Rapid communication, often between widely separated regions of the nervous system, is accomplished by excitable cells that encode and transmit information via electrical signals. Voltage-gated sodium channels play a key role in electrical excitability of cells in vertebrate and invertebrate nervous systems. With the long term goal of understanding the regulation of electrical excitability during normal development and in pathologically altered conditions, the current proposal utilizes Drosophila, an organism in which a combination of physiological and molecular techniques can be employed, to examine the function and regulation of voltage-gated sodium channels, encoded by the para gene, in primary neurons. Our preliminary studies have shown that alternative splicing of the para sodium channel pre-mRNA, in the region encoding the first cytoplasmic loop, results in generation of four mRNAs that differ in their potential for phosphorylation by protein kinase A (PKA). Correlation of para mRNA profiles with whole cell electrophysiological recordings, in single embryonic neurons, suggests that one of the isoforms containing a PKA site (encoded by exon a) is necessary for sodium current expression. The first study in this proposal will determine if sodium channel activity in Drosophila neurons can be modulated by phosphorylation and if this is correlated with expression of para mRNA isoforms encoding specific PKA sites. In a second series of experiments we will examine the effects, of selectively decreasing or eliminating specific splice variants implicated in regulating channel function, on sodium current expression. This will be accomplished by generating transgenic flies expressing heat-shock inducible ribozymes targeted to specific splice variants or exposure of cultured neurons to splice variant-specific antisense oligonucleotides. Sodium channel function, in mutants known to alter cAMP-dependent phosphorylation, will be examined to determine the effects of endogenously altered levels of phosphorylation on channel function. In the third section, correlative studies to identify additional regions of alternative splicing in the para gene involved in function, will be initiated. Since our long term interest is regulation of electrical excitability, the last series of studies is aimed at identification of genes encoding neuronal potassium and calcium channels. The technique of expression profiling will be used to screen for expression of candidate ion channel genes in embryonic neurons. Once we have identified the genes encoding the calcium and potassium channels, techniques developed in the previous sections will be applied to the study of regulation of these currents. To examine how expression of voltage-gated ion channel genes is coordinately regulated to result in cell-specific firing properties, mRNA expression profiles of cells with unique firing properties will be compared. The results of these studies will increase our understanding of the roles of alternative splicing and phosphorylation in mediating ion channel function, as well as how coordinate regulation of ion channel gene expression influences the ability of cells in the developing nervous system to communicate through electrical impulses.
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