VOLTAGE GATED ION CHANNELS--STRUCTURE & EXPRESSION

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Ion channels are the fundamental elements of nerve and muscle excitability and, as such, are fundamental to all animal behavior. A comprehensive understanding of these important molecules must relate molecular structure and design to the biophysical and physiological roles of these proteins. An ideal system of analysis would permit the cloning of ion channel genes and the reintroduction of the cloned genes in a variety of cell types for various studies of synthesis and biophyiscal function. The general aim of this proposal is to develop this comprehensive system in Drosophila for manipulating and studying ion channels. The specific aims of this proposal are: (1) The elucidation of the DNA sequence and genomic organization of a putative Drosophila ion channel gene. The first gene to be studied was isolated with a cDNA probe to the vertebrate sodium channel and partial sequencing has shown that it has close homology with this channel. The gene maps cytogenetically to a chromosomal region where mutations confer behavioral and physiological defects. (2) A. The introduction of a cloned ion channel gene into the germ line of Drosophila by P element-mediated gene transfer, and B. The experimental induction of ion channel gene expression by constructing a hybrid gene which puts an ion channel structural gene under control of the heat shock promoter, a strong promoter which can induce gene expression in most cell types. Animals transformed with this gene will be assayed for: (a) inducibility of mRNA; (b) insertion of protein into membranes; (c) voltage-gated ion conductance. One ultimate aim of these experiments will be to positively identify the voltage gating mechanism of this molecule; a portion of the gene coding for a string of positively charged amino acids hypothesized to be gating charges will be altered by site-directed mutagenesis and the resulting biophysical phenotypes analyzed by voltage and patch clamp techniques. (3) Investigating the hypothesis that ion channels are members of an extended gene family and that shared homology will provide a way in Drosophila to clone and analyze a variety of ion channel genes.