Robert L. Barchi

Primary structure and functional expression of a mammalian skeletal muscle sodium channel

We describe the isolation and characterization of a cDNA encoding the alpha subunit of a new voltage-sensitive sodium channel, microI, from rat skeletal muscle. The 1840 amino acid microI peptide is homologous to alpha subunits from rat brain, but, like the protein from eel electroplax, lacks an extended (approximately 200) amino acid segment between homologous domains I and II. Northern blot analysis indicates that the 8.5 kb microI transcript is preferentially expressed in skeletal muscle. Sodium channels expressed in Xenopus oocytes from synthetic RNA encoding microI are blocked by tetrodotoxin and mu-conotoxin at concentrations near 5 nM. The expressed sodium channels have gating kinetics similar to the native channels in rat muscle fibers, except that inactivation occurs more slowly.

Identification of a mutation in the gene causing hyperkalemic periodic paralysis

DNA from seven unrelated patients with hyperkalemic periodic paralysis (HYPP) was examined for mutations in the adult skeletal muscle sodium channel gene (SCN4A) known to be genetically linked to the disorder. Single-strand conformation polymorphism analysis revealed aberrant bands that were unique to three of these seven patients. All three had prominent fixed muscle weakness, while the remaining four did not. Sequencing the aberrant bands demonstrated the same C to T transition in all three unrelated patients, predicting substitution of a highly conserved threonine residue with a methionine in a membrane-spanning segment of this sodium channel protein. The observation of a distinct mutation that cosegregates with HYPP in two families and appears as a de novo mutation in a third establishes SCN4A as the HYPP gene. Furthermore, this mutation is associated with a form of HYPP in which fixed muscle weakness is seen.

Sodium Channel Mutations in Paramyotonia Congenita Uncouple Inactivation from Activation

Mutations in the adult human skeletal muscle Na+ channel alpha subunit cause the disease paramyotonia congenita. Two paramyotonia congenita mutations, R1448H and R1448C, substitute histidine and cysteine for arginine in the S4 segment of domain 4. These mutations, expressed in a cell line, have only small effects on the activation of Na+ currents, but mutant channels inactivate more slowly with less voltage dependence than wild-type channels and exhibit an enhanced rate of recovery from inactivation. Increase of extracellular pH made the rate of inactivation of R1448H similar to that of R1448C, suggesting that this residue has an extracellular location and that its charge is important for normal inactivation. Analysis of single-channel data reveals that mutant channels inactivate normally from closed states, but poorly from the open state. The data suggest a critical role for the S4 helix of domain 4 in coupling between activation and inactivation.

Primary structure and expression of a sodium channel characteristic of denervated and immature rat skeletal muscle

The alpha subunit of a voltage-sensitive sodium channel characteristic of denervated rat skeletal muscle was cloned and characterized. The cDNA encodes a 2018 amino acid protein (SkM2) that is homologous to other recently cloned sodium channels, including a tetrodotoxin (TTX)-sensitive sodium channel from rat skeletal muscle (SkM1). The SkM2 protein is no more homologous to SkM1 than to the rat brain sodium channels and differs notably from SkM1 in having a longer cytoplasmic loop joining domains 1 and 2. Steady-state mRNA levels for SkM1 and SkM2 are regulated differently during development and following denervation: the SkM2 mRNA level is highest in early development, when TTX-insensitive channels predominate, but declines rapidly with age as SkM1 mRNA increases; SkM2 mRNA is not detectable in normally innervated adult skeletal muscle but increases greater than 100-fold after denervation; rat cardiac muscle has abundant SkM2 mRNA but no detectable SkM1 message. These findings suggest that SkM2 is a TTX-insensitive sodium channel expressed in both skeletal and cardiac muscle.

Mutations in an S4 segment of the adult skeletal muscle sodium channel cause paramyotonia congenita

The periodic paralyses are a group of autosomal dominant muscle diseases sharing a common feature of episodic paralysis. In one form, paramyotonia congenita (PC), the paralysis usually occurs with muscle cooling. Electrophysiologic studies of muscle from PC patients have revealed temperature-dependent alterations in sodium channel (NaCh) function. This observation led to demonstration of genetic linkage of a skeletal muscle NaCh gene to a PC disease allele. We now report the use of the single-strand conformation polymorphism technique to define alleles specific to PC patients from three families. Sequencing of these alleles defined base pair changes within the same codon, which resulted in two distinct amino acid substitutions for a highly conserved arginine residue in the S4 helix of domain 4 in the adult skeletal muscle NaCh. These data establish the chromosome 17q NaCh locus as the PC gene and represent two mutations causing the distinctive, temperature-sensitive PC phenotype.

Protein Components of the Purified Sodium Channel from Rat Skeletal Muscle Sarcolemma

Abstract: Sensitive detection systems have been used to study the protein components of the sodium channel purified from rat skeletal muscle sarcolemma. This functional, purified sodium channel contains at least three subunits on 7–20% gradient sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis: a large glycoprotein which migrates anomalously in the high‐molecular‐weight range, a 45,000 molecular weight polypeptide, and a third protein often seen as a doublet at 38,000. The large glycoprotein runs as a diffuse band and stains very poorly with Coomassie blue, but is adequately visualized with silver staining or iodination followed by autoradiography. This glycoprotein exhibits anomalous electrophoretic behavior in SDS‐polyacrylamide gels. The apparent molecular weight of the center of the band varies from ∼230,000 on 13% acrylamide gels to ∼130,000 on 5% gels; on 7–20% gradient gels a value of 160,000 is found. Plots of relative migration versus gel concentration suggest an unusually high apparent free solution mobility. Lectin binding to purified channel peptides separated by gel electrophoresis indicates that the large glycoprotein is the only subunit that binds either concanavalin A or wheat germ agglutinin, and this component has high binding capacity for both lectins. The smaller channel components run consistently at 45,000 and 38,000 molecular weight in a variety of gel systems and do not appear to be glycosylated.

TTX-sensitive and TTX-insensitive sodium channel mRNA transcripts are independently regulated in adult skeletal muscle after denervation

The expression of mRNA encoding the TTX-sensitive (SkM1) and TTX-insensitive (SkM2) voltage-dependent sodium channels in adult skeletal muscle is independently regulated. In normal skeletal muscle, only the SkM1 message is expressed and the level varies with muscle fiber type. After surgical denervation, the steady-state SkM1 mRNA level declines transiently, but returns to control levels within 5 days. Expression of SkM2 transcripts is markedly activated, reaching a peak 3 days after axotomy and then declining to a maintained level at approximately 30% of peak. Chemical denervation with botulinum toxin results in higher levels of SkM2 mRNA, which by 7 days posttreatment are 7-fold greater than levels in paired axotomized muscles. SkM2 expression subsequently declines as functional reinnervation appears. Quantal acetylcholine release appears to play a major role in suppression of SkM2 expression in adult innervated or reinnervated muscle, whereas nonquantal factors in toxin-treated, but not axotomized, muscle may sustain high level SkM2 mRNA expression.

Chimeric study of sodium channels from rat skeletal and cardiac muscle

Two isoforms of voltage‐dependent Na channels, cloned from rat skeletal muscle, were expressed in Xenopus oocytes. The currents of rSkM1 and rSkM2 differ functionally in 4 properties: (i) tetrodotoxin (TTX) sensitivity, (ii) μ‐conotoxin (μ‐CTX) sensitivity, (iii) amplitude of single channel currents, and (iv) rate of inactivation. rSkM1 is sensitive to both TTX and μ‐CTX. rSkM2 is resistant to both toxins. Currents of rSkM1 have a higher single channel conductance and a slower rate of inactivation than those of rSkM2. We constructed (i) chimeras by interchanging domain 1 (D1) between the two isoforms, (ii) block mutations of 22 amino acids in length that interchanged parts of the loop between transmembrane segments S5 and S6 in both D1 and D4, and (iii) point mutations in the SS2 region of this loop in D1. The TTX sensitivity could be switched between the two isoforms by the exchange of a single amino acid, tyrosine‐401 in rSkM1 and cysteine‐374 in rSkM2 in SS2 of D1. By contrast most chimeras and point mutants had an intermediate sensitivity to μ‐CTX when compared with the wild‐type channels. The point mutant rSkM1 (Y401C) had an intermediate single‐channel conductance between those of the wild‐type isoforms, whereas rSkM2 (C374Y) had a slightly lower conductance than rSkM2. The rate of inactivation was found to be determined by multiple regions of the protein, since chimeras in which D1 was swapped had intermediate rates of inactivation compared with the wild‐type isoforms.

Structure, function and expression of voltage-dependent sodium channels

Voltage-dependent sodium channels control the transient inward current responsible for the action potential in most excitable cells. Members of this multigene family have been cloned, sequenced, and functionally expressed from various tissues and species, and common features of their structure have clearly emerged. Site-directed mutagenesis coupled with in vitro expression has provided additional insight into the relationship between structure and function. Subtle differences between sodium channel isoforms are also important, and aspects of the regulation of sodium channel gene expression and the modulation of channel function are becoming topics of increasing importance. Finally, sodium channel mutations have been directly linked to human disease, yielding insight into both disease pathophysiology and normal channel function. After a brief discussion of previous work, this review will focus on recent advances in each of these areas.

Muscle surface membranes. Preparative methods affect apparent chemical properties and neurotoxin binding

Considerable disagreement exists between results reported by various authors for lipid composition and enzyme activity in purified muscle membrane fractions presumed to be sarcolemma, although an explanation for these discrepancies has not been presented. We have prepared muscle light surface membrane fractions of comparable density (1.050--1.120) by a low-salt sucrose method and by an LiBr-KCl extraction procedure and compared them for density profile, total lipid and cholesterol content, protein composition and ATPase activity. In addition, sodium channels characteristic of excitable membranes have been quantitated in each preparation using [3H]saxitoxin binding assays, and the density of acetylcholine receptors determined in fractions from control and denervated muscle using alpha-[125I]bungarotoxin. Although both fractions contain predominantly surface membrane, the LiBr fraction consistently shows the higher specific activity of p-nitrophenylphosphatase, higher free cholesterol content, and higher density of sodium channels and acetylcholine receptors. The density distribution of sodium channels appears uniform throughout both fractions. Quantitative differences were seen between sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of membrane proteins from the two preparations although most bands are represented in both. A majority of the low-salt sucrose light membrane proteins were accessible in varying degrees to labelling with diazotized diiodosulfanylic acid in intact muscle. These results suggest that light surface membrane fractions may be mixtures of sarcolemma and T-tubular membranes. Using our preparative methods, the LiBr fraction may contain predominantly sarcolemma while low-salt sucrose light membranes may be enriched in T-tubular elements.