The electrically excitable cell membrane of neurons and muscle fibers these cells to receive, process, and transmit information within the nervous system. Electrical excitability is conferred by transmembrane proteins called channels that play the key roles in action potential propagation and synaptic transmission by mediating fluxes of specific ions across the membrane. Despite important recent advances, many unanswered questions remain concerning the structure, function, and regulation of ion channels. The long term goal of this work is to elucidate the molecular mechanisms that underlie membrane excitability by isolating and characterizing mutations in genes that encode ion channels or that otherwise affect their function. Experiments described in this proposal focus on the three mutations: eag (ether a go-go), slo (slowpoke), and nap (no action potential). We have shown that each of these mutations profoundly disrupts membrane excitability owing to specific defects: eag diminishes the function of one class of potassium channels; slo entirely abolishes the function of another class of potassium channels; nap reduces the number and/or function of sodium channels. However, the molecular lesions responsible for these defects are not yet understood. The mutations could alter genes encoding structural components of channels or cause perturbations in one of the cellular mechanisms thought to regulate channel activity or synthesis. Genetic, electrophysiological, and molecular experiments are proposed to elucidate the underlying defects in these mutants. We will complete the cloning of eag and nap, map the molecular limits of these loci, identify their transcripts, isolate and sequences cDNA clones and use this information to characterize the proteins encoded by these genes. We will perform a detailed cytogenetic analysis of slo to isolate new alleles and to assign slo a precise cytological location. Based on the information and new mutants thus generated the molecular analysis of slo will be initiated by chromosome walking or transposon tagging. The new slo alleles will be characterized electrophysiologically to determine whether they display the array of phenotypes expected for defects in a structural gene. Finally, we will identify mutations at new loci by continuing our screens for mutations that (1) suppress or enhance existing membrane excitability mutations or (2) display any locomotor defects such as paralysis or uncoordination. These studies will provide new information on the structure and function of ion channels and on the mechanisms that regulate their synthesis, assembly, membrane insertion, nd modulation. Since various human disorder are known to be associated with perturbations in the function of ion channels, the information we obtain in Drosophila should have broad biological and medical significance.
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