通过用胰蛋白酶灌注去除鱿鱼巨大轴突中的钠离子通道。

Removal of Na+ channels in squid giant axons by perfusion with trypsin.

作者信息

Carbone E

出版信息

Biochim Biophys Acta. 1982 Dec 8;693(1):188-94. doi: 10.1016/0005-2736(82)90486-2.

Abstract

The irreversible effects of the proteolytic enzyme trypsin on ionic and gating currents of voltage-clamped squid axon membranes have been studied. At physiological pH, internal perfusion of the fibre with trypsin was found to be very effective in removing Na+ channels leaving the potassium system almost unaltered. At T = 13 degrees C the rates of channel-cleavage averaged 1/10 min-1 for the Na+ and 1/128 min-1 for the K+ channel, respectively. As estimated by the decrement of peak sodium conductance, the rate of loss of Na+ channels correlates well with the rate of decrease of the total charge associated with the ON component of gating currents, indicating that trypsin probably interacts with an essential proteic portion of the channel whose removal might prevent both the displacement of gating charges and the subsequent opening of the channel. Intracellular pH remarkably influences the action of the enzyme. A plot of the pH-dependence of the rate of cleavage of Na+ channels suggests the involvement of a positively charged group (either lysine or arginine) in the substrate region of the trypsin catalytic reaction.

摘要

研究了蛋白水解酶胰蛋白酶对电压钳制的鱿鱼轴突膜离子电流和门控电流的不可逆作用。在生理pH值下，发现用胰蛋白酶对纤维进行内部灌注能非常有效地去除Na⁺通道，而钾离子系统几乎不受影响。在T = 13℃时，Na⁺通道和K⁺通道的通道裂解速率分别平均为1/10 min⁻¹和1/128 min⁻¹。根据峰值钠电导的衰减估计，Na⁺通道的丢失速率与门控电流ON成分相关的总电荷减少速率密切相关，这表明胰蛋白酶可能与通道的一个必需蛋白质部分相互作用，去除该部分可能会阻止门控电荷的位移以及通道随后的开放。细胞内pH值显著影响该酶的作用。Na⁺通道裂解速率的pH依赖性曲线表明，在胰蛋白酶催化反应的底物区域中涉及一个带正电荷的基团（赖氨酸或精氨酸）。