I propose to continue our ongoing studies of regulatory protein complexes associated with calcium dependent potassium channels. The focus will be on Slob, a protein from the fruit fly Drosophila that binds to and modulates the large conductance calcium-dependent potassium channel (dSlo) encoded by the fly Slowpoke gene. Slowpoke potassium channels in flies, rodents and humans play a major role in the regulation of neuronal excitability, and in the release of neurotransmitters and neurohormones. This proposal will build on several unexpected findings during the current funding period, most notably that: (1) there are several different Slob mRNA and protein products produced from a single gene by alternative splicing; (2) Slob protein level cycles in a circadian manner, and Slob may participate in the output of the circadian clock; (3) Slob expression is particularly prominent in a small set of identified neurons in the fly brain; and (4) mutant flies in which Slob expression is decreased exhibit locomotor rhythm defects and survive food deprivation much longer than their wild type counterparts. We now will test the hypothesis that Slob, via its modulation of dSlo, influences critical physiological processes such as feeding and the circadian control of locomotor activity. Heterologous cell expression will be used to define the binding interactions and ion channel modulatory activity of different Slob splice variants, and the patterns of expression and circadian cycling of the different variants of Slob will be investigated in vivo. The role of Slob in the modulation of dSlo current in fly brain neurons in vivo, and in circadian rhythms and feeding, will be explored by the construction of transgenic flies and the use of physiological and behavioral assays. We are fortunate to have available a unique combination of molecular, biochemical, genetic and electrophysiological tools that can be brought to bear on this fundamental problem of the role of an ion channel regulatory complex in the control of behavior in an intact organism.
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