Ledoux J, Chartier D, Leblanc N
Department of Physiology, University of Montreal, and Research Centre, Montreal Heart Institute, Montreal, Canada.
J Pharmacol Exp Ther. 1999 Sep;290(3):1165-74.
The present study was undertaken to investigate the effects of specific inhibitors of calmodulin-dependent protein kinase II (CamKII) on macroscopic voltage-dependent K(+) current (K(V)) recorded from rabbit portal vein smooth muscle cells. Inhibition of L-type Ca(2+) current facilitation by 1 microM KN-62, a blocker of CamKII, was first demonstrated and provided evidence for functional CamKII activity in this preparation. KN-93, another specific and more potent inhibitor of CamKII in the rat brain, suppressed K(V) and enhanced the rate of inactivation in a dose-dependent manner, in cells dialyzed with both low (0.1 mM) and high (10 mM) EGTA pipette solution. Prolonged dialysis with 10 microM of a synthetic peptide inhibitor of CamKII (fragment 281-301) had little effect on K(V) and did not prevent the inhibitory action of KN-93 on the current. The estimated IC(50) for inhibiting peak and late currents during 250-ms steps to +60 mV (holding potential = -60 mV) were 2.9 and 0.27 microM, respectively. KN-93 also induced slight shifts of the steady-state activation (-7 mV) and inactivation (-6 mV) curves. KN-62, and KN-92, an inactive analog of KN-93, produced effects similar to those of KN-93. In current clamp experiments, 5 microM KN-93 depolarized the myocytes from a control resting membrane potential of -42.3 +/- 2.8 mV to -28.5 +/- 1.4 mV, an effect that was partially reversible after washout (-34.4 +/- 1.3 mV, n = 6). In conclusion, blockers of CamKII produce nonspecific inhibitory effects on K(V) that warrant cautious use of these compounds in physiological experiments designed to assess the role of CamKII.
本研究旨在探讨钙调蛋白依赖性蛋白激酶II（CamKII）的特异性抑制剂对兔门静脉平滑肌细胞记录的宏观电压依赖性钾电流（K(V)）的影响。首先证实了1 μM KN-62（一种CamKII阻滞剂）对L型钙电流促进作用的抑制，这为该制剂中功能性CamKII活性提供了证据。KN-93是大鼠脑中另一种特异性更强的CamKII抑制剂，在用低（0.1 mM）和高（10 mM）EGTA移液管溶液透析的细胞中，以剂量依赖性方式抑制K(V)并提高失活速率。用10 μM的CamKII合成肽抑制剂（片段281-301）进行长时间透析对K(V)影响很小，且不能阻止KN-93对电流的抑制作用。在250 ms阶跃至+60 mV（保持电位=-60 mV）期间抑制峰值电流和晚期电流的估计IC(50)分别为2.9和0.27 μM。KN-93还引起稳态激活曲线（-7 mV）和失活曲线（-6 mV）的轻微偏移。KN-62以及KN-93的无活性类似物KN-92产生了与KN-93类似的效应。在电流钳实验中，5 μM KN-93使心肌细胞从对照静息膜电位-42.3±2.8 mV去极化至-28.5±1.4 mV，洗脱后该效应部分可逆（-34.4±1.3 mV，n = 6）。总之，CamKII阻滞剂对K(V)产生非特异性抑制作用，这使得在旨在评估CamKII作用的生理实验中谨慎使用这些化合物。
