Mutually exclusive exon splicing of type III brain sodium channel alpha subunit RNA generates developmentally regulated isoforms in rat brain.

We have identified two exons of the type III rat brain sodium channel alpha subunit gene that undergo mutually exclusive alternative RNA splicing to produce mRNAs coding either for an isoform predominant in neonatal brain (IIIN) or a different isoform (IIIA) predominant in the adult. These exons are 92 base pairs in length and encode amino acids 203-232, which correspond to part of the S3 and most of the S4 transmembrane segments within domain I and the extracellular loop between them. Despite 21 nucleotide differences between the exons, only a single amino acid at position 209 is altered, specifying either aspartic acid (IIIA) or serine (IIIN). As evidence that these isoforms are generated via alternative splicing, we demonstrate that both exons are encoded within the type III gene. The nucleotide sequences of the neonatal and adult type III exons and the intervening intron as well as the developmental regulation of this splicing are nearly identical in the type II sodium channel gene. The conservation of the exon/intron structure and of the developmentally regulated patterns of expression of the type II and III sodium channel genes suggests that alternative mRNA splicing of this exon may play a substantial role in modulating sodium channel function during brain development by alteration of a single amino acid.

Brain and heart sodium channel subtype mRNA expression in rat cerebral cortex.

The expression of mRNAs coding for the alpha subunit of rat brain and rat heart sodium channels has been studied in adult and neonatal rat cerebral cortex using the reverse transcription-polymerase chain reaction (RT-PCR). Rat brain sodium channel subtype I, II, IIA, and III sequences were simultaneously amplified in the same PCR using a single oligonucleotide primer pair matched to all four subtype sequences. Identification of each subtype-specific product was inferred from the appearance of unique fragments when the product was digested with specific restriction enzymes. By using this RT-PCR method, products arising from mRNAs for all four brain sodium channel subtypes were identified in RNA extracted from adult rat cerebral cortex. The predominant component was type IIA with lesser levels of types I, II, and III. In contrast, the type II and IIA sequences were the predominant RT-PCR products in neonatal rat cortex, with slightly lower levels of type III and undetectable levels of type I. Thus, from neonate to adult, type II mRNA levels decrease relative to type IIA levels. Using a similar approach, we detected mRNA coding for the rat heart sodium channel in neonatal and adult rat cerebral cortex and in adult rat heart. These results reveal that mRNAs coding for the heart sodium channel and all four previously sequenced rat brain sodium channel subtypes are expressed in cerebral cortex and that type II and IIA channels may be differentially regulated during development.