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

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.