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

20 Citations (Scopus)

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
Pages (from-to)619-624
Number of pages6
JournalBiochemical Journal
Volume277
Issue number3
StatePublished - Sep 6 1991
Externally publishedYes

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Glyburide
Cricetinae
Cells
Cell Line
Labeling
glibenclamide receptor
Adenosine Triphosphate
Sulfonylurea Receptors
Derivatives
Membranes
Tolbutamide
Detergents
Adenosine Diphosphate
Polyacrylamide Gel Electrophoresis
Membrane Proteins
Binding Sites
Light

ASJC Scopus subject areas

  • Biochemistry

Cite this

Niki, I., Welsh, M., Berggren, P. O., Hubbard, P., & Ashcroft, S. J. H. (1991). Characterization of the solubilized glibenclamide receptor in a hamster pancreatic β-cell line, HIT T15. Biochemical Journal, 277(3), 619-624.

Characterization of the solubilized glibenclamide receptor in a hamster pancreatic β-cell line, HIT T15. / Niki, I.; Welsh, M.; Berggren, P. O.; Hubbard, P.; Ashcroft, S. J H.

In: Biochemical Journal, Vol. 277, No. 3, 06.09.1991, p. 619-624.

Research output: Contribution to journalArticle

Niki, I, Welsh, M, Berggren, PO, Hubbard, P & Ashcroft, SJH 1991, 'Characterization of the solubilized glibenclamide receptor in a hamster pancreatic β-cell line, HIT T15', Biochemical Journal, vol. 277, no. 3, pp. 619-624.
Niki I, Welsh M, Berggren PO, Hubbard P, Ashcroft SJH. Characterization of the solubilized glibenclamide receptor in a hamster pancreatic β-cell line, HIT T15. Biochemical Journal. 1991 Sep 6;277(3):619-624.
Niki, I. ; Welsh, M. ; Berggren, P. O. ; Hubbard, P. ; Ashcroft, S. J H. / Characterization of the solubilized glibenclamide receptor in a hamster pancreatic β-cell line, HIT T15. In: Biochemical Journal. 1991 ; Vol. 277, No. 3. pp. 619-624.
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