The inward Na+ current underlying the action potential in nerve is terminated by inactivation. The

Voltage-sensitive sodium channels are responsible for the initiation and propagation of the action potential and therefore are important for neuronal excitability. Complementary DNA clones encoding the beta 1 subunit of the rat brain sodium channel were isolated by a combination of polymerase chain reaction and library screening techniques. The deduced primary structure indicates that the beta 1 subunit is a 22,851-dalton protein that contains a single putative transmembrane domain and four potential extracellular N-linked glycosylation sites, consistent with biochemical data. Northern blot analysis reveals a 1,400-nucleotide messenger RNA in rat brain, heart, skeletal muscle, and spinal cord. Coexpression of beta 1 subunits with alpha subunits increases the size of the peak sodium current, accelerates its inactivation, and shifts the voltage dependence of inactivation to more negative membrane potentials. These results indicate that the beta 1 subunit is crucial in the assembly, expression, and functional modulation of the heterotrimeric complex of the rat brain sodium channel.

The cytoplasmic linker connecting domains Il and IV of the voltage-gated Na+ channel is thought to be involved in fast inactivation. This linker is highly conserved among the various Na+ channels that have been cloned. In the rat brain ILA Na+ channel, it consists of 53 amino acids of which 15 are charged. To investigate the role of this linker in inactivation, we mutated all 15 of the charged residues in various combinations. AU but one of these mutants expressed functional channels, and all of these inactivated with kinetics similar to the wild-type channel. We then constructed a series of deletion mutations that span the HI-IV linker to determine if any region of the linker is essential for fast inactivation. Deletion of the first 10 amino acids completely eliminated fast inactivation in the channel, whereas deletion of the last 10 amino acids had no substantial effect on inactivation. These results demonstrate that some residues in the amino end of the HI-Iv linker are critical for fast Na -channel inactivation, but that the highly conserved positively charged and paired negatively charged residues are not essential.Voltage-gated Na+ channels are responsible for the fast depolarizing phase ofthe action potential. The duration ofthe action potential is at least partly determined by the rate with which Na+ channels enter the fast inactivated state (1).Although the molecular basis of fast Na+-channel inactivation is unknown, Armstrong and Bezanilla (2) proposed that it occurs by a positively charged inactivation particle binding to the cytoplasmic mouth of the channel. The apparent voltage dependence of inactivation would then result from electrostatic interactions between the positively charged inactivation particle and gating charges that move during channel activation. Aldrich and coworkers (3,4) have since shown that the "ball-and-chain" model can explain fast inactivation in voltage-dependent potassium channels.The short cytoplasmic segment that links homologous domains III and IV of the Na+ channel (LIII/Iv) is thought to play a critical role in the fast inactivation process. This conclusion is based on the findings that antibodies directed toward this region inhibit fast inactivation (5,6), that molecular "cuts" (7) and small insertions (8) in LIII/lv slow inactivation, and that phosphorylation of a single serine residue in LIII/Iv by protein kinase C slows inactivation (9).These results are consistent with a model in which part of LIII/Iv functions as an inactivation particle, which is tethered on both sides to the cytoplasmic face of the channel.LIII/Iv is highly conserved in all three rat brain Na+ channels that have been cloned. This linker contains 12 positively charged residues and 3 negatively charged residues, often in pairs and groups of three. It is possible that some or all of these charged residues form a critical part of an inactivation particle. Moorman et al. (10) have shown that mutating 6 of the 12 positively charged residues to either asparagine or glutamate in the slowly inacti...

We have cloned a Kv2 potassium channel from squid optic lobe termed sqKv2. Multiple overlapping sqKv2 cDNA clones differed from one another at specific positions by purine transitions. To test whether the purine transitions were generated by RNA editing, we compared a 360 nucleotide genomic sequence with corresponding cDNA sequences (encoding S4-S6) isolated from individual animals and lying on a single gene and exon. cDNA sequences differed from genomic sequence at 17 positions, resulting in 28 unique sequences. There was invariantly an adenosine in the genomic sequence and a guanosine in the edited cDNA sequences. Two of the edits altered the rates of channel closure and slow inactivation. These results extend selective RNA editing to invertebrate taxa and represents a novel mechanism for the posttranscriptional modulation of voltage-gated ion channels.

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Three synthetic oligodeoxynucleotides complementary to different parts of an RNA encoding a glycine receptor subunit were used to discriminate heterogenous mRNAs coding for glycine receptors in adult and neonatal rat spinal cord. Injection of the three antisense oligonucleotides into Xenopus oocytes specifically inhibited the expression of glycine receptors by adult spinal cord mRNA. In contrast, the antisense oligonucleotides were much less potent in inhibiting the expression of glycine receptors encoded by neonatal spinal cord mRNA. Northern blot analysis revealed that the oligonucleotides hybridized mostly to an adult cord transcript of P10 kilobases in size. This band was also present in neonatal spinal cord mRNA but its density was about one-fourth of the adult cord message. There was no intense band in the low molecular weight position (:2 kilobases), the existence of which was expected from electrophysiological studies with size-fractionated mRNA of neonatal spinal cord. Our results suggest that in the rat spinal cord there are at least three different types of mRNAs encoding functional strychnine-sensitive glycine receptors.Glycine is a major inhibitory neurotransmitter in the mammalian central nervous system and is predominant in the spinal cord. Neuronal inhibition induced by glycine arises from the opening of chloride channels operated by glycine receptors, which can be blocked by a selective antagonist, strychnine (1,2).Many attempts at elucidating the molecular structure of glycine receptors are in progress (3). We (4-11) and others (12-17) have shown that mRNAs extracted from the brain and spinal cord of various animals, including human, induce Xenopus oocytes to express functional voltage-dependent ionic channels and neurotransmitter receptors, including strychnine-sensitive glycine receptors. We have recently shown that the glycine receptor mRNA derived from adult rat spinal cord differed from that of adult rat cerebral cortex in molecular size and that some of the properties of the receptors encoded were also different (18). Moreover, the characteristics of the glycine receptor mRNA were shown to change with postnatal development of the spinal cord (18). Thus, the glycine receptor and its mRNA are not homogenous, but rather it appears that multiple glycine receptor mRNAs exist in the central nervous system.Recently, a cDNA clone, encoding the strychnine binding subunit of the glycine receptor from the spinal cord of 20-day-old rats, was isolated, sequenced, and expressed (19,20). The information derived from that sequence is very useful for distinguishing heterogenous glycine receptor mRNAs. Based on that sequence we prepared three synthetic oligodeoxynucleotides complementary to the mRNA coding for the strychnine binding protein. These antisense nucleotides were used to determine their ability to repress (see refs. 19 and 21) the synthesis of functional glycine receptors in Xenopus oocytes injected with native mRNA, and radiolabeled probes were used to detect glycine receptor mRNAs. MAT...