Schwab A, Geibel J, Wang W, Oberleithner H, Giebisch G
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Membr Biol. 1993 Mar;132(2):125-36. doi: 10.1007/BF00239002.
We studied the mechanism of K+ channel activation by minoxidil-sulfate (MxSO4) in fused Madin-Darby canine kidney (MDCK) cells. Patch-clamp techniques were used to assess single channel activity, and fluorescent dye techniques to monitor cell calcium. A Ca(2+)-dependent inward-rectifying K+ channel with slope conductances of 53 +/- 3 (negative potential range) and 20 +/- 3 pS (positive potential range) was identified. Channel activity is minimal in cell-attached patches. MxSO4 initiated both transient channel activation and an increase of intracellular Ca2+ (from 94.2 +/- 9.1 to 475 +/- 12.6 nmol/liter). The observation that K+ channel activity of excised inside-out patches was detected only at Ca2+ concentrations in excess of 10 mumol/liter suggests the involvement of additional mechanisms during channel activation by MxSO4. Transient K+ channel activity was also induced in cell-attached patches by 10 mumol/liter of the protein kinase C activator 1-oleoyl-2-acetyl-glycerol (OAG). OAG (10 mumol/liter in the presence of 1.6 mmol/liter ATP) increased the Ca2+ sensitivity of the K+ channel in inside-out patches significantly by lowering the Km for Ca2+ from 100 mumol/liter to 100 nmol/liter. The channel activation by OAG was reversed by the protein kinase inhibitor H8. Staurosporine, a PKC inhibitor, blocked the effect of MxSO4 on K+ channel activation. We conclude that MxSO4-induced K+ channel activity is mediated by the synergistic effects of an increase in intracellular Ca2+ and a PKC-mediated enhancement of the K+ channel's sensitivity to Ca2+.
我们研究了硫酸米诺地尔（MxSO4）在融合的犬肾Madin-Darby（MDCK）细胞中激活钾通道的机制。采用膜片钳技术评估单通道活性，并用荧光染料技术监测细胞内钙。鉴定出一种钙依赖性内向整流钾通道，其斜率电导在负电位范围为53±3 pS，在正电位范围为20±3 pS。在细胞贴附式膜片中，通道活性极小。MxSO4引发了短暂的通道激活以及细胞内Ca2+的增加（从94.2±9.1增加到475±12.6 nmol/升）。仅在Ca2+浓度超过10 μmol/升时，才检测到切除的内向外膜片中的钾通道活性，这一观察结果表明，在MxSO4激活通道的过程中还涉及其他机制。蛋白激酶C激活剂1-油酰-2-乙酰甘油（OAG）（10 μmol/升）也在细胞贴附式膜片中诱导了短暂的钾通道活性。OAG（在1.6 mmol/升ATP存在下为10 μmol/升）通过将Ca2+的米氏常数从100 μmol/升降低到100 nmol/升，显著提高了内向外膜片中钾通道对Ca2+的敏感性。OAG对通道的激活作用被蛋白激酶抑制剂H8逆转。蛋白激酶C抑制剂星形孢菌素阻断了MxSO4对钾通道激活的作用。我们得出结论，MxSO4诱导的钾通道活性是由细胞内Ca2+增加和蛋白激酶C介导的钾通道对Ca2+敏感性增强的协同作用介导的。
