Characterization of the solubilized glibenclamide receptor in a hamster pancreatic beta-cell line, HIT T15.
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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Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, U.K.
Welsh
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Berggren
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Hubbard
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Ashcroft
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SJ
D000345:Affinity Labels; D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D005905:Glyburide / Q000378:metabolism*; D007515:Islets of Langerhans / Q000737:chemistry*; D010777:Photochemistry; D015221:Potassium Channels / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D011955:Receptors, Drug / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D014044:Tolbutamide / Q000378:metabolism
D000345:Affinity Labels; D001549:Benzamides; D015221:Potassium Channels; D011955:Receptors, Drug; C067662:glibenclamide receptor; C030516:meglitinide; D014044:Tolbutamide; D005905:Glyburide