The long-term goal of this project is to discover new methods to stimulate Ca2+-dependent exocytosis of neurotransmitters or hormones by tuning cellular excitability and thereby the intracellular Ca2+ signal. These new methods will then be used to develop useful therapeutic strategies to combat human diseases. Our immediate goal here is to develop specific reagents to increase membrane excitability to stimulate Ca2+- dependent exocytosis of insulin from pancreatic ? cells. Elevated glucose concentrations increase metabolism and cytosolic ATP in ? cells, which in turn inhibits the ATP-sensitive K+ (KATP) channel. Decreased KATP channel activity depolarizes the cell membrane, activating voltage-gated Ca2+ (CaV) channels. The CaV-mediated Ca2+ influx elevates intracellular Ca2+ concentration ([Ca2+]in), triggering insulin release. A KATP channel of the ? cell consists of the inward-rectifier K+ channel Kir6.2 (KCNJ11) and the modulatory subunit sulphonylurea receptor, SUR1 (ABCC8). Sulphonylureas, a major class of antidiabetic drugs, are generally believed to stimulate insulin secretion by acting on SUR1 and thereby indirectly suppressing Kir6.2 activity. Conceptually, Kir6.2 itself should then represent a more direct, and potentially effective antidiabetic drug target. It is noteworthy that several studies suggest that SUR1 also plays other roles besides inhibiting Kir6.2, such as interacting with syntaxin, a key protein involved in exocytosis. Furthermore, the sulphonylurea glibenclamide appears to promote exocytosis of insulin also by KATP-independent mechanisms. Additionally, in the past decade, many gain-of-function mutations of Kir6.2 or SUR1 (lowering KATP's ATP sensitivity) were found to underlie most cases of permanent neonatal diabetes mellitus (PNDM). Most of these patients have since been successfully treated with sulphonylureas, instead of the traditional insulin therapy. However, some PNDM patients with certain mutations are unresponsive to sulphonylureas. In principle, these patients might be treatable with a suitable Kir6.2 inhibitor. Unfortunately, to date, there are no Kir6.2 inhibitors available. To obtain experimental evidence for judging, on scientific grounds, if serious resources should be invested in developing a clinically useful Kir6.2 inhibitor to treat diabetes, we propose to: i. develop a prototype specific Kir6.2 inhibitor and thereby demonstrate the feasibility of such an endeavor, ii. investigate whether a Kir6.2 inhibitor can stimulate adequate insulin secretion, and iii. apply this inhibitor to test the hypothesis that a sulphonylurea stimulates insulin secretion also via KATP-independent mechanisms. If this hypothesis is correct, a Kir6.2 inhibitor may then not have all the side effects of sulphonylureas. We will perform our studies with a combination of electrophysiology, Ca2+ imaging, protein biochemistry, molecular biology, cell biology, and histology. Our proposal constitutes an innovative, proof-of-concept study of a novel antidiabetic strategy; its significance is underscored by the fact that 350 million people worldwide have diabetes, a growing pandemic that calls for the development of new, effective therapeutic strategies.