Leu Val Met Phe Ile Tyr Ser Ile Phe Gly Met Ala Asn Phe Ala Tyr 
1665               1670                1675                1680 

Val Lys Trp Glu Ala Gly Ile Asp Asp Met Phe Asn Phe Gln Thr Phe 
               1685                1690                1695 

Ala Asn Ser Met Leu Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly Trp 
           1700                1705                1710 

Asp Gly Leu Leu Ser Pro Ile Leu Asn Thr Gly Pro Pro Tyr Cys Asp 
       1715                1720                1725 

Pro Thr Leu Pro Asn Ser Asn Gly Ser Arg Gly Asp Cys Gly Ser Pro 
   1730                1735                1740 

Ala Val Gly Ile Leu Phe Phe Thr Thr Tyr Ile Ile Ile Ser Phe Leu 
1745               1750                1755                1760 

Ile Val Val Asn Met Tyr Ile Ala Ile Ile Leu Glu Asn Phe Ser Val 
               1765                1770                1775 

Ala Thr Glu Glu Ser Thr Glu Pro Leu Ser Glu Asp Asp Phe Asp Met 
           1780                1785                1790 

Phe Tyr Glu Ile Trp Glu Lys Phe Asp Pro Glu Ala Thr Gln Phe Ile 
       1795                1800                1805 

Glu Tyr Ser Val Leu Ser Asp Phe Ala Asp Ala Leu Ser Glu Pro Leu 
   1810                1815                1820 

Arg Ile Ala Lys Pro Asn Gln Ile Ser Leu Ile Asn Met Asp Leu Pro 
1825               1830                1835                1840 

Met Val Ser Gly Asp Arg Ile His Cys Met Asp Ile Leu Phe Ala Phe 
               1845                1850                1855 

Thr Lys Arg Val Leu Gly Glu Ser Gly Glu Met Asp Ala Leu Lys Ile 
           1860                1865                1870 

Gln Met Glu Glu Lys Phe Met Ala Ala Asn Pro Ser Lys Ile Ser Tyr 
       1875                1880                1885 

Glu Pro Ile Thr Thr Thr Leu Arg Arg Lys His Glu Glu Val Ser Ala 
   1890                1895                1900 

Met Val Ile Gln Arg Ala Phe Arg Arg His Leu Leu Gln Arg Ser Leu 
1905               1910                1915                1920 

Lys His Ala Ser Phe Leu Phe Arg Gln Gln Ala Gly Ser Gly Leu Ser 
               1925                1930                1935 

Glu Glu Asp Ala Pro Glu Arg Glu Gly Leu Ile Ala Tyr Val Met Ser 
           1940                1945                1950 

Glu Asn Phe Ser Arg Pro Leu Gly Pro Pro Ser Ser Ser Ser Ile Ser 
       1955                1960                1965 

Ser Thr Ser Phe Pro Pro Ser Tyr Asp Ser Val Thr Arg Ala Thr Ser 
   1970                1975                1980 

Asp Asn Leu Gln Val Arg Gly Ser Asp Tyr Ser His Ser Glu Asp Leu 
1985               1990                1995                2000 

Ala Asp Phe Pro Pro Ser Pro Asp Arg Asp Arg Glu Ser Ile Val 
               2005                2010                2015 

 
           
             3  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer  
             
           
            3 

gatgagaaga tggcaaactt cc                                              22 

 
           
             4  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer  
             
           
            4 

gctctggatc cccgggggtg cc                                              22 

 
           
             5  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer  
             
           
            5 

cacccccggg gatccagagc                                                 20 

 
           
             6  
             19  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer  
             
           
            6 

ttcagtgtgt cctggccag                                                  19 

 
           
             7  
             33  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer  
             
           
            7 

ttcctcatcg tggttaacct gtacattgcc atc                                  33 

 
           
             8  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer  
             
           
            8 

ggagatgatg atgtaggtgg                                                 20 

 
           
             9  
             25  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer  
             
           
            9 

cgagagccac cgcgcatcac tgctg                                           25 

 
           
             10  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer  
             
           
            10 

ctctcccccg ctgtgctgtt ttc                                             23