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ATP-sensitive K-channels in HIT T15 β-cells studied by patch-clamp methods, 86Rb efflux and glibenclamide binding

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Abstract

ATP-sensitive K-channels in the cloned β-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 β-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 μM glibenclamide. Isolated membranes showed dose-dependent saturable binding of [3H]glibenclamide to both high (K d=1.12 nM) and low (K d=136 nM) affinity binding sites. We estimate about 5000 high-affinity binding sites per cell. [3H]-glibenclamide binding was inhibited by tolbutamide (IC50=125 μM) but was not affected by diazoxide. ADP (0.5 or 1.0 mM) markedly reduced binding; other nucleotides tested were ineffective.

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Authors and Affiliations

  1. Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, OX3 9DU, Headington, Oxford, Great Britain

    I. Niki & S. J. H. Ashcroft

  2. University Laboratory of Physiology, Parks Rd., OX1 3PT, Oxford, Great Britain

    R. P. Kelly & Frances M. Ashcroft

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  1. I. Niki
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  2. R. P. Kelly
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  3. S. J. H. Ashcroft
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  4. Frances M. Ashcroft
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Niki, I., Kelly, R.P., Ashcroft, S.J.H. et al. ATP-sensitive K-channels in HIT T15 β-cells studied by patch-clamp methods, 86Rb efflux and glibenclamide binding. Pflugers Arch. 415, 47–55 (1989). https://doi.org/10.1007/BF00373140

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  • DOI: https://doi.org/10.1007/BF00373140

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