Differences in action potential signalling in nerve and muscle are due, in part, to the existence of voltage-sensitive sodium channels with distinct functional and pharmacological properties. The broad goal of this grant has been to elucidate the molecular basis of diversity of the mammalian sodium channel, by exploiting the combined strengths of molecular biology and electrophysiology: We propose to: 1) further define the biological properties of a unique DNA element, termed Repressor Element 1 (RE1), which silences the type II promoter in cells which do not express the endogenous type II sodium channel gene. The precise nucleotides required for RE1 activity will be resolved by standard DNA-binding and cell transfection assays, as well as by analysis of neurons of mice infected with recombinant Herpes simplex viruses containing the type II silencer element. 2) to characterize the transcription factor which binds to the type II RE1 sequence, termed REST. Using the partial cDNA we have already isolated, we will construct a full-length cDNA encoding REST to test several hypotheses about the mechanism used to silence the type II sodium channel gene. We will test, by RNA hybridization and immunoprecipitation studies, the hypothesis that type II-expressing cells lack REST mRNA and protein. We will also test the hypothesis that REST mediates the silencing of two other genes containing RE1 sequences, SCG10 and synapsin I, by co-transfection of full-length REST cDNA and RE1-containing reporter genes, into cell types which lack the factor. Whether REST is required for repression of the endogenous type II gene will be examined by generating fibroblasts cell lines stably expressing antisense REST RNA. The appearance of sodium current in the selected fibroblast cell lines will be detected electrophysiologically, and confirmed by RNA hybridization or PCR methods. Because REST will likely represent a new class of transcription factor, we will identify, using mobility-shift and transfection studies using chimeric fusion genes, the DNA-binding and functional domains in the protein. 3) We will use immunoprecipitation and protein overlay assays to see if REST associates with other factors in nuclear extracts, and characterize another negatively-acting element in the type II gene, termed RE2. 4) We will further characterize the up-regulation by Nerve growth factor of a newly described sodium channel mRNA, termed Peripheral Neuron I (PN1), co-expressed with the type II sodium channel gene in PC12 cells. We will clone a full-length cDNA for PN1 as a first step toward resolving the mechanism for its gene regulation by neuronal growth factor. The gene encoding the adult type skeletal muscle sodium channel, the initial sequence and cDNA cloning of which was supported by this grant, is defective in several types of human myotonias. The studies described herein will contribute to further understanding of this gene family.
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