(taken from the application) It is now well established that the most distal stages of the beta cell insuli secretory machinery involves plasma membrane (PM) ion channels (L-type voltage-dependent Ca2+ channel, sulfonoylurea receptor- SUR/K/ATP, and K/Ca channels) and exocytotic proteins, collectively called SNAREs on the PM and insulin secretory granules (SG). Much of the recent attention has been directe towards the structure and function of the recently cloned SUR/K/ATP, and Ca2+ channels on the PM, and the role of SNARE proteins in insulin SG exocytosis. I contrast, little is known about beta cell K/Ca channel regulation, or the role of SURs on the SGs, which comprise 90% of beta cellular SUR. This proposal hypothesizes that SNARE proteins directly modulate beta cell K/Ca activity and insulin SG-SUR. Towards the first aim of elucidating the mechanisms by which SNARE proteins regulate the beta cell K/Ca channel function, preliminary patch clamp data hav been generated to demonstrate that overexpression of a mutant SNARE protein in insulinoma HIT cells sensitized the K/Ca channel to closure. This resulted in potentiation of glucose-evoked insulin secretion, as closure of K/Ca maintains the PM in a depolarized state after glucose stimulation. Using patch clamp methods, the studies proposed are threefold, including: 1) to identify the functional domains within this SNARE protein which interact with the K/Ca channel in a manner that amplifies insulin secretion, and 2) to explore for other SNARE proteins which might interact with this mutant SNARE to form complexes that further modulate the K/Ca channel, 3) to apply the insights gained from these studies in beta cell lines to a) normal islet beta cells, to amplify the insulin secretory response, and thereby identify potential novel therapeutic targets for drug development; and b) diabetic islet beta cells, to explore for possible distortions of SNARE-K/Ca interactions which might be a basis for the insulin secretory defects of diabetes. Towards the second aim of elucidating the functional interactions of SNAREs an insulin SG-SUR, the hypotheses are that these interactions may serve: 1) to effect SG-SG (homotypic) fusion as a mechanism which enhances insulin exocytosis, or 2) to recruit SURs from this reserve pool in the SGs to the PM where SURs can exert its action. For the proposed studies, the required antibody and cDNA probes (many already subcloned into expression vectors) have been obtained or generated. The combination of molecular biological (to manipulate the expression of these proteins), biochemical (to identify protein binding interactions), morphologic (to detect cellular translocation) and functional-patch clamp methods (to elucidate functional interactions) are in place.
