ATP-sensitive K-channels in HIT T15 beta-cells studied by patch-clamp methods, 86Rb efflux and glibenclamide binding.
ATP-sensitive K-channels in the cloned beta-cell line HIT T15 were studied by patch-clamp methods; by measurement of 86Rb efflux; and by [3H]glibenclamide binding to isolated membrane preparations. In inside-out patches a 50 pS K-channel was found which was blocked by ATP or tolbutamide applied to the intracellular membrane surface. A minimum estimate of about 500 channels per beta-cell was obtained by combining whole-cell and single-channel data. The rate of efflux of 86Rb from 86RbCl-loaded HIT cells was markedly increased by intracellular ATP-depletion; 86Rb-efflux was progressively inhibited by increasing concentrations of glibenclamide or tolbutamide. In non-ATP-depleted cells, diazoxide elicited a concentration-dependent stimulation of 86Rb-efflux which was completely blocked by 1 microM glibenclamide. Isolated membranes showed dose-dependent saturable binding of [3H]glibenclamide to both high (Kd = 1.12 nM) and low (Kd = 136 nM) affinity binding sites. We estimate about 5000 high-affinity binding sites per cell. [3H]-glibenclamide binding was inhibited by tolbutamide (IC50 = 125 microM) but was not affected by diazoxide. ADP (0.5 or 1.0 mM) markedly reduced binding; other nucleotides tested were ineffective.
Niki
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Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, Great Britain.
Kelly
R P
RP
Ashcroft
S J
SJ
Ashcroft
F M
FM
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002461:Cell Line, Transformed; D002463:Cell Membrane Permeability / Q000502:physiology; D006224:Cricetinae; D004553:Electric Conductivity; D005905:Glyburide / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism*; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D012414:Rubidium Radioisotopes / Q000378:metabolism*; D014044:Tolbutamide / Q000494:pharmacology
D015221:Potassium Channels; D012414:Rubidium Radioisotopes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014044:Tolbutamide; D005905:Glyburide