Na channels perform the central role in the generation and propagation of action potentials, which are the primary form of electrical signalling in the nervous system. The long term objective of the proposed work is to elucidate the molecular mechanisms that underlie neural signalling. Here, the investigator focuses on analysis of the structure, function, and regulation of two Na channel structural genes in Drosophila, called para and Dsc. These Na channel genes comprise a small family whose members appear to be differentially utilized and could subserve physiologically distinct functions. The goals are to understand the respective functions of these two Na channel genes in the Drosophila nervous system, the basis of their differential expression, and the phenotypic consequences of mutations. The investigator will approach these goals using a combination of genetic, molecular, and electrophysiological techniques. To characterize Dsc, cDNAs representing the entire open reading frame will be isolated and sequenced; null mutations will be generated by imprecise excision of P element insertions within the gene; and behavioral and electrophysiological phenotypes of these mutants alone and in various double mutant combinations will be characterized. To characterize further the spatial and developmental expression of para and Dsc and to elucidate some of the regulatory mechanisms governing their expression, the investigator will: raise para- and Dsc-specific antisera for immunolocalization of the corresponding channel polypeptides; examine the cellular distribution of the large array of para splice forms using exon- specific probes for in situ hybridization, splice-dependent reporter constructs, or single-cell PCR analysis; and identify the upstream transcriptional regulatory sequences of para for use in germline transformation experiments. To explore structure-function relationships of para and Dsc, the investigator will determine the exact lesion in a large collection of in vivo-generated para mutations using a sensitive SSCP technique in combination with sequence analysis. Electrophysiological recording of Na currents in cultured Drosophila neurons and in heterologous expression systems will be used to compare the properties of channels encoded by wild-type and mutant alleles of para and Dsc as well as by the different splice variants of para. Finally, the investigator proposes to use a PCR-based strategy to search for additional Na channel genes in Drosophila that will be targeted for mutagenesis and phenotypic analysis. Because para and Dsc are the only neuronal Na channel genes in any organism that have been mutated in situ, these genes provide unique opportunities to obtain novel insights into the molecular mechanisms of Na channel expression, function, and regulation, in vivo. A number of human hereditary neuromuscular diseases are known to be associated with perturbation in the structure or function of ion channels, including Na channels. Therefore, the results obtained from these studies could have direct significance for the understanding and possible treatment of these disorders.
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