Valenzuela C, Bennett P B
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Biophys J. 1994 Jul;67(1):161-71. doi: 10.1016/S0006-3495(94)80465-5.
Single cardiac Na+ channels were investigated after intracellular proteolysis to remove the fast inactivation process in an attempt to elucidate the mechanisms of channel gating and the role of slow inactivation. Na+ channels were studied in inside-out patches excised from guinea-pig ventricular myocytes both before and after very brief exposure (2-4 min) to the endopeptidase, alpha-chymotrypsin. Enzyme exposure times were chosen to maximize removal of fast inactivation and to minimize potential nonspecific damage to the channel. After proteolysis, the single channel current-voltage relationship was approximately linear with a slope conductance of 18 +/- 2.5 pS. Na+ channel reversal potentials measured before and after proteolysis by alpha-chymotrypsin were not changed. The unitary current amplitude was not altered after channel modification suggesting little or no effect on channel conductance. Channel open times were increased after removal of fast inactivation and were voltage-dependent, ranging between 0.7 (-70 mV) and 3.2 (-10 mV) ms. Open times increased with membrane potential reaching a maximum at -10 mV; at more positive membrane potentials, open times decreased again. Fast inactivation appeared to be completely removed by alpha-chymotrypsin and slow inactivation became more apparent suggesting that fast and slow inactivation normally compete, and that fast inactivation dominates in unmodified channels. This finding is not consistent with a slow inactivated state that can only be entered through the fast inactivated state, since removal of fast inactivation does not eliminate slow inactivation. The data indicate that cardiac Na+ channels can enter the slow inactivated state by a pathway that bypasses the fast inactivated state and that the likelihood of entering the slow inactivated state increases after removal of fast inactivation.
为了阐明通道门控机制和慢失活的作用，对单个心脏钠通道进行了细胞内蛋白酶解处理以去除快速失活过程。在豚鼠心室肌细胞的内向外膜片中，研究了在极短时间（2 - 4分钟）暴露于内肽酶α-糜蛋白酶前后的钠通道。选择酶暴露时间以最大程度地去除快速失活并最小化对通道的潜在非特异性损伤。蛋白酶解后，单通道电流-电压关系近似线性，斜率电导为18±2.5 pS。用α-糜蛋白酶处理前后测量的钠通道反转电位没有变化。通道修饰后单位电流幅度未改变，表明对通道电导影响很小或没有影响。去除快速失活后通道开放时间增加且依赖电压，范围在0.7（-70 mV）至3.2（-10 mV）毫秒之间。开放时间随膜电位增加，在-10 mV时达到最大值；在更正的膜电位下，开放时间再次减少。α-糜蛋白酶似乎完全去除了快速失活，慢失活变得更加明显，这表明快速失活和慢失活通常相互竞争，并且在未修饰的通道中快速失活占主导。这一发现与只能通过快速失活状态进入的慢失活状态不一致，因为去除快速失活并没有消除慢失活。数据表明心脏钠通道可以通过绕过快速失活状态的途径进入慢失活状态，并且去除快速失活后进入慢失活状态的可能性增加。
