The general goal of these studies is to use mutants of Drosophila melanogaster to study the structure, function, and genetic regulation of the voltage-sensitive sodium channel. Previous work has defined 4-6 different genetic loci which affect saxitoxin-binding to sodium channels or which affect propagation of action potentials. The current application aims to determine which of these genetic loci code for actual structural components of the channel and which are involved in channel regulation or processing. One goal of these studies is to purify the sodium channel from Drosophila to determine the size and polypeptide composition of this channel. Another goal is to clone a Drosophila sodium channel gene on the basis of homology with the Alpha-subunit cDNA sequence of the channel from the electric eel. The cloned Drosophila gene will be mapped by in situ hybridization to salivary gland polytene chromosomes to determine if it corresponds to any of the loci previously shown to affect sodium channels. Mutants affecting sodium channel structural components will be useful for structure/function analysis of the channel. The third goal of these studies is to determine whether the insect-specific polypeptide toxin from the scorpion Androctonus australis binds specifically to Drosophila sodium channels. If it does, mutants which affect saxitoxin binding will be analyzed for effects on binding of this scorpion toxin. Other studies will focus on genetic, behavioral, and biochemical analysis of second site mutations which either enhance or suppress the behavioral phenotypes of mutants known to affect saxitoxin-binding. Specifically, we will study an enhancer of seizure alleles and a suppressor of the no action potential mutant. This approach should allow us to identify new gene products which interact with the sodium channel or control its expression. Finally, a pharmacological screening strategy is proposed which should result in the isolation of mutations causing overproduction of sodium channels. This screen will involve selection of lines which are resistant to sodium channel antagonists and sensitive to channel agonists. These studies should define genetic mechanisms for the regulation of cell excitability and therefore will provide information of fundamental importance for understanding hereditary diseases affecting the nervous system.
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