A member of the inwardly rectifying potassium channel family was cloned here. The channel, called BIR (Kir6.2), was expressed in large amounts in rat pancreatic islets and glucose-responsive insulin-secreting cell lines. Coexpression with the sulfonylurea receptor SUR reconstituted an inwardly rectifying potassium conductance of 76 picosiemens that was sensitive to adenosine triphosphate (ATP) (IKATP) and was inhibited by sulfonylureas and activated by diazoxide. The data indicate that these pancreatic beta cell potassium channels are a complex composed of at least two subunits--BIR, a member of the inward rectifier potassium channel family, and SUR, a member of the ATP-binding cassette superfamily. Gene mapping data show that these two potassium channel subunit genes are clustered on human chromosome 11 at position 11p15.1.

We have cloned an isoform of the sulfonylurea receptor (SUR), designated SUR2. Coexpression of SUR2 and the inward rectifier K+ channel subunit Kir6.2 in COS1 cells reconstitutes the properties of K(ATP) channels described in cardiac and skeletal muscle. The SUR2/Kir6.2 channel is less sensitive than the SUR/Kir6.2 channel (the pancreatic beta cell KATP channel) to both ATP and the sulfonylurea glibenclamide and is activated by the cardiac K(ATP) channel openers, cromakalim and pinacidil, but not by diazoxide. In addition, SUR2 binds glibenclamide with lower affinity. The present study shows that the ATP sensitivity and pharmacological properties of K(ATP) channels are determined by a family of structurally related but functionally distinct sulfonylurea receptors.

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.

Stimulus-secretion coupling is an essential process in secretory cells in which regulated exocytosis occurs, including neuronal, neuroendocrine, endocrine, and exocrine cells. While an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) is the principal signal, other intracellular signals also are important in regulated exocytosis. In particular, the cAMP signaling system is well known to regulate and modulate exocytosis in a variety of secretory cells. Until recently, it was generally thought that the effects of cAMP in regulated exocytosis are mediated by activation of cAMP-dependent protein kinase (PKA), a major cAMP target, followed by phosphorylation of the relevant proteins. Although the involvement of PKA-independent mechanisms has been suggested in cAMP-regulated exocytosis by pharmacological approaches, the molecular mechanisms are unknown. Newly discovered cAMP-GEF/Epac, which belongs to the cAMP-binding protein family, exhibits guanine nucleotide exchange factor activities and exerts diverse effects on cellular functions including hormone/transmitter secretion, cell adhesion, and intracellular Ca(2+) mobilization. cAMP-GEF/Epac mediates the PKA-independent effects on cAMP-regulated exocytosis. Thus cAMP regulates and modulates exocytosis by coordinating both PKA-dependent and PKA-independent mechanisms. Localization of cAMP within intracellular compartments (cAMP compartmentation or compartmentalization) may be a key mechanism underlying the distinct effects of cAMP in different domains of the cell.

The oxidation of glucose represents a major source of metabolic energy for mammalian cells. However, because the plasma membrane is impermeable to polar molecules such as glucose, the cellular uptake of this important nutrient is accomplished by membrane-associated carrier proteins that bind and transfer it across the lipid bilayer. Two classes of glucose carriers have been described in mammalian cells: the Na(+)-glucose cotransporter and the facilitative glucose transporter. The Na(+)-glucose cotransporter transports glucose against its concentration gradient by coupling its uptake with the uptake of Na+ that is being transported down its concentration gradient. Facilitative glucose carriers accelerate the transport of glucose down its concentration gradient by facilitative diffusion, a form of passive transport. cDNAs have been isolated from human tissues encoding a Na(+)-glucose-cotransporter protein and five functional facilitative glucose-transporter isoforms. The Na(+)-glucose cotransporter is expressed by absorptive epithelial cells of the small intestine and is involved in the dietary uptake of glucose. The same or a related protein may be responsible for the reabsorption of glucose by the kidney. Facilitative glucose carriers are expressed by most if not all cells. The facilitative glucose-transporter isoforms have distinct tissue distributions and biochemical properties and contribute to the precise disposal of glucose under varying physiological conditions. The GLUT1 (erythrocyte) and GLUT3 (brain) facilitative glucose-transporter isoforms may be responsible for basal or constitutive glucose uptake. The GLUT2 (liver) isoform mediates the bidirectional transport of glucose by the hepatocyte and is responsible, at least in part, for the movement of glucose out of absorptive epithelial cells into the circulation in the small intestine and kidney. This isoform may also comprise part of the glucose-sensing mechanism of the insulin-producing beta-cell. The subcellular localization of the GLUT4 (muscle/fat) isoform changes in response to insulin, and this isoform is responsible for most of the insulin-stimulated uptake of glucose that occurs in muscle and adipose tissue. The GLUT5 (small intestine) facilitative glucose-transporter isoform is expressed at highest levels in the small intestine and may be involved in the transcellular transport of glucose by absorptive epithelial cells. The exon-intron organizations of the human GLUT1, GLUT2, and GLUT4 genes have been determined. In addition, the chromosomal locations of the genes encoding the Na(+)-dependent and facilitative glucose carriers have been determined. Restriction-fragment-length polymorphisms have also been identified at several of these loci.(ABSTRACT TRUNCATED AT 400 WORDS)