Hilgemann D W, Matsuoka S, Nagel G A, Collins A
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
J Gen Physiol. 1992 Dec;100(6):905-32. doi: 10.1085/jgp.100.6.905.
Sodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (Kd) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (d) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (tau approximately 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased. (i) Kinetics of the sodium-dependent inactivation and its dependence on major experimental variables are well described by a simple two-state inactivation model assuming one fully active and one fully inactive exchanger state, whereby the transition to the inactive state takes place from a fully sodium-loaded exchanger conformation with cytoplasmic orientation of binding sites (E1.3Ni).
采用巨膜片法从豚鼠心室肌细胞分离出内面向外的膜片，记录钠钙交换电流。胞质钠激活后，外向交换电流呈指数衰减（钠依赖性失活）。研究了失活的动力学和机制。（a）失活速率和峰值电流幅度均强烈依赖温度（Q10 = 2.2）。（b）胞质pH从6.8升高到7.8可减弱电流衰减，并使胞质钙对交换电流二次激活的表观解离常数（Kd）从9.6 μM 变为< 0.3 μM。（c）失活过程中，在膜电位范围从 -120 到 60 mV 内，交换电流幅度同步下降，表明失活过程中交换体的电压依赖性未改变。在胞质钙二次调节和胰凝乳蛋白酶激活过程中，交换电流的电压依赖性也未改变。（d）在细胞外钠浓度为150 mM 和细胞外钙浓度为2 mM 时，施加胞质钠后外向交换电流衰减相似。在胞质游离钙浓度为2 - 5 μM 时去除胞质钠，内向交换电流分两个阶段产生，溶液变化过程中的快速阶段，以及指示从钠依赖性失活恢复的缓慢阶段（时间常数约为4 s）。（e）在零转运条件下，施加胞质钙后内向电流在溶液切换时间内完全激活且不衰减。（f）去除胞质钠后内向电流的缓慢恢复阶段在零转运条件下也存在。（g）豚鼠心肌细胞膜中钠依赖性失活对膜电位几乎没有依赖性。（h）随着细胞外钙浓度降低，外向电流的钠依赖性失活速率和程度减弱。（i）假设存在一个完全激活和一个完全失活的交换体状态，通过一个简单的两态失活模型可以很好地描述钠依赖性失活的动力学及其对主要实验变量的依赖性，即从结合位点面向胞质的完全钠负载交换体构象（E1.3Ni）转变为失活状态。
