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.

Expression of diverse Na+ channel messenger RNAs in rat myocardium. Evidence for a cardiac-specific Na+ channel.

This study examined the diversity of Na+ channel gene expression in intact cardiac tissue and purified myocardial cells. The screening of neonatal rat myocardial cell cDNA libraries with a conserved rat brain Na+ channel cDNA probe, resulted in the isolation and characterization of a putative rat cardiac Na+ channel cDNA probe (pCSC-1). The deduced amino acid sequence of pCSC-1 displayed a striking degree of homology with the eel, rat brain-1, and rat brain-2 Na+ channel, thereby identifying pCSC-1 as a related member of the family of Na+ channel genes. Northern blot analysis revealed the expression of a 7-kb CSC-1 transcript in rat cardiac tissue and purified myocardial cells, but little or no detectable expression of CSC-1 in rat brain, skeletal muscle, denervated skeletal muscle, or liver. Using RNase protection and Northern blot hybridization with specific rat brain Na+ channel gene probes, expression of the rat brain-1 Na+ channel was observed in rat myocardium, but no detectable expression of the rat brain-2 gene was found. This study provides evidence for the expression of diverse Na+ channel mRNAs in rat myocardium and presents the initial characterization of a new, related member of the family of Na+ channel genes, which appears to be expressed in a cardiac-specific manner.

Selective induction of brain type II Na+ channels by nerve growth factor.

Cells derived from a rat pheochromocytoma (PC12 cells) can generate an action potential only upon treatment with nerve growth factor. Using electrophysiological methods, we found that the appearance of action potentials in nerve growth factor-treated PC12 cells can be explained by an increase in the density of Na+ channels. The functional properties of Na+ channels in PC12 cells are similar to those described for peripheral nerves but appear to be different from Na+ channels synthesized in Xenopus oocytes injected with brain type II Na+ -channel mRNA. To determine if PC12 cells express the brain type II Na+ -channel gene, we performed RNase-protection analyses using probes that can distinguish between the brain type I and type II Na+ -channel mRNAs. The results from these studies indicate that undifferentiated PC12 cells express the type II but not the type I Na+ -channel gene. Treatment with nerve growth factor increases expression of the type II Na+ -channel gene but has no effect on type I gene expression. Our findings suggest that Na+ -channel excitability in PC12 cells is due to the specific induction of the brain type II gene by nerve growth factor.

Regulation of muscle sodium channel transcripts during development and in response to denervation.

We have recently described the cloning and functional expression of a new sodium channel subtype, microI, isolated from a denervated rat skeletal muscle cDNA library. In studies described here, we have used RNase protection and Northern blot analyses to examine the expression of microI mRNA in different tissues and in neonatal, adult, and adult denervated muscle. We found that microI transcripts were not expressed in brain or heart, or in the myogenic cell line L6, even after differentiation to myotubes. Transcripts for microI were present at low levels in neonatal skeletal muscle and increased to maximum levels in adult tissue, paralleling the expression of tetrodotoxin (TTX)-sensitive sodium currents. Surprisingly, denervation of adult muscle was also followed by a rise in microI mRNA, at a time when TTX-insensitive currents reappear. These results show that expression of this channel subtype is regulated by tissue type, development, and innervation.

Modulation of sodium-channel mRNA levels in rat skeletal muscle.

Action potentials in many types of excitable cells result from changes in permeability to Na ions. Although these permeability changes in nerve and muscle are mediated by voltage-gated Na channels that are functionally similar, we found that the Na-channel gene expressed in skeletal muscle is different from the genes coding for two Na channels (type I and type II) in brain. Despite the structural differences between muscle and brain Na-channel genes, a cDNA clone derived from rat brain hybridizes to skeletal muscle Na-channel mRNA of approximately 9.5 kilobases. We used this cDNA probe to measure changes in Na-channel mRNA levels in skeletal muscle during development and following denervation. By blot hybridization analysis of electrophoretically fractionated RNA, we found that Na-channel mRNA can be detected as early as embryonic day 17 and that mRNA levels increase 2-fold between birth and postnatal day 35. Denervation of adult muscle causes a further 2- to 3-fold increase in muscle Na-channel mRNA levels, suggesting that expression of Na-channel genes in fast-twitch muscle may be regulated by the state of innervation.