Cell, Vol. 68, 769-774,

February

21, 1992, Copyright

0 1992 by Cell Press

Temperature-Sensitive Mutations in the Ill-IV Cytoplasmic Loop Region of the Skeletal Muscle Sodium Channel Gene in Paramyotonia Congenita Andrea I. McClatchey,’ Peter Van den Bergh,3 Margaret A. Pericak-Vance,4 Wendy Raskind,5 Christine Verellen3 Diane McKenna-Yasek,2 Keshav Rao,6 Jonathan L. Haines,’ Thomas Bird,5s7 Robert H. Brown, Jr.,* and James F. Gusella’ ‘Molecular Neurogenetics Laboratory *Day Neuromuscular Research Massachusetts General Hospital and Harvard Medical School Charlestown, Massachusetts 02129 3Department of Neurology University of Louvain Cliniques Universitaires Saint-Luc 1200 Brussels Belgium 4Division of Neurology Department of Medicine Duke University Durham, North Carolina 27710 SDepartment of Medicine University of Washington Seattle, Washington 98195 6Department of Neurology University of Connecticut Health Center Farmington, Connecticut 06032 7Division of Neurology Seattle Veterans Affairs Hospital Seattle, Washington 98195

Summary Paramyotonia congenita (PMC), a dominant disorder featuring cold-induced myotonia (muscle stiffness), has recently been genetically linked to a candidate gene, the skeletal muscle sodium channel gene SCN4A. We have now established that SCN4A is the disease gene in PMC by identifying two different single-base coding sequence alterations in PMC families. Both mutations affect highly conserved residues in the III-IV cytoplasmic loop, a portion of the sodium channel thought to pivot in response to membrane depolarization, thereby blocking and inactivating the channel. Abnormal function of this cytoplasmic loop therefore appears to produce the Na+ current abnormality and the unique temperature-sensitive clinical phenotype in this disorder.

sensitive Na+ gating can be achieved in vitro by expressing only the a subunit (Nodaet al., 1986a; Goldin et al., 1986; Suzuki et al., 1988; Auld et al., 1988). To date, distinct a subunits have been identified for four brain sodium channels, along with separate skeletal and cardiac mUSCle SOdium channels (Noda et al., 1986b; Kayano et al., 1988; Auld et al., 1988; Rogartet al., 1989; Trimmer et al., 1989; Kallen et al., 1990). All share a common structure of fOUr similar domains, each consisting of six membrane-spanning regions (Figure 1). The four domains, which are thought to surround the channel pore, are connected by intracellular loops of varying sizes. Genetic linkage studies implicate SCN4A, the locus encoding the human skeletal muscle sodium channel a subunit, as the site of the primary defect in both paramyotonia congenita (PMC) (lod score i = 35.36 at r?l= 0.0) and hyperkalemic periodic paralysis (HPP) (i = 33.58 at 6 = O.O), two autosomal dominant disorders characterized by episodes of abnormal muscle membrane excitability (Fontaine et al., 1990; Koch et al., 1991 a, 1991 b; PtaEek et al., 1991 a, 1991 b; McClatchey et al., 1992). Clinically, the two disorders are distinct (Engel, 1986). PMC involves a mutation that leads to cold-induced myotonia (stiffness due to muscle membrane hyperexcitability). By contrast, HPP involves periodic attacks of muscle paralysis associated with elevated serum K’. In both disorders, electrophysiological studies have revealed an abnormal, tetrodotoxin-blockable Na’ current in diseased muscle. In HPP, depolarization of the muscle membrane appears to result from incomplete channel inactivation in the presence of high K+ (Lehmann-Horn et al., 1987a, 1991; Cannon et al., 1991). By contrast, in PMC an increased Na+ flux is produced by cooling the muscle below 27% (Lehmann-Horn et al., 1987b). The fact that at least two different neuromuscular disorders may involve molecular defects in SCN4A presents a unique opportunity to correlate a disturbance in channel function with a structural change in a specific domain of the sodium channel protein. Mutations in transmembrane segments have been described recently in HPP (PtaEek et al., 1991~; Rojas et al., 1991) but no SCN4A mutations have been reported in PMC. Here, we identify two PMC mutations which, unlike the HPP lesions, implicate improper function of the III-IV cytoplasmic loop of the sodium channel in this cold-sensitive disorder. To our knowledge, this is the first delineation of a precise molecular defect for a temperature-sensitive human disorder.

Introduction Results Voltage-gated sodium channelson the surface of excitable cells are responsible for the initiation and propagation of action potentials (Barchi, 1988; Catterall, 1988). Consequently, there has been considerable interest in delineating their molecular structure and functional domains. Mammalian sodium channels consist of a large a subunit and one or two smaller 8 subunits. However, voltage-

A Gly-Val Change in the III-IV Cytoplasmic Loop We have previously used haplotype analysis with (dGdA), and (dGdT), repeat polymorphisms at the SCN4A locus to forecast that both disorders display allelic heterogeneity, with at least two independent mutations predicted to have occurred in each (McClatchey et al., 1992). We have also

Cell 770

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1 Proposed

Structure

of the Skeletal

Muscle

Sodium

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and Location

of the Paramyotonia

Congemta

Alterations

The sodium channel consists of four homologous domains, each consisting of SIX transmembrane segments, connected by intracellular linkers Cyloplasmic loop III-IV has been implicated in both activation and inactivation of sodium channel gating. The two candidate PMC mutations in the III-IV loop and the HPP mutations in S5 of domain II and in S6 of domain IV (PtGek et al., 1991c; Rojas et al., 1991) are shown (Modified from the brain channel model of Catterall, 1988, and Kandel et al., 1991.)

discerned most of the genomic structure of SCN4A to facilitate the search for mutations (A. I. M. et al., unpublished data). Using genomic polymerase chain reaction (PCR) to amplify SCN4A exons followed by direct sequence analysis of the uncloned amplified product, we scanned for mutations in lymphoblast DNAfrom members of two independent PMC families. We first identified a base pair change that cosegregated with the disorder in a PMC family of Belgian origin (Figure 2). The mutation, a G-T transversion (Figure 3), produces a Gly-Val change in the highly conserved intracellular loop connecting domains Ill and IV (Figure 1) of the sodium channel. The affected Gly is one of a pair of glycines that is present at this position in all known sodium channels. For affected individual II-2 of the Belgian pedigree, no other deviation in comparison with several normal individuals was found when the 80% of the sodium channel coding sequence for which the intron-exon structure has been determined was scanned, including the regions surrounding both reported HPP mutations. The same mutation was subsequently detected in the only two available affected individuals in an independent North American pedigree, ascertained by myotonia in a distribution of muscles characteristic of PMC. Both pedigrees possessed the same SCN4A haplotype on the disease chromosome, GA1 IGT6.

a frequency of 25% in the normal population (McClatchey et al., 1992), we scanned 22 nondisease GAl/GTG chromosomes by PCR amplification of genomic DNA followed by direct sequencing. None displayed the candidate mutation. In addition, some affected individuals from the Belgian and North American families were homozygous for the GAlIGT6 haplotype but were heterozygous for the G-T transversion. Overall, the mutation was absent from 79 control chromosomes representing the ten known dinucleotide repeat haplotypes for SCN4A, and from an addi-

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Absence of the Candidate Mutation from Control Chromosomes To establish whether the base change was a polymorphism associated with the GAlIGT6 haplotype, which has

Figure

2. Belgian

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PMC Pedigree

A three-generation pedigree is shown. Individuals known symbols.

described in Van den Bergh et al. (1991) to suffer from PMC are shown as shaded

Sodium 771

Channel

Mutations

in Paramyotonia

Congenita

Figure 3. Detection PMC Pedigree

AGCTAGCTAGCTAGCT /.‘/4 ./_.. :"t.i b...' ..I -. -

of Mutation

in the Belgian

DNA sequence analysis of the sodium channel gene region surrounding a putative PMC mutaAGCTAGCTAGCT tion is shown. Direct sequencing of an un2 cloned PCR product containing a 105 bp exon of the skeletal muscle sodium channel gene was performed as described in Experimental Procedures. The panel at the left shows antisense seauence corresoondina to the left-hand portion ofthe exon shown in Figure 4, for indiII-2 III-l II-I II-3 II-2 III-1 c viduals II-Z, Ill-l, II-I, and h-3, respectively, from Figure 2. The right-hand panel shows the same region in the sense direction for individuals 11-2, Ill-l, and an unrelated normal control. All unaffected individuals display homozygous sequence while the affected individuals are heterozygous, possessing one chromosome with normal sequence and one bearing a G-T transversion (C-A antisense) reflecting transmission of the mutation from affected mother II-2 to her affected daughter, Ill-l. This mutation was seen in several independently generated PCR products from these affected individuals and from affected individuals in a second North American family with features of PMC. It was absent from a total of 140 control chromosomes assayed in the same manner.

tional 61 normal chromosomes of undetermined haplotype, many of which were probably GAlIGT6, given its prevalence. Of the 140 non-PMC chromosomes tested, 37 were from the normal Belgian population and 4 were disease chromosomes from independent HPP families, A Thr-Met Change in the Same Vicinity in an Independent PMC Pedigree In a larger North American PMC family, the disorder cosegregates (lod score i = 2.71 at I?I= 0.0) with a different SCN4A haplotype, GA4/GT8. The six affected members of this family did not possess the G-T transversion, but did reveal a discrete change in the same region of the III-IV cytoplasmic loop. A C-T transition (Figure 4) altered a Thr-Met, 6 amino acids beyond the site of the Gly-Val change in the other PMC families (data not shown). This second candidate mutation affects an amino acid residue that is also common to all known sodium channels. The

HUMAN

SKEL.

Discussion Several lines of evidence have indicated that the III-IV intracellular loop is crucial for normal sodium channel function. An analogous cytoplasmic structure in potassium channels appears to act as a “ball and chain” to block the channel pore (Hoshi et al., 1990). Antibodies to the III-IV intracellular domain of the sodium channel slow inactivation in both rat muscle cells and rat brain neurons (Vassilev et al., 1988,

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change cosegregated with PMC in all members of the pedigree and was absent from 52 control chromosomes. The GA41GT8 haplotype is extremely rare in the normal population (