The voltage-dependent sodium channel is the basis of electrical excitability in mammalian nerve and muscle cells. This proposal decribes experiments to elucidate the structure of the rat sodium channel, to measure expression of the sodium channel gene in response to neuronal growth factors, and to identify and characterize regulatory sequences in the gene. The structural gene (cDNA) for the rat muscle sodium channel will be isolated from a bacteriophage expression library by screening with polyclonal and monoclonal antibodies directed against purified sodium channel proteins. The nucleotide sequence of the muscle cDNA clones will be used to deduce the primary structure of the sodium channel proteins. Northern blot and S1 nuclease hybridization assays will be performed to determine whether muscle and nerve sodium channels are identical. A sensitive assay system for measuring changes in sodium channel mRNA production will be established using an NGF-inducible cell line (PC-12) and the characterized cDNA clones as probes. These probes will be used in Northern blot assays to detect changes in sodium channel mRNA levels after treatment of PC-12 cells with NGF. If an effect on transcription of the sodium channel gene is observed, experiments will be performed to identify the NGF-responsive sequences in the gene. These studies involve constructing expression vectors containing the sodium channel gene fused to bacterial gene sequences. The recombinants will be introduced into PC-12 cells and assayed for fusion gene products in the presence and absence of NGF. Because cAMP may be involved in the mechanism by which NGF exerts its effects on PC-12 cells, cAMP will also be tested for its ability to regulate expression of the sodium channel gene. These experiments will provide a framework for understanding the molecular mechanisms underlying the development of excitability in neuronal cells.
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