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The beta-cell glibenclamide receptor is an ADP-binding protein.

ABSTRACT: The effects of ADP on [3H]glibenclamide binding to membranes and whole cells, the activity of the ATP-sensitive K+ channel (K-ATP channel), intracellular Ca2+ concentration and insulin secretion were studied in a hamster pancreatic beta-cell line, HIT T15. ADP dose-dependently inhibited [3H]glibenclamide binding to membranes and to whole cells in a competitive manner. ADP-agarose also inhibited the binding to whole cells. The activity of the K-ATP channel was assayed by measuring 86Rb efflux from whole cells. ADP inhibited the 86Rb efflux elicited either by diazoxide or by ATP depletion. In the presence, but not in the absence, of extracellular Ca2+, ADP evoked a rapid and sustained increase in intracellular Ca2+ concentration as estimated with the fluorescent dye quin 2. Insulin release from HIT cells was also increased by 0.5-2 mM-ADP in the presence of 0.5 mM-glucose. These effects of ADP on glibenclamide binding, K-ATP channel activity and insulin release were specific for ADP, and were not reproduced by any other nucleotide so far tested. The present findings strongly suggest that ADP and sulphonylureas have common binding sites on the extracellular side of beta-cell plasma membranes, where they inhibit the activity of the K-ATP channel, resulting in an increase in intracellular Ca2+ concentration and insulin release.

SUBMITTER: Niki I

PROVIDER: S-EPMC1131498 | biostudies-other | 1990 Jun

REPOSITORIES: biostudies-other

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Glibenclamide binding to sulphonylurea receptor subtypes: dependence on adenine nucleotides.

Project description:1: ATP-sensitive K(+) channels are composed of pore-forming subunits Kir6.2 and of sulphonylurea receptors (SURs); the latter are the target of the hypoglycaemic sulphonylureas like glibenclamide. Here, we report on the negative allosteric modulation by MgATP and MgADP of glibenclamide binding to SUR1 and to SUR2 mutants with high glibenclamide affinity, SUR2A(Y1206S) and SUR2B(Y1206S). 2: ATP, in the presence of an ATP-regenerating system to oppose hydrolysis during incubation, inhibited glibenclamide binding to SUR1 and SUR2B(Y1206S) by approximately 60%, to SUR2A(Y1206S) by 21%). Inhibition curves for the SUR2(Y1206S) isoforms were monophasic with IC(50) values of 5-10 microM; the curve for SUR1 was biphasic (IC(50) values 4.7 and 1300 microM). 3: Glibenclamide inhibition curves for ADP, performed in the presence of an ATP-consuming system to oppose ATP formation from ADP, were generally shifted rightwards and showed positive cooperativity, in particular with the SUR2(Y1206S) isoforms. 4: In the absence of the coupled enzyme systems, inhibition curves of MgATP or MgADP were generally shifted leftwards. This indicated synergy of MgATP and MgATP in acting together. 5: Coexpression of SUR1 and SUR2B(Y1206S) with Kir6.2 reduced both potency and efficacy of ATP in inhibiting glibenclamide binding; this was particularly marked for Kir6.2/SUR1. 6: The data show (a) that the inhibitory effects of ATP and ADP on glibenclamide binding differ from one another, (b) that they depend on the SUR subtype, and (c) that they are weakened by coexpression with Kir6.2.

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Characterization of the solubilized glibenclamide receptor in a hamster pancreatic beta-cell line, HIT T15.

Project description:The glibenclamide receptor, a putative ATP-sensitive K+ channel in the hamster pancreatic beta-cell line HIT T15, was solubilized by using the zwitterionic detergent CHAPS. [3H]Glibenclamide binding was dependent on the incubation time and on the concentration of soluble membrane protein. Over 80% of [3H]glibenclamide bound could be displaced with 1 microM non-labelled glibenclamide. The curve relating specific binding to the concentration of [3H]glibenclamide (1-20 nM) showed saturation kinetics. Scatchard analysis suggested a single class of non-interacting binding sites with a Kd of 3.3 nM and a Bmax. of 90 fmol/mg of protein. [3H]Glibenclamide binding to solubilized membranes was inhibited by glibenclamide, tolbutamide and meglitinide. The relative potency of these agents on binding of [3H]glibenclamide to solubilized membranes was similar to that observed with microsomal preparations and paralleled their effects on K-ATP channel activity, measured as 86Rb efflux. These data show that the sulphonylurea receptor in the pancreatic beta-cell can be solubilized in an active form retaining specificity for sulphonylureas. ADP, which inhibits [3H]glibenclamide binding to microsomal preparations or intact HIT beta-cells, did not inhibit binding to the solubilized receptor. Incubation of intact HIT beta-cells with 125I-glibenclamide derivative followed by exposure to u.v. light resulted in covalent labelling of a peptide of 65 kDa on SDS/PAGE. The extent of labelling increased with 125I-glibenclamide derivative concentration (1-20 nM) and was inhibited in the presence of excess unlabelled glibenclamide.

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The ht beta gene encodes a novel CACCC box-binding protein that regulates T-cell receptor gene expression.

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