The Actions of a Novel Potent Islet β-Cell–Specific ATP-Sensitive K⁺ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1

Abstract

Islet β-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 β-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 β-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.