A novel, voltage-gated sodium channel cDNA, designated NaCh6, has been isolated from the rat central and peripheral nervous systems. RNase protection assays showed that NaCh6 is highly expressed in the brain, and NaCh6 mRNA is as abundant or more abundant than the mRNAs for previously identified rat brain sodium channels. In situ hybridization demonstrated that a wide variety of neurons express NaCh6, including motor neurons in the brainstem and spinal cord, cerebellar granule cells, and pyramidal and granule cells of the hippocampus. RT-PCR and/or in situ hybridization showed that astrocytes and Schwann cells express NaCh6. Thus, this sodium channel is broadly distributed throughout the nervous system and is shown to be expressed in both neurons and glial cells.

Sodium (Na) channel cDNAs were synthesized from RNA isolated from rat brain, cardiac muscle, and skeletal muscle. Partial cDNAs coding for the largest cytoplasmic loop of the Na channel were amplified with PCR. Sequence analysis of these cDNAs revealed that Na channel cDNAs originally described as brain genes were also expressed in both cardiac and skeletal muscle. Some of these cDNAs were isoforms that differed by insertions or deletions and can be explained by alternative choices of a 5' splice site. Southern blot analysis of genomic DNA confirmed the presence of introns in this region of the gene. Transcripts of multiple isoforms were detected with RNase protection in brain, heart, and skeletal muscle. Several conclusions can be drawn from the data. (1) Some rat sodium channel genes are transcribed in all excitable tissues studied here: brain, cardiac muscle, and skeletal muscle. (2) Each of these three tissues expresses multiple sodium channel genes. (3) Alternative splicing of sodium channel transcripts occurs in these tissues. (4) Expression of multiple genes and alternative splicing of the transcripts is responsible for at least seven different sodium channel mRNAs in skeletal muscle.

The voltage-activated Na channel (NaCh) is an integral membrane protein that is enriched at the neuromuscular end plate. Using loose-patch voltage-clamp and immunofluorescence, we have found that the aggregation of NaChs occurs late, during maturation of the neuromuscular junction. A decline in expression of embryonic NaCh mRNA and increase in adult NaCh mRNA precedes the onset of aggregation, and the appearance of functional adult NaChs coincides with NaCh aggregation. We tested the possibility that only the adult NaCh subtype could aggregate during development and found that both the embryonic and adult isoforms become concentrated at the synapse. The NaCh is the first postsynaptic membrane protein shown to become clustered postnatally, and the mechanism producing this aggregation appears to be different from the process producing aggregation of other synaptic proteins.

1. Loose-patch voltage-clamp recordings were made from rat and mouse skeletal muscle fibres denervated for up to 6 weeks. Innervated muscles possessed a Na+ current density of 107 + 3-3 mA cm-2 in endplate membrane, and 6'3 + 0-6 mA cm-2 in extrajunctional membrane. This high concentration of Na+ channels at the endplate was gradually reduced following denervation. After 6 weeks of denervation, the endplate Na+ channel concentration was reduced by 40-50 %, and the density of Na+ channels in extrajunctional membrane was increased by about 30 %.2. The tetrodotoxin (TTX)-resistant form of the Na+ channel appeared after 3 days of denervation and comprised -43 % of the endplate Na+ channels 5-6 days after denervation. Subsequently, TTX-resistant Na+ channels were reduced in density to -25% of the postjunctional Na+ channels and remained at this level up to 6 weeks after denervation.3. RNase protection analysis showed that mRNA encoding the TTX-resistant Na+ channel was virtually absent in innervated muscle, rose > 50-fold after 3 days of denervation, then decreased by 95% 6 weeks after denervation. The density of TTX-resistant Na+ channels correlated qualitatively with changes in mRNA levels.4. These results suggest that the density of Na+ channels at neuromuscular junctions is maintained by two mechanisms, one influenced by the nerve terminal and the other independent of innervation.