Adult Skeletal Muscle Sodium Channel Research Articles

Severe phenotypes of paralysis periodica paramyotonia are associated with the Met1592Val mutation in the voltage-gated sodium channel gene ( SCN4A) in a Chinese family

Paralysis periodica paramyotonia (PPP) is caused by mutation of the adult skeletal muscle sodium channel gene’s alpha (α)-subunit (SCN4A). Here, we report four generations of a Chinese family affected by a remarkably severe form of PPP with progressive myopathy. Routine electromyograms (EMG) showed myotonic discharge and after a long exercise test, compound motor action potential amplitudes were markedly decreased by 40–55%. Muscle biopsy revealed obvious vacuolar changes. Moreover, genetic analysis revealed the Met1592Val mutation in the α-subunit, SCN4A. The patients showed a striking clinical and electrophysiological improvement during treatment with acetazolamide. Thus, our findings showed that mutation of Met1592Val in the SCN4A gene is associated with aggressive development of PPP characterized by severe vacuolar myopathy.

A carboxy-terminal α-helical segment in the rat skeletal muscle voltage-dependent Na + channel is responsible for its interaction with the amino-terminus

Cytoplasmic segments of the adult rat skeletal muscle sodium channel α-subunit (rSkM1) comprise a major portion (∼40%) of the total protein and are involved in channel functions both general, such as inactivation, and isoform-specific, for example, protein kinase A modulation. Far ultraviolet circular dichroism measurements of synthetic peptides and overexpressed fusion proteins containing individual channel cytoplasmic segments suggest that cytoplasmic domains of rSkM1 contain ordered secondary structures even in the absence of adjoining transmembrane segments. Intrinsic fluorescence experiments with a nested set of carboxy-terminal deletion proteins confirm a specific interaction between the channel’s amino- and carboxy-termini and identify residues 1716–1737 in the carboxy-terminus as the region that binds to the amino-terminus. Circular dichroism measurements suggest that this same region is organized as an α-helix and that electrostatic forces may contribute to this association. The interaction of the amino- and carboxy-termini is not accompanied by secondary structure changes detectable by circular dichroism spectroscopy, but a decrease in intrinsic fluorescence indicates that this association is accompanied by a change in the environment of Trp 1617.

Rapid and Slow Voltage-Dependent Conformational Changes in Segment IVS6 of Voltage-Gated Na + Channels

Mutations in segment IVS6 of voltage-gated Na + channels affect fast-inactivation, slow-inactivation, local anesthetic action, and batrachotoxin (BTX) action. To detect conformational changes associated with these processes, we substituted a cysteine for a valine at position 1583 in the rat adult skeletal muscle sodium channel α-subunit, and examined the accessibility of the substituted cysteine to modification by 2-aminoethyl methanethiosulfonate (MTS-EA) in excised macropatches. MTS-EA causes an irreversible reduction in the peak current when applied both internally and externally, with a reaction rate that is strongly voltage-dependent. The rate increased when exposures to MTS-EA occurred during brief conditioning pulses to progressively more depolarized voltages, but decreased when exposures occurred at the end of prolonged depolarizations, revealing two conformational changes near site 1583, one coupled to fast inactivation, and one tightly associated with slow inactivation. Tetraethylammonium, a pore blocker, did not affect the reaction rate from either direction, while BTX, a lipophilic activator of sodium channels, completely prevented the modification reaction from occurring from either direction. We conclude that there are two inactivation-associated conformational changes in the vicinity of site 1583, that the reactive site most likely faces away from the pore, and that site 1583 comprises part of the BTX receptor.

Introduction

Introduction

Lack of sodium channel mutation in an Italian family with paramyotonia congenita.

To conduct the genotype-phenotype correlation in a family in which several individuals share clinical and electrophysiologic features of paramyotonia congenita (PC). PC, hyperkalemic periodic paralysis (HyperPP), and potassium-aggravated myotonias form the group of hereditary sodium channelopathies. Each of these disorders is associated with different point mutations in SCN4A, the gene encoding the alpha-subunit of the adult human skeletal muscle sodium channel. However, in HyperPP families, evidence of a causative gene different from SCN4A has been found. We conducted direct clinical examination, electrophysiologic (EMG/electroneurographic) and cardiologic studies, as well as laboratory screening in several affected and nonaffected members of the family. We performed the genotype-phenotype correlation by microsatellite linkage and cDNA-mutation analyses of the SCN4A gene. Affected members in this family showed clinical and electrophysiologic features typical of PC. The disease phenotype segregated with the chromosomal region that includes the SCN4A gene. Analysis of the entire cDNA sequence of the SCN4A gene in the index case disclosed a G3826A transition, which results in the Val1276Ile substitution. However, PCR-single-stranded confirmation polymorphism and direct sequencing analysis of the segment coding for Val-1276 on genomic DNA confirmed the G3826A transition in the index case but was negative in 11 affected members of the family; however, neither mutations nor aberrant splicings causative of the PC phenotype in this family were found on SCN4A. The existence of a second gene different from SCN4A that can give rise to a clinical PC phenotype can be speculated upon.

A novel mutation in the gene for the adult skeletal muscle sodium channel alpha-subunit (SCN4A) that causes paramyotonia congenita of von Eulenburg.

Paramyotonia congenita (PMC) of von Eulenburg is an autosomal dominant muscular disease characterized by exercise- and cold-induced myotonia and weakness. To date, 18 missense mutations in the adult skeletal muscle sodium channel alpha-subunit (SCN4A) gene have been identified to cause a spectrum of muscular diseases, including PMC of von Eulenburg, PMC without cold paralysis, potassium-aggravating myotonia, and hyperkalemic periodic paralysis. However, no obvious correlations can be made between the location or nature of amino acid substitutions in SCN4A and its clinical phenotypes. To describe clinical and genetic features of a family with PMC of von Eulenburg. A Japanese family with cold-induced myotonia and weakness was diagnosed as having PMC of von Eulenburg. This phenotype was identified to be caused by a novel mutation that substituted a glutamic acid residue for a highly conserved glycine residue in the fourth transmembrane segment (S4) of domain IV. This predicted a decrease in positive charge specific for the S4. In addition to the G1456E identified in this study, 4 mutations that cause a decrease in positive charge in the S4/D4 are associated with the phenotype of PMC of von Eulenburg. This provides an important genotype-phenotype correlation in sodium channelopathies.

The position of the fast-inactivation gate during lidocaine block of voltage-gated Na+ channels.

Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na+ channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na+ channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has been difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For that reason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substituted at phenylalanine 1304 in the rat adult skeletal muscle sodium channel alpha subunit (rSkM1) with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of the fast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence of site 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. More significantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from a slowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activation pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action.

Definition of epitopes for monoclonal antibodies developed against purified sodium channel protein: implications for channel structure.

To test sodium channel structural models, we defined the epitopes for nineteen independently cloned monoclonal antibodies previously generated against purified, detergent-solubilized, adult rat skeletal muscle sodium channel protein using channel proteolysis, synthetic peptides, and fusion proteins. All identified epitopes were continuous and unique to the skeletal muscle subtype alpha-subunit. Of the nineteen independent clones, seventeen had epitopes located either in the origin of the amino-terminus or in the interdomain 2-3 region while only two antibodies had epitopes located in the mid-portion of the interdomain 1-2 region. No immunogenic regions were identified on the alpha-subunit's extracellular regions, interdomain 3-4 segment, or carboxyl-terminus or on channel beta-subunits. While immune tolerance may explain the lack of immunogenicity of extracellular regions, the lack of immunogenicity of most of the channel's cytoplasmic mass may be due to segment inaccessibility from organization of these regions as globular domains, to insertion of parts of these regions into the membrane phase, or to interaction with other protein elements. The definition of monoclonal antibody epitopes allows us to reinterpret previously reported monoclonal antibody competition studies, providing independent support for our model of sodium channel cytoplasmic domain structure. In addition, these data suggest additional testable hypotheses concerning the interactions of the sodium channel amino- and carboxyl-termini with each other as well as with other protein elements.

The familial periodic paralyses and nondystrophic myotonias

The periodic paralyses are divided into hypokalemic, hyperkalemic, and paramyotonic forms ( 1 Ptáček L.J Johnson K.J Griggs R.C Genetics and physiology of the myotonic muscle disorders. NEJM. 1993; 328: 482-489 Crossref PubMed Scopus (138) Google Scholar ). A normokalemic form has been included in the literature but most (if not all) patients with this disorder have hyperkalemic periodic paralysis. Over the past decade, a combination of electrophysiologic and molecular biologic studies have led to reclassification of the disease (Table 1). All forms of periodic paralysis are either autosomal dominantly inherited or occur as sporadic cases that are probably the result of new mutations. It has become apparent that there are two broad categories of disease, each resulting from mutations in a distinct gene: hyperkalemic periodic paralysis (hyperKPP) and hypokalemic periodic paralysis (hypoKPP). In hyperKPP, the disease results from mutations in a skeletal muscle, voltage-gated sodium channel gene ( 2 Ptáček L.J George Jr, A.L Griggs R.C et al. Identification of a mutation in the gene causing hyperkalemic periodic paralysis. Cell. 1991; 67: 1021-1027 Abstract Full Text PDF PubMed Scopus (334) Google Scholar , 3 Rojas C.V Wang J.Z Schwartz L.S et al. A Met-to-Val mutation in the skeletal muscle Na+ channel alpha-subunit in hyperkalaemic periodic paralysis. Nature. 1991; 354: 387-389 Crossref PubMed Scopus (286) Google Scholar ). The molecular alterations have been defined for most cases. It is becoming clear that a number of disorders once considered separate entities are in fact allelic to hyperkalemic periodic paralysis, including paramyotonia congenita ( 4 Ptáček L.J George Jr, A.L Barchi R.L et al. Mutations in an S4 segment of the adult skeletal muscle sodium channel cause paramyotonia congenita. Neuron. 1992; 8: 891-897 Abstract Full Text PDF PubMed Scopus (219) Google Scholar , 5 McClatchey A.I Van den Bergh P Pericak Vance M.A et al. Temperature-sensitive mutations in the III-IV cytoplasmic loop region of the skeletal muscle sodium channel gene in paramyotonia congenita. Cell. 1992; 68: 769-774 Abstract Full Text PDF PubMed Scopus (199) Google Scholar ), and most recently, a form of myotonia without periodic paralysis that is potassium sensitive ( 6 Ptáček L.J Tawil R Griggs R.C et al. Sodium channel mutations in acetazolamide-responsive myotonia congenita, paramyotonia congenita, and hyperkalemic periodic paralysis. Neurology. 1994; 44: 1500-1503 Crossref PubMed Google Scholar , 7 Heine R Pika U Lehmann Horn F A novel SCN4A mutation causing myotonia aggravated by cold and potassium. Hum Mol Genet. 1993; 2: 1349-1353 Crossref PubMed Scopus (105) Google Scholar ). Remarkably, transient weakness also characterizes a disorder of voltage-gated chloride channels ( 8 Griggs R.C Ptáček L.J The periodic paralyses. Hosp Pract Off Ed. 1992; 27: 123-137 PubMed Google Scholar ).

Linkage of malignant hyperthermia and hyperkalemic periodic paralysis to the adult skeletal muscle sodium channel (SCN4A) gene in a large pedigree

Hyperkalemic periodic paralysis (HPP) is caused by mutations of the adult skeletal muscle sodium channel (SCN4A) gene on chromosome 17. Malignant hyperthermia (MH) is a genetically heterogeneous autosomal-dominant disorder occurring in association with various neuromuscular diseases or without other apparent abnormalities. In some families, MH is associated with mutations of a calcium release channel (RYR1) gene on chromosome 19. In other families, linkage of this disorder to the SCN4A gene on chromosome 17 has been suggested. We report on linkage analysis in a family in which both HPP and MH are inherited as autosomal-dominant traits. Two polymorphisms within the SCN4A locus, an RFLP and a (C-A)n repeat, were typed on multiple family members. The findings were consistent with linkage of the polymorphic markers within the SCN4A gene to both HPP (Zmax = 6.79 at theta = 0.0) and MH (Zmax = 1.76 at theta = 0) in this family. Our data provide further evidence that MH is linked to the SCN4A locus in some families.

Linkage of malignant hyperthermia and hyperkalemic periodic paralysis to the adult skeletal muscle sodium channel (SCN4A) gene in a large pedigree

Linkage of malignant hyperthermia and hyperkalemic periodic paralysis to the adult skeletal muscle sodium channel (SCN4A) gene in a large pedigree

Probing Sodium Channel Cytoplasmic Domain Structure: EVIDENCE FOR THE INTERACTION OF THE rSkM1 AMINO AND CARBOXYL TERMINI

Epitopes for monoclonal antibodies directed against the purified adult rat skeletal muscle sodium channel (rSkM1) were localized using channel proteolysis and fusion proteins. The interactions between these and other monoclonal antibodies with site-specific polyclonal antibodies were used to investigate the spatial relationships among rSkM1 cytoplasmic segments. Competition. between antibodies for binding was performed using a solution-phase assay in which solubilized channel protein retains many of the biophysical characteristics of the rSkM1 protein in vivo. Our results support a model in which: 1) the amino terminus assumes a rigid structure having a fixed orientation with respect to other intracellular segments; 2) the interdomain 2-3 region is centrally located on the cytoplasmic surface of the channel, extends farther into the cytoplasm, and has an intermediate degree of flexibility; 3) the beginning of the amino terminus and end of the carboxyl terminus specifically interact with each other; and 4) domains 1 and 4 are adjacent. The sequences responsible for the interaction of the amino and carboxyl termini were identified by demonstrating the specific binding of a synthetic peptide encompassing the first 30 residues of the rSkM1 amino terminus to a fusion protein containing the rSkM1 carboxyl terminus.

Masseter muscle rigidity associated with glycine1306-to-alanine mutation in the adult muscle sodium channel alpha-subunit gene.

Succinylcholine-induced masseter muscle rigidity (MMR) is a potentially life-threatening complication of anesthesia and is closely correlated with the heterogeneous disorder malignant hyperthermia (MH) susceptibility. MMR also is identified with a variety of neuromuscular disorders, including the myotonias, that are associated with abnormal in vitro contracture test (IVCT) results. Recently, mutations in the adult skeletal muscle sodium channel alpha-subunit gene (SCN4A) have been shown to cause generalized nondystrophic myotonias, some of which are associated with mild nonspecific symptoms. The purpose of the current investigation was to begin to evaluate the molecular genetic relationship between known mutations in the SCN4A gene, MMR, and the results of the IVCT used to diagnose MH-susceptibility. A single extended pedigree of 16 individuals was ascertained through a proband who experienced MMR and whole-body rigidity after succinylcholine administration. Subsequently, four individuals were shown to have a mild form of myotonia on clinical and laboratory examination. IVCT was carried out according to standardized protocols. Mutations in the SCN4A gene were sought in exons 22 and 24 using single-strand conformational analyses. Variability in the SCN4A gene sequence was confirmed by direct DNA sequence analyses. Four individuals with myotonia were shown to carry a guanine-to-cytosine mutation at nucleotide position 3917 of the reported SCN4A sequence. This DNA mutation was coinherited with MMR and an abnormal IVCT result in this family. Previous studies have demonstrated that the glycine1306-to-alanine substitution is associated with a mild clinical syndrome referred to as myotonia fluctuans. The current report provides direct evidence that succinylcholine-induced MMR, whole-body rigidity, and an abnormal IVCT result are associated with a mutation in the SCN4A gene.

International Malignant Hyperthermia Workshop and Symposium

International Malignant Hyperthermia Workshop and Symposium

Localization of Epitopes for Monoclonal Antibodies Directed against the Adult Rat Skeletal Muscle Sodium Channel (rSkM1) Using Polymerase Chain Reaction, Fusion Proteins, and Western Blotting

Localization of Epitopes for Monoclonal Antibodies Directed against the Adult Rat Skeletal Muscle Sodium Channel (rSkM1) Using Polymerase Chain Reaction, Fusion Proteins, and Western Blotting

Myotonia levior is a chloride channel disorder.

The group of dominant non-dystrophic myotonias, comprising disorders characterized by clinically similar forms of myogenic muscle stiffness, is genetically inhomogeneous. Dominant myotonia congenita (Thomsen's disease) is linked to CLCN1, the gene encoding the major muscle chloride channel, localized on chromosome 7q35. In contrast, dominant myotonias sensitive to potassium are caused by point mutations in SCN4A on chromosome 17q, the gene for the alpha subunit of the adult skeletal muscle sodium channel. No linkage or molecular genetic data are as yet available on 'myotonia levior' characterized by milder symptoms and later onset of myotonia than in Thomsen's disease, and absence of muscle hypertrophy. We report a CLCN1 Gln-552-Arg substitution for a family with dominant inheritance previously diagnosed to have myotonia levior. Thus, this disorder appears as a variant of Thomsen's disease due to mutations leading to low clinical expressivity. In addition, we report a novel Ile-290-Met CLCN1 mutation for a typical Thomsen pedigree. In another family previously diagnosed as having Thomsen's disease, we unexpectedly found a CLCN1 14 bp deletion known to cause recessive myotonia, and a rare Trp-118-Gly polymorphism.

The skeletal muscle sodium and chloride channel diseases.

The cause of several familial muscular diseases have recently been linked to mutations within skeletal muscle sodium and chloride channel genes. Thomsen's and Becker's diseases are autosomal dominant and recessive, respectively, and are caused by at least seven different mutations in the CLCN1 (ClC-1) skeletal muscle chloride channel gene on chromosome 7q35. Hyperkalaemic periodic paralysis, paramyotonia congenita and a small heterogeneous group of related 'pure' myotonias are autosomal dominant disorders and are due to at least 16 different mutations in the SCN4A (SkM1) adult skeletal muscle sodium channel gene on chromosome 17q23-25. There is generally little correlation between the position of a mutation in the channel and the phenotype. Indeed, identical sodium channel mutations in unrelated subjects and sometimes in different members of the same family can have different clinical expressions. It seems, however, that mutations of the inactivation gate (ID3-4 loop) of the sodium channel tend to produce paramyotonia or pure, sometimes severe, myotonia and respond most favourably to the same medications (tocainide and mexiletine). The structure and polarity of substituted amino acids at a mutation site, especially in highly evolutionally conserved regions of the gene, are undoubtedly important to the expression of a channel disease and may partly explain phenotypic variability. In addition, genetic polymorphisms elsewhere, either in the gene or other channel-related loci, and the net effect of other types of muscle ion channels on the electrical potential of the plasma membrane probably contribute to disease expression.

Sodium channel mutations in acetazolamide‐responsive myotonia congenita, paramyotonia congenita, and hyperkalemic periodic paralysis

Hyperkalemic periodic paralysis (hyperKPP) and paramyotonia congenita (PC) are genetic muscle disorders sharing the common features of myotonia and episodic weakness. In hyperKPP, patient symptoms and signs are worsened by elevated serum potassium, whereas in PC, muscle cooling exacerbates the condition. There are patients in whom features of both hyperKPP and PC are present. These diseases result from molecular alterations in the adult skeletal muscle sodium channel. This report summarizes our sodium channel mutation analysis in 25 families with hyperKPP and PC. We also report the putative disease-causing mutation in acetazolamide-responsive myotonia congenita, a related disease in which myotonia is worsened by potassium but in which episodic weakness does not occur. This missense mutation (I1160V) occurs at a very highly conserved position in the sodium channel, cosegregates with the disease, and was not present in any of a large panel of normal DNAs. Electrophysiologic characterization of specific mutations will lead to better understanding of the biophysics of this voltage-gated ion channel.

Pathophysiology of sodium channelopathies. Studies of sodium channel expression by quantitative multiplex fluorescence polymerase chain reaction

The inherited diseases hyperkalemic periodic paralysis and paramyotonia congenita are caused by mutations in the adult skeletal muscle sodium channel gene. To determine if differences in the expression patterns of the adult and cardiac/fetal sodium channel genes could explain some clinical features of these disorders, we developed a novel mRNA quantitation strategy called quantitative multiplex fluorescent polymerase chain reaction (QMF-PCR). This assay tests the relative levels of multiple mRNA species simultaneously using automated sequenators. We show validation of this method by competitive-PCR and RNase protection. Developmental studies of sodium channel mRNAs in humans and mice by QMF-PCR showed that the adult sodium channel mRNA quickly increased, while the cardiac/fetal sodium channel mRNA slowly decreased similarly in both limb and diaphragm muscle. We find that the adult sodium channel gene expression is predominant in fetal and neonatal muscle of both humans and mice: adult isoform mRNA concentration in fetal muscle was 8.4 x 10(-6) micrograms/micrograms of total RNA; cardiac/fetal isoform mRNA was 2.0 x 10(-6) micrograms/micrograms; and actin mRNA was 3.4 x 10(-3) micrograms/micrograms. Our results suggest that differential sodium channel gene expression correlates with age of onset of disease, but not with diaphragm involvement, in patients with hyperkalemic periodic paralysis.

Molecular and genetic characterisation of German families with paramyotonia congenita and demonstration of founder effect in the Ravensberg families

Eighteen German families with a history of paramyotonia congenita (PC) were characterised by genetic and mutational analysis at the SCN4A locus, which encodes the alpha-subunit of the adult skeletal muscle sodium channel. We concentrated our analysis primarily on these families to test the hypothesis that a predominance of one common mutation occurs in all German PC families and that this mutation arose in a common ancestor originating in the North-West of the country. The present eighteen PC families exhibit two different mutations (R1448C and R1448H) on various SCN4A dinucleotide repeat haplotypes and therefore the majority of the mutations probably occurred independently. However, the R1448H mutation is extremely frequent in the North-West of Germany (Ravensberger Land) on a specific SCN4A microsatellite haplotype, indicating a founder effect within this subpopulation. Our results suggest that the R1448C/R1448H mutations are by far the most common to be associated with the PC phenotype in the German population.