In type II diabetes, the insulin secreting beta cells of pancreatic islets fail to secrete insulin in sufficient quantities to maintain normal blood glucose levels. The resulting hyperglycemia can Iead to many serious complications. Therefore, understanding the mechanisms that mediate insulin secretion could lead to new therapies to prevent the onset and complications of Type II diabetes. Two Sub-classes of L-type Calcium channels, Cav1.2 and Cav1.3 are expressed in pancreatic beta cells. A """"""""knock in"""""""" method to introduce Cav1.2 and Cav1.3 mutant channels that are insensitive to the dihydropyridine (DHP) class of L-type channel blockers into the insulinoma cell line INS-1 has been described. In this system, the endogenous L-type channels can be """"""""shut off"""""""" with DHP drugs, thus pharmacologically isolating either Cav1.2 of Cav1.3 channels. Using this system, it is shown that Cav1.3 but not Cav1.2 channels can mediate glucose-stimulated insulin secretion, but that both channels can mediate KCI stimulated insulin secretion. Furthermore, the intracellular loop between homologous domains III and IV (II-III loop) of Cav1.3 but not Cav1.2 completely inhibits glucose-stimulated insulin secretion when over-expressed in INS-1 cells. To elucidate the mechanism and structural determinants of the specificity of Cav1.3 coupling glucose-stimulated insulin secretion in INS-1 cells, this proposal will: 1. Determine the mechanism whereby glucose stimulated insulin secretion is specifically coupled to Cav1.3. 2. Identify the molecular determinants of the preferential coupling of Cav1.3 to glucose-stimulated insulin secretion. 3. Identify proteins that interact with intracellular domains of Cav1.3 and mediate the specificity for glucose-induced insulin secretion. 4. Define functional roles for the distal C-terminal tails of Cav 1.2 and Cav1.3 in INS-1 cells. Although much of this work will be done in the INS-1 cell model, adenovirus vectors will be used to introduce mutant channels and channel fragments into primary rat beta cells to repeat key experiments in this more physiologically relevant system. This proposal will utilize techniques such as patch clamp whole-cell electrophysiology, confocal microscopy, and total internal reflection microscopy. This proposal is consistent with the PI's long-term goal of understanding L-type calcium channel modulation and cellular function.
