Alternative splicing of paralytic regulates functional properties of voltage-gates sodium channels in Drosophila

WEI-HSIANG LIN; DUNCAN E. WRIGHT; NARA I MURARO; RICHARD BAINES

Nueva York

Congreso; 13th biennial Neurobiology of Drosophila meeting; 2009

Cold Spring Harbor Laboratory

The voltage-gated sodium channel in Drosophila is encoded by a single gene, termed paralytic (para). Currently 11 alternatively-spliced exons have been reported that serve to increase functional channel diversity. However, which exon compositions are present at specific developmental stages and how each transcript contributes to alter channel properties are not well understood. In order to determine the most common splice isoforms present in late stage 17 embryos, RT-PCR was used to isolate 50 clones of the complete open reading frame. Comparing the exon compositions of these sequences revealed 27 unique splice isoforms. Exons i ,b, d, f and L are present in over 75% of clones, whilst exons j, c, e, h and k are uncommon in isoforms of this stage, being present in less than 25% of clones. The channel properties of the most common variants were determined by two-electrode voltage clamping following expression in Xenopus oocytes. Isoforms containing spliced exons j and e activate at more depolarized membrane potentials, while those containing exon f activate at hyperpolarsied potentials. By contrast, splice variants containing exon h inactivate at more depolarized membrane potentials. Finally, we show that isoforms containing exon k have a significantly smaller non-inactivating persistent sodium current compared to L. Interestingly, transcripts lacking all cytoplasmic alternatively-spliced exons still produce functional channels indicative that splicing may influence channel kinetics not only through change to protein structure, but also by allowing differential modification (i.e. phosphorylation, binding of co-factors etc). These data imply that the capacity for fine-tuning of the properties of voltage-gated sodium currents, in response to changing demands, might be regulated by expressing differing para sodium channel splice variants in the Drosophila CNS.