Holevinsky K O, Fan Z, Frame M, Makielski J C, Groppi V, Nelson D J
Department of Neurology, University of Chicago, Illinois 60637.
J Membr Biol. 1994 Jan;137(1):59-70. doi: 10.1007/BF00234998.
We describe the activation of a K+ current and inhibition of a Cl- current by a cyanoguanidine activator 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 KATP was confirmed in single channel experiments on isolated ventricular myocytes. The effects of U83757 were examined in the clonal smooth muscle cell line A10 using voltage-sensitive dyes and digital fluorescent imaging techniques. Exposure of A10 cells to U83757 (10 nM to 1 microM) produced a rapid membrane hyperpolarization as monitored by the membrane potential-sensitive dye bis-oxonol ([diBAC4(3)], 5 microM). The U83757-induced hyperpolarization was antagonized by glyburide and tetrapropylammonium (TPrA) but not by tetraethlyl-ammonium (TEA) or charybdotoxin (ChTX). The molecular basis of the observed hyperpolarization was studied in whole-cell, voltage-clamp experiments. Exposure of voltage-clamped cells to U83757 (300 nM to 300 microM) produced a hyperpolarizing shift in the zero current potential; however, the hyperpolarizing shift in reversal potential was associated with either an increase or decrease in membrane conductance. In solutions where EK = -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 membrane conductance was observed. In experiments in which a decrease in conductance was observed, the reversal potential of the U83757-sensitive current was approximately 0 mV, suggesting that U83757 might be acting as a Cl- channel blocker as well as a K+ channel opener. In experiments in which Cl- current activation was specifically brought about by cellular swelling and performed in solutions where Cl- was the major permeant ion, U83757 (300 nM to 300 microM) produced a dose-dependent current inhibition. Taken together these results (i) demonstrate the presence of a K(+)-selective current which is sensitive to KATP channel openers in A10 cells and (ii) indicate that the hyperpolarizing effects of K+ channel openers in vascular smooth muscle may be due to both the inhibition of Cl- currents as well as the activation of a K(+)-selective current.
我们描述了在平滑肌细胞系A10中，ATP敏感性钾通道（KATP）的氰基胍激活剂对钾电流的激活作用以及对氯电流的抑制作用。在分离的心室肌细胞的单通道实验中，证实了匹那地尔类似物U83757作为KATP激活剂的功效。使用电压敏感染料和数字荧光成像技术，在克隆平滑肌细胞系A10中检测了U83757的作用。用膜电位敏感染料双氧杂萘醇（[diBAC4(3)]，5 μM）监测发现，将A10细胞暴露于U83757（10 nM至1 μM）会导致细胞膜迅速超极化。格列本脲和四丙基铵（TPrA）可拮抗U83757诱导的超极化，但四乙铵（TEA）或蝎毒素（ChTX）则不能。在全细胞膜片钳实验中研究了观察到的超极化的分子基础。将膜片钳细胞暴露于U83757（300 nM至300 μM）会使零电流电位产生超极化偏移；然而，反转电位的超极化偏移与膜电导的增加或减少有关。在EK = -82 mV且ECl = 0 mV的溶液中，在观察到膜电导增加的实验中，U83757敏感电流的反转电位约为 -70 mV。在观察到电导降低的实验中，U83757敏感电流的反转电位约为0 mV，这表明U83757可能既是一种钾通道开放剂，也是一种氯通道阻滞剂。在通过细胞肿胀特异性引起氯电流激活且在氯是主要通透离子的溶液中进行的实验中，U83757（300 nM至300 μM）产生了剂量依赖性电流抑制。综上所述，这些结果（i）证明了A10细胞中存在对KATP通道开放剂敏感的钾选择性电流，（ii）表明钾通道开放剂在血管平滑肌中的超极化作用可能是由于氯电流的抑制以及钾选择性电流的激活。
