Kim Y C, Kim S J, Kang T M, Suh S H, So I, Kim K W
Department of Physiology and Biophysics, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Korea.
Pflugers Arch. 1997 Aug;434(4):346-53. doi: 10.1007/s004240050407.
The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (ICCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. ICCh was induced by carbachol (CCh, 50 microM) at a holding potential of -30 mV or -60 mV. ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited ICCh concentration dependently in a reversible manner (53 +/- 8.6% at 1 microM, mean +/- SE, n = 11). In addition, amplitudes of ICCh were only 37 +/- 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca2+ channel current. The peak value of Ba2+ current at 0 mV was reduced to 35 +/- 7.4% (n = 9) by 3 microM of ML-7. As ICCh is known to have an intracellular Ca2+ dependence, we tried to exclude the possibility that ML-7 inhibited ICCh indirectly via suppression of Ca2+ current and the similar inhibitory effects of ML-7 on ICCh were confirmed under the following conditions: (1) clamp of membrane potential at -60 mV; (2) clamp of intracellular [Ca2+] to 1 microM by 10 mM BAPTA; (3) pre-inhibition of Ca2+ channel by verapamil. Different from the effects on ICCh, ML-7 barely inhibited the same cationic current induced by guanosine 5'-O-(3-thiotriphosphate) (GTP[gammaS], 0.2 mM) in the pipette solution. These results suggest that a Ca2+/calmodulin-MLCK-dependent pathway can modulate the activation of ICCh in guinea-pig gastric antral myocytes.
采用全细胞膜片钳技术研究了肌球蛋白轻链激酶抑制剂对豚鼠胃窦肌细胞毒蕈碱刺激激活的非选择性阳离子电流（ICCh）的影响。在-30 mV或-60 mV的钳制电位下，卡巴胆碱（CCh，50 μM）可诱导ICCh产生。肌球蛋白轻链激酶（MLCK）的化学抑制剂ML-7以浓度依赖性且可逆的方式抑制ICCh（1 μM时抑制率为53±8.6%，均值±标准误，n = 11）。此外，向微电极溶液中添加MLCK的肽抑制剂后，ICCh的幅度仅为每日对照值的37±2.7%。另一方面，ML-7对电压门控性Ca2+通道电流有抑制作用。3 μM的ML-7可使0 mV时Ba2+电流的峰值降低至35±7.4%（n = 9）。由于已知ICCh具有细胞内Ca2+依赖性，我们试图排除ML-7通过抑制Ca2+电流间接抑制ICCh的可能性，并且在以下条件下证实了ML-7对ICCh具有类似的抑制作用：（1）将膜电位钳制在-60 mV；（2）用10 mM BAPTA将细胞内[Ca2+]钳制在1 μM；（3）用维拉帕米预抑制Ca2+通道。与对ICCh的作用不同，ML-7几乎不抑制微电极溶液中5'-O-(3-硫代三磷酸)鸟苷（GTP[γS]，0.2 mM）诱导的相同阳离子电流。这些结果表明，Ca2+/钙调蛋白-MLCK依赖性途径可调节豚鼠胃窦肌细胞中ICCh的激活。
