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

I. Niki, M. Welsh, P. O. Berggren, P. Hubbard, S. J.H. Ashcroft

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

The glibenclamide receptor, a putative ATP-sensitive K+ channel in the hamster pancreatic β-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 μM 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 K(d) of 3.3 nM and a B(max) 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 β-cell can be solubilized in an active form retaining specificity for sulphonylureas. ADP, which inhibits [3H]glibenclamide binding to microsomal preparations or intact HIT β-cells, did not inhibit binding to the solubilized receptor. Incubation of intact HIT β-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.

Original languageEnglish (US)
Pages (from-to)619-624
Number of pages6
JournalBiochemical Journal
Volume277
Issue number3
DOIs
StatePublished - 1991

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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