Intracellular ATP-sensitive K+ channels in mouse pancreatic beta cells: against a role in organelle cation homeostasis

Diabetologia. 2006 Jul;49(7):1567-77. doi: 10.1007/s00125-006-0257-9. Epub 2006 May 12.

Abstract

Aims/hypothesis: 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/interpretation: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / metabolism*
  • ATP-Binding Cassette Transporters / physiology
  • Animals
  • Calcium / metabolism
  • Cells, Cultured
  • Homeostasis
  • Hydrogen-Ion Concentration
  • Insulin-Secreting Cells / metabolism*
  • Insulin-Secreting Cells / ultrastructure
  • Ion Transport
  • Membrane Potential, Mitochondrial
  • Mice
  • Multidrug Resistance-Associated Proteins / metabolism*
  • Multidrug Resistance-Associated Proteins / physiology
  • Organelles / metabolism
  • Potassium / metabolism*
  • Potassium Channels, Inwardly Rectifying / metabolism*
  • Potassium Channels, Inwardly Rectifying / physiology
  • Receptors, Drug
  • Secretory Vesicles / metabolism
  • Sulfonylurea Receptors
  • Tissue Distribution

Substances

  • ATP-Binding Cassette Transporters
  • Abcc8 protein, mouse
  • Kir6.2 channel
  • Multidrug Resistance-Associated Proteins
  • Potassium Channels, Inwardly Rectifying
  • Receptors, Drug
  • Sulfonylurea Receptors
  • Potassium
  • Calcium