Information is encoded and transmitted in the nervous system in the form of electrical impulses or action potentials. Neurons are characterized by an electrically excitable membrane that enables them to receive, process and relay these impulses. Transmembrane proteins, called ion channels, form voltage-sensitive, ion-specific pores in these cells and carry out the key functions in nerve signalling by mediating the fluxes of particular ions across the membrane. Sodium channels in particular perform the central role in the generation and propagation of action potentials. Despite important recent advances, many unanswered questions remain concerning the structure, function and regulation of sodium channels. The long term goal of the work proposed here is to elucidate the function and regulation of sodium channels in Drosophila by use of a combined genetic and molecular approach. We will concentrate on the para locus, mutations of which cause lethality or temperature-sensitive paralysis correlated with a block in nerve conduction. We have cloned the para locus, determined the sequence of the encoded protein and demonstrated that para is a sodium channel structural gene. The existence of other putative sodium channel loci in Drosophila besides para suggests that, as in mammals, these genes comprise a small family whose members are differentially utilized and may subserve physiologically distinct functions. To understand the function and regulation of para in the Drosophila nervous system we now propose to characterize its expression and the distribution of its gene product and to examine the perturbations caused by mutations at para and two other loci (nap and tip-E) that apparently interfere with expression or function or para. To accomplish these goals we will use NOrthern blot and nuclease S1 experiments to determine the abundance and developmental profile of para transcripts in wild type. The normal spatial distribution of para transcripts will be characterized by tissue in situ hybridization. para- specific antibodies will be raised against synthetic peptides or para-lacZ fusion proteins and used for immunolocalization of the paraprotein in the nervous system and to identify it on Western blots. To elucidate the phenotypic effects of para, nap and tip-E mutations, similar experiments will be carried out on these mutants to determine their effect on the structure, abundance or distribution of the para transcript or protein. Finally, we will develop germline transformation for para by use of a hybrid genomic/cDNA fusion gene to pave the way for future experiments that will utilize site-directed mutagenesis for more detailed studies of para function and regulation in vivo. Because para is the only sodium channel structural gene in any organism that has been mutated in situ, it provides a unique opportunity to obtain novel insights into to molecular mechanisms of sodium channel function, expression and regulation in vivo. Since a number of human neurogenetic disease are known to be associated with perturbations in the function of ion channels, the information we obtain may have significant implications for the understanding and treatment of these disorders.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Neurology C Study Section (NEUC)
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University of Wisconsin Madison
Schools of Earth Sciences/Natur
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