title: Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis.
abstract: OBJECTIVE
  ATP-sensitive K(+) (K(ATP)) channels located on the beta cell plasma membrane play a critical role in regulating insulin secretion and are targets for the sulfonylurea class of antihyperglycaemic drugs. Recent reports suggest that these channels may also reside on insulin-containing dense-core vesicles and mitochondria. The aim of this study was to explore these possibilities and to test the hypothesis that vesicle-resident channels play a role in the control of organellar Ca(2+) concentration or pH.
  
  
  METHODS
  To quantify the subcellular distribution of the pore-forming subunit Kir6.2 and the sulfonylurea binding subunit SUR1 in isolated mouse islets and clonal pancreatic MIN6 beta cells, we used four complementary techniques: immunoelectron microscopy, density gradient fractionation, vesicle immunopurification and fluorescence-activated vesicle isolation. Intravesicular and mitochondrial concentrations of free Ca(2+) were measured in intact or digitonin-permeabilised MIN6 cells using recombinant, targeted aequorins, and intravesicular pH was measured with the recombinant fluorescent probe pHluorin.
  
  
  RESULTS
  SUR1 and Kir6.2 immunoreactivity were concentrated on dense-core vesicles and on vesicles plus the endoplasmic reticulum/Golgi network, respectively, in both islets and MIN6 cells. Reactivity to neither subunit was detected on mitochondria. Glibenclamide, tolbutamide and diazoxide all failed to affect Ca(2+) uptake into mitochondria, and K(ATP) channel regulators had no significant effect on intravesicular free Ca(2+) concentrations or vesicular pH.
  
  
  CONCLUSIONS
  A significant proportion of Kir6.2 and SUR1 subunits reside on insulin-secretory vesicles and the distal secretory pathway in mouse beta cells but do not influence intravesicular ion homeostasis. We propose that dense-core vesicles may serve instead as sorting stations for the delivery of channels to the plasma membrane.
authors.affiliation: Henry Wellcome Laboratories for Integrated Cell Signalling and Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol, BS8 1TD, UK.
authors.forename: A
authors.initials: A
authors.lastname: Varadi
authors.forename: A
authors.initials: A
authors.lastname: Grant
authors.forename: M
authors.initials: M
authors.lastname: McCormack
authors.forename: T
authors.initials: T
authors.lastname: Nicolson
authors.forename: M
authors.initials: M
authors.lastname: Magistri
authors.forename: K J
authors.initials: KJ
authors.lastname: Mitchell
authors.forename: A P
authors.initials: AP
authors.lastname: Halestrap
authors.forename: H
authors.initials: H
authors.lastname: Yuan
authors.forename: B
authors.initials: B
authors.lastname: Schwappach
authors.forename: G A
authors.initials: GA
authors.lastname: Rutter
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D050417:Insulin-Secreting Cells / Q000378:metabolism* / Q000648:ultrastructure; D017136:Ion Transport; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism* / Q000502:physiology; D015388:Organelles / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism* / Q000502:physiology; D011955:Receptors, Drug; D022142:Secretory Vesicles / Q000378:metabolism; D064233:Sulfonylurea Receptors; D014018:Tissue Distribution; D000099161:Kcnj11 Channel
chemical_list: D018528:ATP-Binding Cassette Transporters; D002118:Calcium; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011188:Potassium; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D000099161:Kcnj11 Channel; C577508:Abcc8 protein, mouse