title: Targeting of voltage-gated K+ and Ca2+ channels and soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins to cholesterol-rich lipid rafts in pancreatic alpha-cells: effects on glucagon stimulus-secretion coupling.
abstract: Pancreatic alpha-cells secrete glucagon in response to low glucose to counter insulin actions, thereby maintaining glucose homeostasis. The molecular basis of alpha-cell stimulus-secretion coupling has not been fully elucidated. We investigated the expression of voltage-gated K(+) (K(V)) and Ca(2+) (Ca(V)) channels, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in pancreatic alpha-cells and examined their targeting to specialized cholesterol-rich lipid rafts. In alpha-cells, we detected the expression of K(V)4.1/4.3 (A-type current), K(V)3.2/3.3 (delayed rectifier current), Ca(V)1.2 (L-type current), Ca(V)2.2 (N-type current), and the SNARE (synaptosomal-associated protein of 25 kDa, syntaxin 1A, and vesicle-associated membrane protein 2) and SNARE-associated proteins (Munc-13-1 and Munc-18a). We also detected caveolin-2, a structural protein of cholesterol-rich lipid rafts. Of these proteins, caveolin-2, K(V)4.1/4.3, Ca(V)1.2, and SNARE proteins (syntaxin 1A, synaptosomal-associated protein of 25 kDa, and vesicle-associated membrane protein 2) target to lipid raft domains on alpha-cell plasma membranes. Disruption of lipid rafts by depletion of membrane cholesterol with methyl-beta-cyclodextrin decreased the association of K(V)4.1/4.3, Ca(V)1.2, and SNARE proteins with lipid rafts. This resulted in inhibition of A-type K(V) currents and enhancement of glucagon secretion from alpha-cells. Consistently, capacitance measurements of exocytosis of single alpha-cells showed enhanced exocytosis after membrane cholesterol depletion. Taken together, our results demonstrate the association of K(V)4, Ca(V)1.2, and SNARE proteins with lipid rafts in pancreatic alpha-cells. Glucagon secretion from alpha-cells is regulated by lipid rafts, and the dissociation of SNARE proteins from cholesterol-rich lipid raft domains enhances glucagon secretion.
authors.affiliation: Department of Medicine, University of Toronto, Ontario, Canada M5S 1A8.
authors.forename: Fuzhen
authors.initials: F
authors.lastname: Xia
authors.forename: Yuk M
authors.initials: YM
authors.lastname: Leung
authors.forename: Gregory
authors.initials: G
authors.lastname: Gaisano
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Gao
authors.forename: Yi
authors.initials: Y
authors.lastname: Chen
authors.forename: Jocelyn E Manning
authors.initials: JE
authors.lastname: Fox
authors.forename: Alpana
authors.initials: A
authors.lastname: Bhattacharjee
authors.forename: Michael B
authors.initials: MB
authors.lastname: Wheeler
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
authors.forename: Robert G
authors.initials: RG
authors.lastname: Tsushima
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000502:physiology*; D020864:Calcium Channels, N-Type / Q000502:physiology; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology; D005934:Glucagon / Q000378:metabolism*; D050416:Glucagon-Secreting Cells / Q000378:metabolism / Q000502:physiology*; D005947:Glucose / Q000494:pharmacology; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D018408:Patch-Clamp Techniques; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism; D051672:Shal Potassium Channels / Q000235:genetics / Q000502:physiology*; D012995:Solubility; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D050827:Syntaxin 1 / Q000378:metabolism
chemical_list: C514350:CACNA1C protein, mouse; C500963:Cacna1b protein, mouse; C503279:Cacna1b protein, rat; D020746:Calcium Channels, L-Type; D020864:Calcium Channels, N-Type; D050600:SNARE Proteins; D051672:Shal Potassium Channels; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D005934:Glucagon; D002784:Cholesterol; D005947:Glucose