This project studies G protein-gated inward rectifier K+ channels (GIRKs), continuing electrophysiological studies and also emphasizing biochemical studies of interactions between GIRKs and other molecules of signal transduction. (1) We shall address the physiological significance of the GIRK-integrin interaction. We shall study possible colocalization of the two molecules with confocal immunocytochemistry, study the identity of the integrin(s), and test for interactions between other inward rectifier K channels (non G protein-gated) and integrins. We shall test the hypothesis that the interaction also affects signal transduction by integrins. (2) We shall address the hypothesis that cells lacking normal RGS protein function will display postsynaptic responses. We shall study the detains of RGS action at the single-channel level, compare effects of RGS proteins on the kinetics of modulation at either GIRK or Ca/2+ channels, and use BIAcore measurements to address the hypothesis of ternary complexes among G proteins, RGS proteins, and GIRK channels. (3) We shall investigate surface exposure and topology of residues in GIRK channels using incorporated biocytin, domain structure and domain organization using site-specific nitrobenzyl-induced photochemical proteolysis of the main chain (SNIPP), and kinetic aspects of signal transduction using flash decaging of phosphorylatable tyrosine residues. (4) We shall investigate the hypothesis that the pathophysiology of the weaver mutation arises because of regenerative Na+ fluxes. We shall simultaneously conduct Ca2+ and Na+ imaging and electrophysiological investigations on weaver granule cells in culture following sudden activation of the GIRK channels. We shall investigate the hypothesis that some cells are spared because weaver channels appear in the cell membrane but are inactivated or inhibited. We shall attempt to understand the basis for activation by intracellular Na+. G protein-gated K+ channels (GIRKs) control the strength and frequency of the heartbeat, some responses to drugs of therapy and abuse, and some aspects of insulin secretion.
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