ATP-sensitive K ؉ (K ATP ) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated K ATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K ؉ ion-selective pore of the channel. K ATP channels couple cell metabolism to membrane potential in many tissues (1-7). Classical K ATP channels comprise two subunits: a receptor [SUR1 (8), SUR2A (9), or SUR2B (10)] of sulfonylureas such as glibenclamide and tolbutamide, widely used to treat noninsulin-dependent diabetes mellitus, and an inward rectifier K ϩ channel member, Kir6.2 (11,12). The pancreatic beta cell K ATP channel comprises SUR1 and Kir6.2 (11, 12), while the skeletal muscle and cardiac K ATP channel comprises SUR2A and Kir6.2 (9). The pancreatic beta cell K ATP channels, as ATP and ADP sensors, have been thought to play a critical role in the regulation of glucose-and sulfonylurea-induced insulin secretion (13). In fact, mutations of the SUR1 or Kir6.2 gene are known to cause familial hypoglycemia associated with unregulated insulin secretion (14-18). However, recent studies suggest that both glucose and the sulfonylureas might have additional effects distal to those on the K ATP channels (19-21). In addition, although different roles of the K ATP channels in the various tissues, including cytoprotection in heart and brain ischemia and excitability of muscles and neurons, have been proposed (22,23), no direct evidence has been available. To clarify the physiological roles of K ATP channels in various cellular functions directly, we generated K ATP channel-deficient mice by disruption of the Kir6.2 gene. In the present study, we have focused on the role of the K ATP channels in pancreatic beta cell function. Our data clearly demonstrate that both glucose-and sulfonylureainduced insulin secretion depend critically on K ATP channeldependent pathway, and also suggest that the K ATP channels in skeletal muscle are involved in insulin action. MATERIALS AND METHODSTargeting the Kir6.2 Gene. The Kir6.2 gene was cloned from a 129͞Sv mouse genomic DNA library (Stratagene) by using its cDNA probe. A targeting vector was constructed by inserting the neomycin-resistance gene at the XhoI site in Kir6.2. The herpes simplex virus thymidine kinase gene was inserted downstream (Fig. 1 A). The targeting vector was introduced into E14 embryonic stem (ES) cells by electroporation. The homologous recombinant clone was identified by Southern blot analysis, and homozygous mice (Kir6.2 Ϫ͞Ϫ ) were generated by the standard procedures.Electrophysiology and Measurements of Intracellular Calcium Concentrations ([Ca 2؉ ] i ). Pancreatic islets were isolated by collagenase digestion method (24), and dispersed islet cells were cultured in DMEM supplemented with 10% fetal bovine serum, plated into 3.5-cm dishes containing Cellocate Coverslips (Eppendorf), and incubated at 37°C for 24-72 hr before experiments. The whole-cell recordings, single-channel recordings, and measurements of [Ca 2ϩ ] i in single p...
We have cloned a cDNA encoding a novel protein of 302 amino acids (designated Noc2, no C2 domain) that has 40.7% amino acid identity with and 77.9% similarity to the N-terminal region of rabphilin-3A, a target molecule of Rab3A. However, unlike rabphilin-3A, Noc2 lacks two C2 domains that are thought to interact with Ca
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