ORIGINAL ARTICLES

Primary Structure of the Adult Human Skeletal Muscle Voltage-Dependent Sodium Channel Alfred L. George, Jr, M D , W Jeffrey Komisarof,* Roland G. Kallen, MD, PhD,*$ and Robert L. Barchi, MD, PhD,*O

The gene encoding the principal voltage-dependent sodium channel expressed in adult human skeletal muscle (SCN4A) has recently been linked to the pathogenesis of human hyperkalemic periodic paralysis and paramyotonia congenita. We report the cloning and nucleotide sequence determination of the normal product of this gene. The 7,823 nucleotide complementary DNA, designated hSkM1, encodes a 1,836 amino acid protein that exhibits 92% identity with the tetrodotoxin-sensitive rat skeletal muscle sodium channel alpha subunit, but lower homology with either the human heart sodium channel or with other sodium channels from immature rat muscle or rat brain. Specific hSkMl RNA transcripts are expressed in adult human skeletal muscle but not in heart, brain, or uterus. The SCN4A gene product, hSkM1, is the human homologue of rSkM1, the tetrodotoxin-sensitive sodium channel characteristic of adult rat skeletal muscle. This structural information should provide the necessary backdrop for identifying and evaluating mutations affecting the function of this channel in the periodic paralyses. George AL Jr, Komisarof J, Kallen RG, Barchi RL.Primary structure of the adult human skeletal muscle voltage-dependent sodium channel. Ann Neurol 1992;3I: 131- 137

Voltage-dependent sodium channels are responsible for the rapid membrane depolarization that characterizes the initial phase of an action potential in nerve and muscle. In skeletal muscle, sodium channels in the sarcolemma are essential for conducting electrical impulses generated at the neuromuscular junction along the muscle fiber surface and into the transverse tubular system, thus forming a necessary link in the chain of events leading to muscle contraction. Loss of sodium channel activity will lead to muscle paralysis despite normal nerve, neuromuscular junction, and contractile protein function. Although the sodium channels in adult skeletal muscle share many attributes with sodium channels in other excitable membranes, they are kinetically, pharmacologically, and immunologically distinct from sodium channels in brain, heart, and immature skeletal muscle (1, 21. The sodium channel protein from adult rat skeletal muscle consists of an approximately 260,000-molecular weight alpha subunit that contains within it all the elements necessary to form a functional channel {3]. A 38,000-molecular weight beta subunit, the role of which remains unknown, is stoichiometrically associated with the alpha subunit in muscle (41. The primary

structure of the alpha subunit from rat muscle has been determined previously from its complementary D N A (cDNA) sequence { S } ; it resembles in organization the sequence of related channels from rat brain {6, 71, heart 181, eel electroplax [9}, and Drosophilu species

From the 'David Mahoney Institute of Neurological Sciences, and the Departments of ?Medicine, $Biochemistry and Biophysics, and §Neurology, University of Pennsylvania, Philadelphia, PA.

Address correspondence to D r George, 215 Stemmler Hall, 36th & Hamilton Walk, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.

[.lOl. The periodic paralyses are a group of rare, dominantly inherited disorders of skeletal muscle characterized by intermittent weakness or paralysis associated with muscle membrane depolarization. Characteristic shifts in serum potassium concentration associated with these episodes have provided grounds for dividing these disorders into hyperkalemic and hypokalemic forms. Over the past few years, evidence has accumulated that associates an abnormal sodium current with the membrane depolarization, conduction failure, and paralysis seen in these disorders (1 1-14}. The pharmacological characteristics of this abnormal current in hyperkalemic periodic paralysis and in paramyotonia congenita specifically implicate the voltage-dependent sodium channel as the source of this current. O n the basis of sequence homology with the rat skeletal muscle sodium channel, the gene encoding the human adult skeletal muscle sodium channel, desig-

Received Oct 25, 1991, and in revised form Nov 4.Accepted for publication Nov 4, 1991.

Copyright 0 1992 by the American Neurological Association 131

nated SCN4A, was identified and localized to chromosome 17q23.1-25.3 [15, 161. Genetic evidence has been presented recently that links an abnormality in this gene to the expression of certain forms of hyperkalemic periodic paralysis and of paramyotonia congenita [ 17-207. We describe the complete primary strucrure of the SCN4A gene product, a voltage-dependent sodium channel alpha subunit isoform that is expressed in adult skeletal muscle. The availability of this information should facilitate rhe search for mutations that affect sodium channel function in diseases such as the periodic paralyses. Materials and Methods

94°C 1 minute, 45°C 2 minutes, and 72°C 2 minutes; 3 0 cycles at 94°C I minute, 55°C 2 minutes, and 72°C 3 minutes; and a final extension at 72°C 10 minutes. Reaction mixtures (100 pL) contained the following: approximatc:ly 100 ng cDNA; 50 pmol of each primer; 100 niM of each dcoxynucleoside triphosphate; 10 mM Tris-hydrochloride (pH 8.3); 50 mM potassium chloride; 1.5 mM (HSP-1) or 3.0 mM (HSP-2) magnesium chloride; 0.0017J gelatin; a1nd 2.’j U AmpliTaq D N A polymerase (Perkin-Elmer-Cerus Corp,). PCR products were size-fractionated on 1$i agxosc gels, transferred to nylon membranes, and hybridized with nested cDNA probes. Specifically hybridizing PCK products o f expected length were isolated from preparative 1% agarose gels by the method of Vogelstein and Gillespie i271 using Glassmilk (BIO 101). Gel-purified PCR products were digested with Sal1 and Not 1, ligated t o pBluescript, and used to transform competent E.irhevicbia ioli (DHSu) Anchored PCRs were performed essentially as described by Frohman and associates 1281 except that random hexamers were used to prime first-strand cDNA synthesis from human muscle RNA, and a sodium channel-specific amplification primer (HSP-3amp, 5’-ggggcggccgcTCAGGCA& AAGACAGTGAG-3’) was used. Amplified products giving specific hybridization signals to a nested probe were subcloned into pBluescript by the methods described. Transformants resulting from anchored PCR experiments were subjected to a final colony hybridization screen using a nested probe. Nucleotide sequence was determined entirely o n both strands ofcDNAs C6b, HSP-1, HSP-2, HSP-I, and 1AI by the dideoxynucleotide chain termination method employing Scquenase 2.0 (United States Biochemical Corp, Cievelanti, OH) as previously described [ 2 3 ] . Nucleotide sequence an?biguities were resolved by using a modified Sequenasc protocol 1291 and formamide-containing gels. Nucleotitlc SCIquence analysis was performed using the Intclligcnetics (Mountain View, CA) molecular biology programs.

Human skeletal muscle and other tissues were procured from cadaveric transplant organ donors by the National Disease Research Interchange (Philadelphia, PA) or as quick frozen surgical pathology specimens from the Hospital of the University o f Pennsylvania. Total R N A was extracted using the method of C h i r p i n and associates {Zl], and p:)ly-A+ R N A was isolated by olih.odeoxythymictine (oligo-(dT))cellulose chromatography 1221. I n vitro transcription of antisense complementary R N A (cRNA) probes and Northern blot analyses were performed as previously described 1231. A size-selected oligo-(dT) primed AgtlO human muscle cDNA library (generously provided by Hansel1 Stedman, MD; 1243)and a random hexamerioligo-(dT) primed AZAPII human muscle cDNA library (made in collaboration with Stratagene Cloning Systems, La Jolla, CA) were screened using probes derived from the rat skeletal muscle sodium channel cDNA, rSkM1 C S ] . Primary screenings (5-6 X lo5 recombinants) were performed using the technique of Benton and Davis { 2 5 ] with nylon membranes (Biotrans, ICN). Hybridizations were performed at 42°C for 18 hours in 50% formamide, 0.9 M sodium chloride, 0.1 M sodium phosphate, 5 m N ethylenediaminctctraacetic acid, 1% SDS, 5 X Denhardt’s solution, 0.15 mgiml salmon sperm D N A , and filters washed with 0.9 M sodium chloride, 0.09 M sodium Results citrate, 0.19; SDS at 63°C. The cDNA inserts from plaqueFive independent cDNA clones were isolated b y Iipurified clones were either subcloned from AgtlO into the brary screening, and 3 cDNA segments were generEmRI site of pBluescript or were rescued from AZAPII as ated by PCR (Fig 1). Clones C6b and H2a originated pBluescript phagcimids containing the cDNA segment using in the A g t l O human muscle cDNA library, which was the R408 helper phage [26). screened with an antisense cRNA probe representing For polymerase chain reactions (PCRs), 1 to 3 pg of huthe full-length rSkM1 sequence. Clones 1A1, 6A2, and man skeletal musc-le poly-A R N A was reverse transcribed 8A2 were isolated from the XZAPIl human muscle using random hexamers ( 10 pmol/pg R N A ) and ribonuclease cDNA library, which was screened using a cDNA H MMLV reverse transcriptase (Bethesda Research Laboratories, Bethesda, MD) according to the enzyme supplier’s probe consisting of nucleotides 367 to 4407 of rSkM 1. protocol. The following oligonucleotide pairs were used to Because these 5 clones did not provide a full-length amplify two overlapping cDNA fragments, HSP1 and sequence, PCR was used to complete the cloning of HSP-2 (underlined portion represents sodium channelhSkM1. Both HSP-I and HSP-2 were generated b y specific sequence; remaining sequence in lower case letters PCR using flanking primer sequences designed on the was added to provide restriction endonuclease sites): basis of sequences of the partial-length cDNA clones 8A2-1, 5‘-mtcgacCATC?TCGCCGTGGTGGGCAT-3‘ , 8A2, and and C6B- 1, 5 ‘ - ~ ~ ~ ~ C R ~ ~ ~ C ~ C - T T G C C G A A G G lTACl T C:GA A -Cbb. Anchored PCR was used to obtain HSP-3. The identity, orientation, and approximate a - 3 ’ for HSP-I; 1 A l - I , 5‘-ggggtcgac-CTGGAACTGposition of all cDNAs were defined in part by alignGCTGGACTTCAG-3’ and 8A2-2, 5’-ggggcggccgcment with the nucleotide sequence of rSkM1. TGTAGTCAGCTGTGCT-3’ for HSP-2. Amplifications The complete nucleotide sequence of hSkM1 (7,825 were performed in a Perkin-Elmer-Cetus thermal cycler as follows: iiiitial denaturation at 94°C 8 minutes; 3 cydes at base pairs [bp]; Fig 2) consists of 77 bp of a 5‘-untrans+

132 Annals of Neurology

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Coding Region

- IAI

8AZ

HSP-3

6AZ

C@ HZ

HSP-I

HSP-2

Fig 1. hSkMl complementaty DNAs. Designation of indiuidual complementary DNAs is as follows (nucleotide I zs the jirst base zn the start codon,: HSP-3 = 623 base paws Ibp) Inucleotides - 77 to 546); IAl = 623 bp (94 t o 717); HSP-2 = 2,338 bp (576 t o 29141, 8A2 = 832 bp (2097 to 2929); 6A2 = 637 bp (2121 to 2758); HSP-I = 2,435 bp (21 36 t o 45711, C6b = 3.543 bp (4203 t o 7746); and H2a = 3,209 bp (4537 t o 7746).

lated (UT) sequence, an open reading frame of 5,508 bp, and a 2,238-bp 3'-UT region that contains a consensus polyadenylation signal sequence (AATAAA) that is located 17 bp 5' of a poly-(dA) tail. The translation start site (ATG) is located at position 78 to 80, and an in-frame termination codon (TAG) is present at position 5586 to 5588 in the complete hSkM1 sequence. A cytosine at position 4937 is absent in clone Cbb, but is present in both H2a and a genomic D N A clone encompassing this region and therefore is likely to be a cloning artifact. The primary structure of hSkM1 (see Fig 2) consists of 1,836 amino acids and has a calculated molecular mass of 208,172 daltons. The predicted secondary structure of hSkM1 is consistent with current models of sodium channel topology with four large (234-323 amino acids) homologous repeat domains, each containing 6 potential membrane-spanning alpha-helical segments (SI-S6), including a positively charged amphipathic S4 helix. In addition, the region between transmembrane segments S5 and S6 in each of the repeat domains contains two segments (SS1, SS2) that are homologous to those postulated to form part of the ion pore in other sodium channels [30]. Comparisons made between hSkM 1 and each of the previously cloned sodium channels from human heart, rat muscle and brain, and eel electroplax reveal a consistent pattern of primary structure homology that is most prominent within the repeat domains and the interdomain 3 to 4 region (Table). In contrast, the regions between domains 1 to 2 and 2 to 3 are not well conserved except between hSkM1 and rSkM1. In addition, the length of interdomain 1 to 2 is only 116 residues, similar to both rSkM1 and the eel electroplax sodium channels but much shorter than sodium channels in brain, heart, and immature muscle. No significant homology exists between the hSkM1 3'-UT region and between any previously cloned sodium

channel or other nucleotide sequence registered in GenBank (Los Alamos, NM). The primary sequence of hSkM1 was surveyed for consensus sites of posttranslational modification. There are 15 potential sites for N-linked glycosylation; 1I sites occur in regions of hSkM1 predicted to be extracellular, and 5 (asparagine residues at positions 214, 271, 362, 1191, and 1205) are conserved in all previously cloned rat and eel sodium channels. The highest density of sites is in the region between helices S 5 and S6 in repeat domain 1. In addition, there are two potential sites for cyclic nucleotide-dependent phosphorylation (serine residues at positions 25 1 and 1511); neither are conserved among all sodium channels. Steady-state levels of hSkM1 messenger R N A transcripts in various human tissues were examined by Northern blot analysis. An hSkM 1-specific antisense cRNA probe derived from the 3'-UT region hybridized specifically with a 8.5 to 9.0 kb transcript present in total R N A isolated from adult human skeletal muscle, but not in RNA isolated from human brain, heart (atrium and ventricle), myometrium, liver, or spleen (Fig 3). These results are consistent with tissue-specific expression of hSkM1 in adult skeletal muscle. Discussion Paroxysmal muscle weakness in the hereditary periodic paralyses is a manifestation of abnormal sarcolemmal permeability to sodium ions that episodically renders the membrane electrically unexcitable. At the onset of weakness, muscle cells become depolarized by a noninactivating, inwardly directed sodium current that in different forms of the disease can be triggered by cooling or by changes in extracellular potassium concentration [ll-13, 31). In both hyperkalemic periodic paralysis and paramyotonia congenita, this abnormal sodium current is blocked by tetrodotoxin ('ITX), a specific inhibitory ligand of voltage-dependent sodium channels [13, 141. These observations strongly suggest that the noninactivating sodium current responsible for membrane depolarization and weakness in these disorders arises from a subpopulation of functionally abnormal sodium channels in affected skeletal muscle. Voltage-dependent sodium channels comprise a multigene family with at least 6 structurally distinct isoforms known to exist in mammalian brain, skeletal muscle, and heart [5-7, 23, 32-34]. In skeletal muscle, two sodium channel isoforms are differentially expressed with respect to developmental stage and state of innervation of the muscle [23, 35, 361. In developing muscle (i.e., myoblast to early myotube stage), the predominant sodium channel isoform is relatively insensitive to TTX. With maturation of the muscle fiber, the 'ITX-insensitive sodium channel isoform disappears, and a TTX-sensitive sodium channel with George et al: Human Skeletal Muscle Na+ Channel

133

-

If1

lVS2

Arc

wi

ATC TTC MC TTC

I l a ASP ASP LEI Vhc A m Vhe

I

4563 45w 4617 4644 4671 46m KC TTC GGC MC ACC h i c ATC IGC CCI ITC wb IIC ACC ACG TCG GCC GCC TGC wc GCG c i c CIC MC ccc AIC CTC MC AGC CGC ccc CCA wc TGT CLC ccc MC C ~ Gwc u c CCG f f i c ACC ACT GIC MC bti Phc Glr A m SCr I I C I I C C I S L N Phr b l u llr i h r l h r Ser #I* G I ? Irp I s p G I Y Lu L N A m VI I l e LN A mr.S Gly PID Pro asp C l s ASP Pro L m L.u Glu Isn P r 6 G I ? l h r ELI V.1 Lw GI"

wb

ClU l h r

Fig 2. Nucleotide and deduced amino acid sequence of the adult human skeletal muscle sodium channel, hSkM I. Nucleotides are numbered in the 5' t o 3' direction beginning with the first base of the initiating methionine codon. Nucleotides on the >'side of 6a.w number 1 are indirated by negutke numbers. The 3' terminal sequence is followed by a poi'ydeoxyadenosine tract. The deduced amino acid sequence of hSkM1 is shown beneath the nucleotide sequence. The positions of putative trunsmembrane segments ( S l to S6) and the 4 homologous repeat domains (1-4) are indicated. Consensus sites for N-linked ghcosylation are indicated by circles (filled circles represent sites that are conserved in all known sodium channel sequences). Differences observed among the individual clones are as follows: A (1A l ) o r G (HSP-21 at nucleotide 607; G IHSP-1) o r A (HSP-2) at nucleotide 25 78. The resulting amino acid substitutions are also shown.

slightly larger single-channel conductance is expressed. Following surgical denervation or chronic neuromuscular block, the TTX-insensitive sodium channel reemerges 1353. The adult skeletal muscle sodium channel isoform can also be distinguished from both the immature muscle channel and sodium channels in brain by its unique sensitivity to pconotoxin 137). The primary structures of both rat skeletal muscle sodium channel isoforms have been previously deduced from cDNA sequence, and preliminary evidence has indi-

cated the presence of homologous proteins in human skeletal muscle 116, 333. Preliminary sequence information of the human adult skeletal muscle sodium channel (hSkM1) has facilitated the mapping of its genetic locus on human chromosome 17 (SCN4A; 17q23.1-25.3) 115, 201. Using a Bgl I1 restriction fragment length polymorphism for the SCN4A locus and other nearby polymorphic markers, several groups have shown significant linkage with hyperkalemic periodic paralysis (both myotonic and nonmyotonic forms) and paramyotonia congenita [ 17-20]. Because no other sodium channel isoform has been mapped to chromosome 17, SCN4A appears likely to be the disease-producing gene in these syndromes. To identify and evaluate specific mutations affecting the structure of the adult skeletal muscle sodium channel in these disorders, it is first desirable to determine the complete primary structure of the normal SCN4A product. The complete deduced primary structure of hSkM 1, the product of the SCN4A locus, is reported herein. The cDNA sequence encodes a sodium channel protein of a size comparable to those found in rat muscle and brain. This protein exhibits the 4 homologous repeat domains, each containing the characteristic posiGeorge

et al: Human

Skeletal Muscle Na+ Channel 155

Honiologiel Betzc,een the Ammo Acid Sequence ~,f hSkM 1 and Other Sodium Channel Isoforms

9Amino Acid

Identity With hSkMl

Sodium Channel Isoform, Ref

N

D1

ID 1-2

D2

ID2-3

Di

1Di-4

D4

c

rSkM1 C5J hH1 1331 RB-I [6] RB-I1 [C.] RB-Ill 171 Eel 191

90

92 64

88

88 9

97

85

90 81

87 56

18 21 20

82 85 88

85

72 12

97 78 90

87 87

8,4 8.4

47

85 79

87

82

40

78

80

31'

54 59 59 61

40

74 61

13

14

22 35

88 88

23

76

17

47

N = amino terminus; D = domain; ID = interdomain region; C = carboxy terminus; rSkM1 = adult rat skeletal muscle sodium channcl; hH1 = human heart sodium channel; RB-I = rat: brain sodium channel I; RB-I1 = rat brain sodium channel 11, RH-111 = rat brain :;odium channel 111; Eel = Electrophorus electricus electric organ sodium channel.

{33]).These comparisons are most striking in the interdomain 1 to 2 and 2 to 3 regions, where amino acid sequence identity between hSkM1 and h H t is much lower (13 and 9%, respectively) than that between hSkMl and rSkMI (88%';see Table). This observation agrees with similar comparisons between rat and human cardiac sodium channel isoforms [ 3 3 ] , and confirms that there is far greater structural similarity between the same isoform in different species than there is between different isoforms expressed in the same tissue of one species. The structural differences between sodium channel isoforms have been conselwed during evolution and are likely to be important physiologically. The availability of the primary sequence of the human adult skeletal muscle sodium channel will facilitate the identification of genetic channel defects underlying the period paralyses and related disorders. F i g 3. Northern blot analysis of the tissue dictribution of hSkM 1 transcripts. Total RNA 110 pa uas size-fractionated O N ~JrnzaldebydetaKarososegels, electroblotted t o nylon membranes. and probed under high stringency with an antisenre complement a y KNA probe corresponding t o nuileotide.r GO70 to 7435. Numbers t o the left of the panel indicate the positinns of size standardi (in kb) run on the same gel. The size of the hSkM 1 transcript in skeletal muscle is dpproximately 8.5 t.7 9.0 kb.

tively charged S 4 amphipathic helix, which is found in all sodium channel sequences reported to date. The large number of potential N-linked glycosylation sites, concentrated especially in the S5 to S6 interhelical region of domain 1, suggest that this protein will be heavily glycosylated. The primary sequence of hSkM 1 is remarkably similar to the hoinologous sodium channel isoform in rat skeletal muscle (rSkM1). T h e degree of primary structure identity that exists between hSkMl and rSkM1 ( 9 2 p1 is significantly greater than that between hSkM 1 and the human cardiac sodium channel, h H 1 (59%;

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This work was supported by grants from rhe National 1nstitutc:s of Healrh (NS-18013 and AR-01862), rhe Muscular Dystrophy Asrociarion (R. L. B. and R. G. K.), rhe Pew Charitable Trust, and the Veterans Administration. A. L. G. IS a Lucille P. Markey Sch'olar, and this work was also supporred in part b y a grant from the Lucille P. Markey Charirable Trust. We are graceful for the technical contributions of Ms Geera Iyer. W e also thank D r Richard Horn, Dr Mohamed Chahine. and the D N A synthesis facility at the Roche Institute for Molecular Biology (Nutley, NJ) for providing oligonucleotides The nucleotide sequence data reporred in this article have been submitred to GenBank and have been assigned the accession number M81758.

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2 1. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979;18:5294-5299 22. Aviv H, Leder P. Purification of biologically active globin messenger R N A by chromatography on oligo-thymidylic acidcellulose. Proc Natl Acad Sci USA 1972;69:1408-1412 23. Kallen RG, Sheng 2,Yang J, et al. Primary structure and expression of a sodium channel characteristic of denervared and immature rat skeletal muscle. Neuron 1990;4:233-242 24. Stedman HH, Eller M, Jullian EH, et al. The human embryonic myosin heavy chain. Complete primary structure reveals evolutionary relationships with other developmental isoforms. J Biol Chem 1990;265:3568-3 5 76 25. Benton WD, Davis RW. Screening lambda-gt recombinant clones by hybridization to single plaques in situ. Science 1977; 196: 180- 182 26. Russel M, Kidd S, Kelley MR. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene 1986;45:3 3 3-3 38 27. Vogelstein B, Gillespie D. Preparative and analytical purification of D N A from agarose. Proc Narl Acad Sci USA 1979;76: 615-619 28. Frohman MA, Dush MK, Martin GR. Rapid production of fulllength cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 1988;85:8998-9002 27. Schuurman R, Keulen W. Modified protocol for D N A sequence analysis using Sequenase 2.0. BioTechniques 1991;10:185 30. Guy H R , Conti F. Pursuing the structure and function of voltage-gated channels. Trends Neurosci 1990;13:201-206 31. Cannon SC, Brown R H , Corey DP. A sodium channel defect in hyperkalemic periodic paralysis: potassium induced failure of inactivation. Neuron 1991;6:619-626 32. Rogart RB, Cribbs L, Muglia L, et al. Molecular cloning of a putative tetrodotoxin-resistant rat heart Na* channel isoform. Proc Natl Acad Sci USA 1989;86:8170-8174 33. Gellens ME, George AL, Chen L, et al. Primary structure and functional expression of the human cardiac tetrodotoxininsensitive volrage-dependent sodium channel. Proc Natl Acad Sci USA 1992 (in press) 34. Sills M N , Xu YC, Baracchini E, et al. Expression of diverse N a + channel messenger RNAs in rat myocardium. Evidence for a cardiac-specific N a + channel. J Clin invest 1989;84:331-336 35. Yang JS-J, Sladky JT, Kallen RG, Barchi RL.TTX-sensitive and TTX-insensitive sodium channel mRNA transcripts are independently regulated in adult skeletal muscle after denervation. Neuron 1991;7:421-427 36. Trimmer JS, Cooperman SS, Agnew WS, Mandel G. Regulation of muscle sodium channel transcripts during development and in response to denervation. Dev Biol 1990;142:360-367 37. Cruz LJ, Gray WR, Olivera BM, et al. Conus geographus toxins that discriminate between neuronal and muscle sodium channels. J Biol Chem 1985;260:9280-9288

George et al: Human Skeletal Muscle Na+ Channel 137