Signalling in the nervous system and in many other cell types in largely accomplished by ion channels. These molecules are water- filled pores in cell membranes that open and close in response to a wide variety of stimuli, such as membrane voltage, neurotransmitter molecules and intracellular messengers. Ions flowing through these channels transmit information either by directly acting as intracellular messengers themselves or by changing the membrane voltage. An important goal of modern neurobiology is to understand the molecular mechanisms by which channel molecules change their conformation in response to changes in membrane voltage. The elucidation of this voltage- dependent gating mechanism is important to our understanding of the basic cellular mechanisms of the nervous system, and their dysfunction during pathological conditions, such as epilespy. The understanding of molecular mechanisms of voltage-gating will require a combination of electrophysiological, molecular and structural techniques. This proposal focuses on an electrophysiological analysis of voltage-gated sodium and potassium channels in an experimental system tht is well suited for molecular manipulation of channel molecules, the fruit-fly Drosophila. Single-channel studies of gating-kinetics will be combined with genetic manipulations to analyze and characterize the gating of normal channels and alterations that occur in specific mutations. A number of Drosophila mutants have been described that are likely candidates for affecting sodium and potassium channels. These will be examined for alterations in channel-gating. Genes tht are strong candidates for altering channel-gating will be studied in detail with genetic and biophysical techniques. These include: the Shaker locus, which changes type A potassium channel gating, the seizure locus, which affects the amplitude of sodium currents in cultured neurns, and a region of the second chromosome that has been shown to be highly homologous to vertebrate sodium channels. The results of these studies will be important in determining the functional alterations that occur due to changes in amino-acid sequence in the mutant proteins.
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