title: The actions of a novel potent islet beta-cell specific ATP-sensitive K+ channel opener can be modulated by syntaxin-1A acting on sulfonylurea receptor 1.
abstract: Islet beta-cell-specific ATP-sensitive K(+) (K(ATP)) channel openers thiadiazine dioxides induce islet rest to improve insulin secretion, but their molecular basis of action remains unclear. We reported that syntaxin-1A binds nucleotide binding folds of sulfonylurea receptor 1 (SUR1) in beta-cells to inhibit K(ATP) channels. As a strategy to elucidate the molecular mechanism of action of these K(ATP) channel openers, we explored the possibility that 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) might influence syntaxin-1A-SUR1 interactions or vice versa. Whole-cell and inside-out patch-clamp electrophysiology was used to examine the effects of glutathione S-transferase (GST)-syntaxin-1A dialysis or green fluorescence protein/syntaxin-1A cotransfection on NNC55-0462 actions. In vitro pull-down binding studies were used to examine NNC55-0462 influence on syntaxin-1A-SUR1 interactions. Dialysis of GST-syntaxin-1A into the cell cytoplasm reduced both potency and efficacy of extracellularly perfused NNC55-0462 in a HEK cell line stably expressing Kir6.2/SUR1 (BA8 cells) and in rat islet beta-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST-syntaxin-1A and its H3 domain inhibited K(ATP) channels previously activated by NNC55-0462. This action on K(ATP) channels is isoform-specific to syntaxin-1A because syntaxin-2 was without effect. Furthermore, the parent compound diazoxide showed similar sensitivity to GST-syntaxin-1A inhibition. NNC55-0462, however, did not influence syntaxin-1A-SUR1 binding interaction. Our results demonstrated that syntaxin-1A interactions with SUR1 at its cytoplasmic domains can modulate the actions of the K(ATP) channel openers NNC55-0462 and diazoxide on K(ATP) channels. The reduced levels of islet syntaxin-1A in diabetes would thus be expected to exert a positive influence on the therapeutic effects of this class of K(ATP) channel openers.
authors.affiliation: Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Betty
authors.initials: B
authors.lastname: Ng
authors.forename: Youhou
authors.initials: Y
authors.lastname: Kang
authors.forename: Chadwick L
authors.initials: CL
authors.lastname: Elias
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.forename: Huanli
authors.initials: H
authors.lastname: Xie
authors.forename: John B
authors.initials: JB
authors.lastname: Hansen
authors.forename: Philip
authors.initials: P
authors.lastname: Wahl
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D003981:Diazoxide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D004594:Electrophysiology; D006801:Humans; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D015394:Molecular Structure; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011955:Receptors, Drug / Q000378:metabolism*; D064233:Sulfonylurea Receptors; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: C521934:6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno(3,2-e)-1,2,4-thiadiazine 1,1-dioxide; C577507:ABCC8 protein, human; D018528:ATP-Binding Cassette Transporters; C577509:Abcc8 protein, rat; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors; D050827:Syntaxin 1; D000255:Adenosine Triphosphate; D003981:Diazoxide