The broad goal of this revised program project grant is to reveal the molecular mechanisms controlling expression of genes encoding voltage-dependent ion channels, and to understand how the expression and subcellular targeting of different members of the gene family contribute to cell-specific firing properties in neurons. The thrust of the program is towards addressing these issues in the peripheral nervous system (PNS), but comparisons between channels in the PNS and CNS are proposed where this information provides specific insights into control mechanisms. Goal l seeks to identify the functional counterparts of specific sodium channel structures in neurons. The strategy will be to compare the functional """"""""fingerprints"""""""" of alpha and accessory sodium channel subunits expressed in Xenopus oocytes with the fingerprints of sodium currents expressed differentially in PC12 cells through stimulation of distinct signal transduction pathways. Goal 2 is to compare the mechanisms controlling sodium channel gene expression in the PNS and CNS. The DNA elements and transcription factors required for neural-specific expression of a sodium channel gene expressed exclusively in PNS neurons will be identified. Receptor domains and cytoplasmic molecular intermediates involved in the up-regulation of sodium channel genes by neuronal growth factors will be identified through multiple biochemical and genetic approaches. Goal 3 is to reveal the molecular mechanisms involved in targeting of sodium and potassium channels to distinct subcellular domains. Targeting in PC12 and MDCK cell lines, and in neurons, will be studied using genetic, biochemical, and immunochemical approaches. All of these goals exploit unique reagents provided by the program investigators. Core facilities will provide support, equipment, reagents, and technical expertise for molecular biology, tissue culture, and administrative functions. This program project has the potential to lead to new strategies for therapeutic treatment of sensory and sympathetic pathologies. The studies also represent an important first step toward an understanding of how ion channels involved in pain pathways are modulated in vivo.
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