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...

Voltage-dependent calcium (Ca2+) channels are involved in many specialized cellular functions, and are controlled by intracellular signals such as heterotrimeric G-proteins, protein kinases and calmodulin (CaM). However, the direct role of small G-proteins in the regulation of Ca2+ channels is unclear. We report here that the GTP-bound form of kir/Gem, identified originally as a Ras-related small G-protein that binds CaM, inhibits high-voltage-activated Ca2+ channel activities by interacting directly with the beta-subunit. The reduced channel activities are due to a decrease in alpha1-subunit expression at the plasma membrane. The binding of Ca2+/CaM to kir/Gem is required for this inhibitory effect by promoting the cytoplasmic localization of kir/Gem. Inhibition of L-type Ca2+ channels by kir/Gem prevents Ca2+-triggered exocytosis in hormone-secreting cells. We propose that the small G-protein kir/Gem, interacting with beta-subunits, regulates Ca2+ channel expression at the cell surface.

Tight glycemic control in individuals with diabetes mellitus is essential to prevent or delay its complications. Present treatments to reduce hyperglycemia mainly target the ATP-sensitive K(+) (K(ATP)) channel of pancreatic beta cells to increase insulin secretion. These current approaches are often associated with the side effect of hypoglycemia. Here we show that inhibition of the activity of cyclin-dependent kinase 5 (Cdk5) enhanced insulin secretion under conditions of stimulation by high glucose but not low glucose in MIN6 cells and pancreatic islets. The role of Cdk5 in regulation of insulin secretion was confirmed in pancreatic beta cells deficient in p35, an activator of Cdk5. p35-knockout mice also showed enhanced insulin secretion in response to a glucose challenge. Cdk5 kinase inhibition enhanced the inward whole-cell Ca(2+) channel current and increased Ca(2+) influx across the L-type voltage-dependent Ca(2+) channel (L-VDCC) upon stimulation with high glucose in beta cells, but had no effect on Ca(2+) influx without glucose stimulation. The inhibitory regulation by Cdk5 on the L-VDCC was attributed to the phosphorylation of loop II-III of the alpha(1C) subunit of L-VDCC at Ser783, which prevented the binding to SNARE proteins and subsequently resulted in a decrease of the activity of L-VDCC. These results suggest that Cdk5/p35 may be a drug target for the regulation of glucose-stimulated insulin secretion.