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...

ATP-sensitive potassium (K(ATP)) channels are present in many tissues, including pancreatic islet cells, heart, skeletal muscle, vascular smooth muscle, and brain, in which they couple the cell metabolic state to its membrane potential, playing a crucial role in various cellular functions. The K(ATP) channel is a hetero-octamer comprising two subunits: the pore-forming subunit Kir6.x (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptor SUR (SUR1 or SUR2). Kir6.x belongs to the inward rectifier K(+) channel family; SUR belongs to the ATP-binding cassette protein superfamily. Heterologous expression of differing combinations of Kir6.1 or Kir6.2 and SUR1 or SUR2 variant (SUR2A or SUR2B) reconstitute different types of K(ATP) channels with distinct electrophysiological properties and nucleotide and pharmacological sensitivities corresponding to the various K(ATP) channels in native tissues. The physiological and pathophysiological roles of K(ATP) channels have been studied primarily using K(ATP) channel blockers and K(+) channel openers, but there is no direct evidence on the role of the K(ATP) channels in many important cellular responses. In addition to the analyses of naturally occurring mutations of the genes in humans, determination of the phenotypes of mice generated by genetic manipulation has been successful in clarifying the function of various gene products. Recently, various genetically engineered mice, including mice lacking K(ATP) channels (knockout mice) and mice expressing various mutant K(ATP) channels (transgenic mice), have been generated. In this review, we focus on the physiological and pathophysiological roles of K(ATP) channels learned from genetic manipulation of mice and naturally occurring mutations in humans.

cAMP is well known to regulate exocytosis in various secretory cells, but the precise mechanism of its action remains unknown. Here, we examine the role of cAMP signaling in the exocytotic process of insulin granules in pancreatic beta cells. Although activation of cAMP signaling alone does not cause fusion of the granules to the plasma membrane, it clearly potentiates both the first phase (a prompt, marked, and transient increase) and the second phase (a moderate and sustained increase) of glucoseinduced fusion events. Interestingly, all granules responsible for this potentiation are newly recruited and immediately fused to the plasma membrane without docking (restless newcomer). Importantly, cAMP-potentiated fusion events in the first phase of glucose-induced exocytosis are markedly reduced in mice lacking the cAMP-binding protein Epac2 (Epac2 ko/ko ). In addition, the small GTPase Rap1, which is activated by cAMP specifically through Epac2 in pancreatic beta cells, mediates cAMP-induced insulin secretion in a protein kinase A-independent manner. We also have developed a simulation model of insulin granule movement in which potentiation of the first phase is associated with an increase in the insulin granule density near the plasma membrane. Taken together, these data indicate that Epac2/Rap1 signaling is essential in regulation of insulin granule dynamics by cAMP, most likely by controlling granule density near the plasma membrane.insulin secretion ͉ total internal reflection fluorescence microscopy ͉ pancreatic beta cell

Although cAMP is well known to regulate exocytosis in many secretory cells, its direct target in the exocytotic machinery is not known. Here we show that cAMP-GEFII, a cAMP sensor, binds to Rim (Rab3-interacting molecule, Rab3 being a small G protein) and to a new isoform, Rim2, both of which are putative regulators of fusion of vesicles to the plasma membrane. We also show that cAMP-GEFII, through its interaction with Rim2, mediates cAMP-induced, Ca2+-dependent secretion that is not blocked by an inhibitor of cAMP-dependent protein kinase (PKA). Accordingly, cAMP-GEFII is a direct target of cAMP in regulated exocytosis and is responsible for cAMP-dependent, PKA-independent exocytosis.

Reaction to stress requires feedback adaptation of cellular functions to secure a response without distress, but the molecular order of this process is only partially understood. Here, we report a previously unrecognized regulatory element in the general adaptation syndrome. Kir6.2, the ion-conducting subunit of the metabolically responsive ATP-sensitive potassium (KATP) channel, was mandatory for optimal adaptation capacity under stress. Genetic deletion of Kir6.2 disrupted KATP channel-dependent adjustment of membrane excitability and calcium handling, compromising the enhancement of cardiac performance driven by sympathetic stimulation, a key mediator of the adaptation response. In the absence of Kir6.2, vigorous sympathetic challenge caused arrhythmia and sudden death, preventable by calcium-channel blockade. Thus, this vital function identifies a physiological role for KATP channels in the heart. I on channels control the electrical potential across the cell membrane of all living organisms. The profile of channel expression within the cell is defined by evolution through natural selection (1, 2). Developed as channel͞enzyme multimers, K ATP channels combine properties of two different classes of protein to adjust rapidly and precisely membrane excitability according to the metabolic state of the cell (3-7). Identified in metabolically active tissues of a broad range of species, K ATP channels were discovered originally in heart muscle where they are expressed in high density (8, 9). Functional cardiac K ATP channels can be formed only through physical association of the pore-forming Kir6.2 subunit with the regulatory sulfonylurea receptor SUR2A (10-12). In this complex, which harbors an intrinsic ATPase activity, nucleotide interaction at SUR2A gates potassium permeation through Kir6.2, a property believed to be responsible for the fine metabolic modulation of membrane potential-dependent cellular functions (7,(13)(14)(15)(16).The physiological role of K ATP channels as metabolic sensors has been understood best in the regulation of hormone secretion in pancreatic ␤-cells and more recently in the hypothalamus (17-21). In the heart, definition of the function of this protein complex thus far has been limited to acute protection against ischemic events (22). In fact, under ischemia, the opening of as few as 1% of K ATP channels is sufficient to produce significant shortening of the cardiac action potential (23), manifested globally by ST-segment elevation on the electrocardiogram (24). Yet, beyond the impact in pathophysiology, a physiological role for the cardiac K ATP channel that supports its maintenance in hearts of many species is lacking (25).The general adaptation syndrome is a ubiquitous reaction vital for self-preservation under conditions of stress such as exertion or fear (26-28). Mediated by a catecholamine surge, this syndrome generates an alteration of physiologic and biochemical functions to sustain a superior level of bodily performance and allows confrontation or escape in response to threat...

Glucose-responsive (GR) neurons in the hypothalamus are thought to be critical in glucose homeostasis, but it is not known how they function in this context. Kir6.2 is the pore-forming subunit of K(ATP) channels in many cell types, including pancreatic beta-cells and heart. Here we show the complete absence of both functional ATP-sensitive K+ (K(ATP)) channels and glucose responsiveness in the neurons of the ventromedial hypothalamus (VMH) in Kir6.2-/- mice. Although pancreatic alpha-cells were functional in Kir6.2-/-, the mice exhibited a severe defect in glucagon secretion in response to systemic hypoglycemia. In addition, they showed a complete loss of glucagon secretion, together with reduced food intake in response to neuroglycopenia. Thus, our results demonstrate that KATP channels are important in glucose sensing in VMH GR neurons, and are essential for the maintenance of glucose homeostasis.

Incretins such as glucagon-like peptide-1 and gastric inhibitory polypeptide/glucose-dependent insulinotropic peptide are known to potentiate insulin secretion mainly through a cAMP/protein kinase A (PKA) signaling pathway in pancreatic ␤-cells, but the mechanism is not clear. We recently found that the cAMP-binding protein cAMP-GEFII (or Epac 2), interacting with Rim2, a target of the small G protein Rab3, mediates cAMP-dependent, PKA-independent exocytosis in a reconstituted system. In the present study, we investigated the role of the cAMP-GEFII⅐Rim2 pathway in incretin-potentiated insulin secretion in native pancreatic ␤-cells. Treatment of pancreatic islets with antisense oligodeoxynucleotides (ODNs) against cAMP-GEFII alone or with the PKA inhibitor H-89 alone inhibited incretinpotentiated insulin secretion ϳ50%, while a combination of antisense ODNs and H-89 inhibited the secretion ϳ80 -90%. The effect of cAMP-GEFII on insulin secretion is mediated by Rim2 and depends on intracellular calcium as well as on cAMP. Treatment of the islets with antisense ODNs attenuated both the first and second phases of insulin secretion potentiated by the cAMP analog 8-bromo-cAMP. These results indicate that the PKA-independent mechanism involving the cAMP-GEFII⅐Rim2 pathway is critical in the potentiation of insulin secretion by incretins.

The inwardly rectifying K(+) channel Kir6.1 forms K(+) channels by coupling with a sulfonylurea receptor in reconstituted systems, but the physiological roles of Kir6.1-containing K(+) channels have not been determined. We report here that mice lacking the gene encoding Kir6.1 (known as Kcnj8) have a high rate of sudden death associated with spontaneous ST elevation followed by atrioventricular block as seen on an electrocardiogram. The K(+) channel opener pinacidil did not induce K(+) currents in vascular smooth-muscle cells of Kir6.1-null mice, and there was no vasodilation response to pinacidil. The administration of methylergometrine, a vasoconstrictive agent, elicited ST elevation followed by cardiac death in Kir6.1-null mice but not in wild-type mice, indicating a phenotype characterized by hypercontractility of coronary arteries and resembling Prinzmetal (or variant) angina in humans. The Kir6.1-containing K(+) channel is critical in the regulation of vascular tonus, especially in the coronary arteries, and its disruption may cause Prinzmetal angina.