Alternative Splicing in the Voltage-Gated Sodium Channel DmNav Regulates Activation, Inactivation, and Persistent Current

Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom Submitted 15 July 2009; accepted in final form 15 July 2009 Abstract Diversity in neuronal signaling is a product not only of differential gene expression, but also of alternative splicing. However, although recognized, the precise contribution of alternative splicing in ion channel transcripts to channel kinetics remains poorly understood. Invertebrates, with their smaller genomes, offer attractive models to examine the contribution of splicing to neuronal function. In this study we report the sequencing and biophysical characterization of alternative splice variants of the sole voltage-gated Na + gene ( DmNa v , paralytic ), in late-stage embryos of Drosophila melanogaster . We identify 27 unique splice variants, based on the presence of 15 alternative exons. Heterologous expression, in Xenopus oocytes, shows that alternative exons j , e , and f primarily influence activation kinetics: when present, exon f confers a hyperpolarizing shift in half-activation voltage ( V 1/2 ), whereas j and e result in a depolarizing shift. The presence of exon h is sufficient to produce a depolarizing shift in the V 1/2 of steady-state inactivation. The magnitude of the persistent Na + current, but not the fast-inactivating current, in both oocytes and Drosophila motoneurons in vivo is directly influenced by the presence of either one of a pair of mutually exclusive, membrane-spanning exons, termed k and L . Transcripts containing k have significantly smaller persistent currents compared with those containing L . Finally, we show that transcripts lacking all cytoplasmic alternatively spliced exons still produce functional channels, indicating that splicing may influence channel kinetics not only through change to protein structure, but also by allowing differential modification (i.e., phosphorylation, binding of cofactors, etc.). Our results provide a functional basis for understanding how alternative splicing of a voltage-gated Na + channel results in diversity in neuronal signaling. Address for reprint requests and other correspondence: R. Baines, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PL, UK (E-mail: Richard.Baines{at}manchester.ac.uk )