Ahn S C, Lee S J, Goo Y S, Sim J H, So I, Kim K W
Department of Physiology and Biophysics, Seoul National University College of Medicine, Korea.
Pflugers Arch. 2001 Jan;441(4):417-24. doi: 10.1007/s004240000446.
The effect of protein kinase C (PKC) on the Ca2+-activated K+ current (IK,Ca) in guinea-pig gastric myocytes was studied using the whole-cell voltage-clamp technique. At a holding potential of 0 mV, IK,Ca, recorded as spontaneous, transient, outwards currents (STOCs), was markedly inhibited, both in mean amplitude (54 +/- 5%) and frequency (60 +/- 8%) by 1 microM phorbol 12, 13 dibutyrate (PDBu, n = 6). These effects were antagonized by pretreatment with 10 nM bisindolylmaleimide I (n = 5), a selective inhibitor of PKC. The possibility that the inhibition of STOCs was due to direct channel inhibition by PKC was addressed using inside-out or open-cell-attached patch-clamp techniques, the latter established using beta-escin. PDBu did not alter the conductance or open probability of the KCa channel in any mode, suggesting that PKC does not inhibit the KCa channel directly. To study the involvement of the Na/Ca exchanger in the inhibition of STOCs by PDBu, its operation was prevented by replacing Na+ in the internal solution by tris(hydroxymethyl)aminomethane (TRIS) and external Na+ by equimolar K+ and Ca2+-activated inwards K+ currents recorded at a holding potential of 0 mV. Neither the mean amplitude (96 +/- 8%) nor the frequency of these currents was inhibited significantly by 1 microM PDBu (n = 5). Like PDBu, 5 microM 2-(2-[4-(4-nitrobenzyloxy)phenyl]ethyl) isothiourea methanesulphonate (KB-R7943), a selective inhibitor of the reverse mode Na/Ca exchanger, also inhibited the mean amplitude (45 +/- 6%) and frequency (26 +/- 2%) of STOCs at the holding potential of 0 mV (n=6). The results suggest that the suppression of STOCs by PKC is mediated by inhibition of the Na/Ca exchanger.
采用全细胞电压钳技术研究了蛋白激酶C（PKC）对豚鼠胃肌细胞中钙激活钾电流（IK,Ca）的影响。在0 mV的钳制电位下，IK,Ca表现为自发、短暂的外向电流（STOCs），1 μM佛波醇12,13 -二丁酸酯（PDBu，n = 6）使其平均幅度（54±5%）和频率（60±8%）均受到显著抑制。这些效应可被10 nM双吲哚马来酰胺I预处理（n = 5）拮抗，双吲哚马来酰胺I是PKC的选择性抑制剂。使用内面向外式或膜片贴附式膜片钳技术探讨了STOCs受抑制是否是由于PKC直接抑制通道，后者是用β - 七叶皂苷建立的。PDBu在任何模式下均未改变KCa通道的电导或开放概率，表明PKC不直接抑制KCa通道。为研究钠钙交换体在PDBu抑制STOCs中的作用，通过用三（羟甲基）氨基甲烷（TRIS）替代内液中的Na+以及用等摩尔K+替代外液中的Na+来阻止其运转，并在0 mV的钳制电位下记录钙激活内向钾电流。1 μM PDBu对这些电流的平均幅度（96±8%）和频率均无显著抑制作用（n = 5）。与PDBu类似，5 μM 2 -（2 - [4 -（4 - 硝基苄氧基）苯基]乙基）异硫脲甲磺酸盐（KB - R7943），一种反向模式钠钙交换体的选择性抑制剂，在0 mV的钳制电位下也抑制了STOCs的平均幅度（45±6%）和频率（26±2%）（n = 6）。结果表明，PKC对STOCs的抑制作用是通过抑制钠钙交换体介导的。
