Gadsby D C, Nakao M, Bahinski A, Nagel G, Suenson M
Laboratory of Cardiac/Membrane Physiology, Rockefeller University, New York, New York 10021.
Acta Physiol Scand Suppl. 1992;607:111-23.
The voltage dependence of transient and steady-state pump currents was examined in guinea pig ventricular myocytes to investigate mechanisms of charge translocation by the Na,K-ATPase. Na/K pump current was determined at approximately 36 degrees C as strophanthidin-sensitive whole-cell current in myocytes voltage clamped and internally dialyzed via wide tipped pipettes containing a pipette perfusion device. External Na ions diminished stationary pump current during forward Na/K cycling in a voltage dependent manner, the inhibition becoming stronger upon hyperpolarization. When Na,K-ATPase activity was restricted to Na translocation steps, stationary pump current was prevented but voltage pulses still elicited large transient pump currents which could be abolished by oligomycin B (> or = 2 micrograms/ml). The transients arose instantaneously on stepping the voltage, and decayed with voltage-dependent approximately single exponential time courses. The decay rates, and their high temperature sensitivity (approximately 200 s-1 at 0 mV at 36 degrees C; approximately 40 s-1 at 20 degrees C), suggest that the charge movements were limited by a conformational change associated with Na deocclusion. Those rates varied asymmetrically with voltage, changing little at positive voltages but increasing roughly exponentially with hyperpolarization (e-fold/approximately 80 mV). Lowering the extracellular [Na] ([Na]o) slowed the relaxation of charge movement at negative potentials but had little effect at positive potentials, and so shifted the rate constant-voltage curve to the left. The implied dependence on [Na]o of the backward rate constant governing pump charge movement accounts satisfactorily for the observed [Na]o sensitivity of stationary outward Na/K pump current, and indicates that the voltage-dependent step somehow involves the release of Na ions to the external medium. However, no strophanthidin-sensitive current was seen, at saturating external [K], when Na,K-ATPase activity was limited to K translocation steps by complete withdrawal of Na ions. But, at very low [Na]o, a weak negative slope appeared in the stationary pump current-voltage relationship at subsaturating, but not at saturating, external [K], indicating an increased apparent affinity for external K at more negative potentials. The results support the existence of a high field access channel through which extracellular Na and K ions must pass before interacting with their binding sites deep within the Na,K-ATPase molecule.
在豚鼠心室肌细胞中研究了瞬态和稳态泵电流的电压依赖性，以探讨钠钾ATP酶的电荷转运机制。钠钾泵电流在约36℃下测定，作为通过含有移液管灌注装置的宽尖端移液管进行电压钳制和内部透析的心肌细胞中对毒毛花苷敏感的全细胞电流。在正向钠钾循环过程中，外部钠离子以电压依赖性方式降低固定泵电流，超极化时抑制作用增强。当钠钾ATP酶活性仅限于钠转运步骤时，固定泵电流被阻断，但电压脉冲仍能引发大的瞬态泵电流，而寡霉素B（≥2μg/ml）可消除这些瞬态电流。瞬态电流在电压阶跃时瞬间出现，并以电压依赖性的近似单指数时间进程衰减。衰减速率及其高温敏感性（36℃时0mV处约为200s-1；20℃时约为40s-1）表明，电荷运动受与钠去封闭相关的构象变化限制。这些速率随电压不对称变化，在正电压下变化很小，但随着超极化大致呈指数增加（每80mV约增加一倍）。降低细胞外[Na]（[Na]o）会减慢负电位下电荷运动的弛豫，但对正电位影响不大，从而使速率常数-电压曲线向左移动。控制泵电荷运动的反向速率常数对[Na]o的隐含依赖性令人满意地解释了观察到的固定外向钠钾泵电流对[Na]o的敏感性，并表明电压依赖性步骤 somehow涉及钠离子向外部介质的释放。然而，当通过完全去除钠离子将钠钾ATP酶活性限制在钾转运步骤时，在饱和外部[K]下未观察到对毒毛花苷敏感的电流。但是，在非常低的[Na]o下，在亚饱和但不饱和的外部[K]下，固定泵电流-电压关系中出现了微弱的负斜率，表明在更负的电位下对外部K的表观亲和力增加。结果支持存在一个高场通道，细胞外钠和钾离子在与钠钾ATP酶分子深处的结合位点相互作用之前必须通过该通道。
