Peluffo R D, Argüello J M, Lingrel J B, Berlin J R
Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
J Gen Physiol. 2000 Jul 1;116(1):61-73. doi: 10.1085/jgp.116.1.61.
Na,K-ATPase containing the amino acid substitution glutamate to alanine at position 779 of the alpha subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+-Na+ exchange activity in the absence of K+. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 +/- 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K+. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K+. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K+-free solutions. Electrogenic Na+-Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+(o)) concentration. At all Na+(o) concentrations tested (3-148 mM), exchange current was maximal at negative membrane potentials (V(M)), but decreased as V(M) became more positive. Analyzing this current at each V(M) with a Hill equation showed that Na+-Na+ exchange had a high-affinity, low-capacity component with an apparent Na+(o) affinity at 0 mV (K0(0.5)) of 13.4 +/- 0.6 mM and a low-affinity, high-capacity component with a K0(0.5) of 120 +/- 13 mM (n = 17). Both high- and low-affinity exchange components were V(M) dependent, dissipating 30 +/- 3% and 82 +/- 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+-Na+ exchange could be explained by Na+(o) acting as a low-affinity K+ congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+(o) binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K-ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47-59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K-ATPase.
α亚基第779位氨基酸由谷氨酸替换为丙氨酸（Glu779Ala）的钠钾ATP酶在无钾条件下支持高水平的钠ATP酶活性和生电钠-钠交换活性。在Glu779Ala酶的微粒体制剂中，钠ATP酶活性半最大激活时的钠浓度为161±14 mM（n = 3）。此外，发现800 mM钠存在和不存在20 mM钾时的酶活性相似。这些结果表明，低亲和力的钠能像钾一样有效地刺激酶的周转。为了进一步深入了解这种酶活性的机制，在无钾溶液灌流期间，用含有115 mM钠的膜片电极对表达Glu779Ala酶的HeLa细胞进行电压钳制。生电钠-钠交换表现为哇巴因可抑制的外向电流，其幅度与细胞外钠（Na+(o)）浓度成正比。在所有测试的Na+(o)浓度（3 - 148 mM）下，交换电流在负膜电位（V(M)）时最大，但随着V(M)变得更正而减小。用希尔方程分析每个V(M)下的该电流表明，钠-钠交换有一个高亲和力、低容量成分，在0 mV时对Na+(o)的表观亲和力（K0(0.5)）为13.4±0.6 mM，以及一个低亲和力、高容量成分，K0(0.5)为120±13 mM（n = 17）。高亲和力和低亲和力交换成分均依赖于V(M)，分别消耗膜电介质的30±3%和82±6%（n = 17）。膜片电极溶液中2 mM游离ADP抑制低亲和力但不抑制高亲和力交换成分。这些结果表明，生电钠-钠交换的高亲和力成分可以用Na+(o)作为低亲和力钾类似物来解释；然而，生电交换的低亲和力成分似乎是由于Na+(o)在膜电介质深处的钠特异性位点结合激活了正向酶循环。建立了钠钾ATP酶的伪六态模型来模拟这些数据以及随附论文（Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47 - 59）的结果。该模型表明，仅细胞外离子依赖性反应动力学的改变就能解释Glu779Ala替换对钠钾ATP酶的影响。
