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In pancreatic β-cells, the predominant voltage-gated Ca2+ channel (CaV1.2) and K+ channel (KV2.1) are directly coupled to SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor) proteins. These SNARE proteins modulate channel expression and gating and closely associate these channels with the insulin secretory vesicles. We show that KV2.1 and CaV1.2, but not KV1.4, SUR1, or Kir6.2, target to specialized cholesterol-rich lipid raft domains on β-cell plasma membranes. Similarly, the SNARE proteins syntaxin 1A, SNAP-25, and VAMP.2, but not Munc-13-1 or n-Sec1, are associated with lipid rafts. Disruption of the lipid rafts by depleting membrane cholesterol with methyl-β-cyclodextrin shunts KV2.1, CaV1.2, and SNARE proteins out of lipid rafts. Furthermore, methyl-β-cyclodextrin inhibits KV2.1 but not CaV1.2 channel activity and enhances single-cell exocytic events and insulin secretion. Membrane compartmentalization of ion channels and SNARE proteins in lipid rafts may be critical for the temporal and spatial coordination of insulin release, forming what has been described as the excitosome complex. [ABSTRACT FROM AUTHOR]

Insulin secretion from pancreatic β-cells is mediated by the opening of Ca[sub v] channels and exocytosis is facilitated by the secretory SNARE protein machinery. We have observed these factors are sensitive to the acute removal of membrane cholesterol. However, less is known about the chronic changes in endogenous cholesterol and its biosynthesis in regulating β-cell stimulus-secretion coupling. We have examined the effects of inhibiting endogenous β-cell cholesterol biosynthesis by using a specific squalene epoxidase inhibitor, NB598. We detected the expression of squalene epoxidase in primary human, rat and mouse islet cells and clonal MIN6 and INS-1 cells. A cholesterol reduction of 40% - 52% from pancreatic islets from mouse and human was observed after being cultured with 10/µM NB598 for 48 hr. We found NB598 significantly inhibited both basal and glucose-stimulated insulin secretion from mouse and human pancreatic islets. Voltage-dependent Ca[sup 2+] (Ca[sub v]) channels were significantly reduced by NB598, an effect which may contribute to the observed impairment of insulin secretion. Rapid photolytic release of intracellular caged Ca[sup 2+]and simultaneous measurements of the changes in membrane capacitance revealed that NB598 also inhibited exocytosis primarily through reducing insulin granule mobilization. These results implicate the direct perturbation of β-cell exocytotic secretory proteins. Furthermore, NB598 accelerated the steady-state voltage dependence of voltage-gated K[sup +] (K[sub v]) channel inactivation, but did not affect the current density or channel activation. These data implicate endogenous cholesterol in pancreatic β-cells plays a critical role in regulating insulin secretion. Two distinct roles of cholesterol are involved in maintaining normal β-cell function. The first is through the functional regulation of Ca[sub v] and K[sub v] channels. The second is cholesterol is critical for the mobilization and fusion of insulin secretory granules with plasma membranes, in part, involving the formation of cholesterol-rich domain within the secretory vesicles and on the plasma membrane. [ABSTRACT FROM AUTHOR]

ATP-sensitive potassium (KATP) channels couple the metabolic status of the cell to its membrane potential to regulate a number of cell actions, including secretion (neurons and neuroendocrine cells) and muscle contractility (skeletal, cardiac, and vascular smooth muscle). KATP channels consist of regulatory sulfonylurea receptors (SUR) and pore-forming (Kir6.X) subunits. We recently reported (Pasyk, E. A., Kang, Y., Huang, X., Cui, N., Sheu, L., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 4234-4240) that syntaxin-1A (Syn-1A), known to mediate exocytotic fusion, was capable of binding the nucleotide binding folds (NBF1 and C-terminal NBF2) of SUR1 to inhibit the KATP channels in insulin-secreting pancreatic islet beta cells. This prompted us to examine whether Syn-1A might modulate cardiac SUR2A/KATP channels. Here, we show that Syn-1A is present in the plasma membrane of rat cardiac myocytes and binds the SUR2A protein (of rat brain, heart, and human embryonic kidney 293 cells expressing SUR2A/Kir6.2) at its NBF1 and NBF2 domains to decrease KATP channel activation. Unlike islet beta cells, in which Syn-1A inhibition of the channel activity was apparently mediated only via NBF1 and not NBF2 of SUR1, both exogenous recombinant NBF1 and NBF2 of SUR2A were found to abolish the inhibitory actions of Syn-1A on KATP channels in rat cardiac myocytes and HEK293 cells expressing SUR2A/Kir6.2. Together with our recent report, this study suggests that Syn-1A binds both NBFs of SUR1 and SUR2A but appears to exhibit distinct interactions with NBF2 of these SUR proteins in modulating the KATP channels in islet beta cells and cardiac myocytes. [ABSTRACT FROM AUTHOR]

Shows that SNARE protein syntaxin 1A binds and modulates rat islet at its cytoplasmic C terminus. Evidence that the SNARE proteins act not only on distinct sites within a K[sup +] (Kv) channel, but also on distinct sites between different voltage-gated Kv channel families; Cell culture and transfections; Use of confocal immunofluorescence microscopy.