J. Membrane Biol. 137, 59-70 (1994) The Journal of Membrane Bioloc , 9 Springer Verlag New York Inc. 1994 ATP-sensitive K + Channel Opener Acts as a Potent CI- Channel Inhibitor in Vascular Smooth Muscle Cells K.O. Holevinsky, 2 Z. Fan, 1 M. Frame, 3 J.C. Makielski, 1 V. Groppi, 3 D.J. Nelson 1,2 IDepartment of Medicine, The University of Chicago, Chicago,Illinois 60637 2Department of Neurology,University of Chicago, Hospital Box 2030, 5841 S. Maryland Avenue, Chicago,Illinois 60637 3Department of CelI Biology,Upjohn Laboratories, Kalamazoo,Michigan 49001 Received: 4 June 1993/Revised:20 September 1993 Abstract. We describe the activation of a K + current and inhibition of a C1- current by a cyanoguanidine ac- tivator of ATP-sensitive K + channels (KATP) in the smooth muscle cell line A10. The efficacy of U83757, an analogue of pinacidil, as an activator of KAT P was confirmed in single channel experiments on isolated ventricular myocytes. The effects of U83757 were ex- amined in the clonal smooth muscle cell line A10 us- ing voltage-sensitive dyes and digital fluorescent imag- ing techniques. Exposure of A10 cells to U83757 (10 nM to 1 ~tM) produced a rapid membrane hyperpolar- ization as monitored by the membrane potential-sensi- tive dye bis-oxonol ([diBAC4(3)], 5 ~tM). The U83757- induced hyperpolarization was antagonized by glyburide and tetrapropylammonium (TPrA) but not by tetraethlyl- ammonium (TEA) or charybdotoxin (ChTX). The mol- ecular basis of the observed hyperpolarization was stud- ied in whole-cell, voltage-clamp experiments. Exposure of voltage-clamped cells to U83757 (300 nM to 300 ~tM) produced a hyperpolarizing shift in the zero current potential; however, the hyperpolarizing shift in rever- sal potential was associated with either an increase or decrease in membrane conductance. In solutions where E K = -82 mV and Ecl = 0 mV, the reversal potential of the U83757-sensitive current was approximately -70 mV in those experiments where an increase in mem- brane conductance was observed. In experiments in which a decrease in conductance was observed, the re- versal potential of the U83757-sensitive current was approximately 0 mV, suggesting that U83757 might be acting as a C1- channel blocker as well as a K +chan- Correspondence to: DJ. Nelson nel opener. In experiments in which C1- current acti- vation was specifically brought about by cellular swelling and performed in solutions where C1- was the major permeant ion, U83757 (300 nM tO 300 gM) pro- duced a dose-dependent current inhibition. Taken to- gether these results (i) demonstrate the presence of a K+-selective current which is sensitive to KAy P channel openers in A10 cells and (ii) indicate that the hyperpo- larizing effects of K + channel openers in vascular smooth muscle may be due to both the inhibition of C1- currents as well as the activation of a K+-selective cur- rent. Key words: Smooth muscle -- Glyburide -- Pinacidil -- Chloride channels -- Membrane potential -- Va- sodilation Introduction Potassium channels that are inhibited by intra,cellular ATP (KATP) were first characterized in vertebrate car- diac muscle (Noma, 1983) and have since been de- scribed in skeletal muscle (Spruce, Standen, & Stanfield, 1985; Quasthoff et al., 1990), and in smooth muscle (Standen et al., 1989). The vasorelaxation in vascular smooth muscle produced by compounds which act as KAT p channel agonists in cardiac and [3-cells has been attributed to specific effects on KAT p channels in smooth muscle (Quast & Cook, 1989; Standen et al., 1989). We have used a potent cyanoguanidine KAT P channel open- er, U83757, which activates KAT P in cardiac w~ntricu- lar myocytes to determine whether KAT P is expressed in cells from the smooth muscle cell line A10. The clon- al line A10 was derived from the thoracic aorta of the