Single-gene mutations can be used to alter any given macromolecule or cellular process in the excitable cells. A proper collection of mutants should enable us to dissect the cellular structures or mechanisms of interest. The proposed research involves Drosophila mutants that have specific defects in membrane currents in nerve and muscle. Three different outward K+ currents have been identified in Drosophila muscles. The transient IA, delayed rectification IK, and Ca++ -dependent IC. These K+ currents are separately altered by Sh, eag, Hk and slo mutations. We will conduct voltage-clamp analysis of these currents in larval muscles to characterize the activation and inactivation properties, ionic selectivity and pharmacological sensitivity of these ionic channels and the mechanisms altered by the mutations. Using the patch-clamp and whole-cell clamp techniques we will extend this analysis to examine the mutational effects on the K+ channels in cultured Drosophila neurons. In addition, the specific alterations in Na current, which is present in nerve but not muscle, caused by nap ts and para ts mutations will be studied. Single-channel and whole-cell current experiments can provide an incisive analysis of the molecular mechanisms altered by these mutations. Previous studies in Drosophila suggest that certain types of channels may be composed of subunits and may share some subunits with other channel types. This possibility will be further investigated by studying the effects of a single-gene mutation on different channels and alterations in a channel type caused by mutations of different genes. The functional relationship among the gene products can be revealed by analysis of inter-actions between different mutations (alleles) of the same gene or mutations of different genes in heterozygous or double-mutant individuals. We will attempt to integrate these functional analysis with the recombinant DNA studies of the alleles of eag, para ts and nap ts in order to relate structure to function for channels and related proteins.
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