Schmid R, Seydl K, Baumgartner W, Groschner K, Romanin C
Institute for Biophysics, University of Linz, Austria.
Biophys J. 1995 Nov;69(5):1847-57. doi: 10.1016/S0006-3495(95)80055-X.
The patch-clamp technique was employed to investigate the response of single L-type Ca2+ channels to the protease trypsin applied to the intracellular face of excised membrane patches from guinea pig ventricular myocytes. Calpastatin and ATP were used to prevent run-down of Ca2+ channel activity monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)-BAYK 8644. Upon application of trypsin (100 micrograms/ml) channel activity was enhanced fourfold and remained elevated upon removal of trypsin, as expected of a proteolytic, irreversible modification. The trypsin effect was not mediated by a proteolytic activation of protein kinases, as evidenced by the insensitivity of this effect to protein kinase inhibitors. Trypsin-modified Ca2+ channels exhibited the usual run-down phanomenon upon removal of calpastatin and ATP. In ensemble average currents trypsin-induced changes of channel function are apparent as a threefold increase in peak current and a reduction in current inactivation. At the single channel level these effects were based on about a twofold increase in both Ca2+ channels' availability and open probability. Neither the actual number of channels in the patch nor their unitary conductance as well as reversal potential was changed by trypsin. The Ca(2+)-induced inactivation was not impaired, as judged by a comparable sensitivity of trypsin-modified Ca2+ channels to intracellular Ca2+. Similarly, trypsin treatment did not affect the sensitivity of Ca2+ channels to phenylalkylmine inhibition. The observed alterations in channel function are discussed in terms of possible structural correlates.
采用膜片钳技术研究了单个L型Ca2+通道对应用于豚鼠心室肌细胞切除膜片内表面的蛋白酶胰蛋白酶的反应。在存在2.5 microM（-）-BAYK 8644的情况下，使用钙蛋白酶抑制蛋白和ATP来防止以96 mM Ba2+作为电荷载体监测的Ca2+通道活性的衰减。应用胰蛋白酶（100微克/毫升）后，通道活性增强了四倍，并且在去除胰蛋白酶后仍保持升高，这符合蛋白水解的、不可逆修饰的预期。胰蛋白酶的作用不是由蛋白激酶的蛋白水解激活介导的，这一作用对蛋白激酶抑制剂不敏感证明了这一点。去除钙蛋白酶抑制蛋白和ATP后，经胰蛋白酶修饰的Ca2+通道表现出通常的衰减现象。在总体平均电流中，胰蛋白酶诱导的通道功能变化表现为峰值电流增加三倍和电流失活减少。在单通道水平上，这些效应是基于Ca2+通道的可用性和开放概率都增加了约两倍。胰蛋白酶既没有改变膜片中通道的实际数量，也没有改变它们的单位电导以及反转电位时。根据经胰蛋白酶修饰的Ca2+通道对细胞内Ca2+的可比敏感性判断，Ca2+诱导的失活没有受损。同样，胰蛋白酶处理也不影响Ca2+通道对苯烷基胺抑制的敏感性。从可能的结构相关性方面讨论了观察到的通道功能变化。
