Koenderink Jan B, Geibel Sven, Grabsch Eva, De Pont Jan Joep H H M, Bamberg Ernst, Friedrich Thomas
Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Marie-Curie-Strasse 15, D-60439 Frankfurt am Main, Germany.
J Biol Chem. 2003 Dec 19;278(51):51213-22. doi: 10.1074/jbc.M306384200. Epub 2003 Oct 7.
Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp, Gln, Ala), Asp804 (Glu, Asn, Ala), and Asp808 (Glu, Asn, Ala). Electrogenic cation transport properties of these 12 mutants were analyzed in two-electrode voltage-clamp experiments on Xenopus laevis oocytes by measuring the voltage dependence of K+-stimulated stationary currents and pre-steady-state currents under electrogenic Na+/Na+ exchange conditions. Whereas mutants D804N, D804A, and D808A hardly showed any Na+/K+ pump currents, the other constructs could be classified according to the [K+] and voltage dependence of their stationary currents; mutants N776A and E779Q behaved similarly to the wild-type enzyme. Mutants E779D, E779A, D808E, and D808N had in common a decreased apparent affinity for extracellular K+. Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the current-voltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na+/Na+ exchange currents are rather voltage-independent and at potentials above -60 mV faster than the wild type. Thus, the characteristic voltage-dependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn776 and Asp804, respectively, decreases the extracellular Na+ affinity.
钠钾ATP酶通过在每个反应周期中将三个钠离子挤出并将两个钾离子导入质膜来介导净生电转运。我们对大鼠钠钾ATP酶跨膜发夹M5 - M6中假定的阳离子配位氨基酸进行了突变：天冬氨酸776（谷氨酰胺、天冬氨酸、丙氨酸）、谷氨酸779（天冬氨酸、谷氨酰胺、丙氨酸）、天冬氨酸804（谷氨酸、天冬酰胺、丙氨酸）和天冬氨酸808（谷氨酸、天冬酰胺、丙氨酸）。在非洲爪蟾卵母细胞的双电极电压钳实验中，通过测量在生电钠/钠交换条件下钾离子刺激的稳态电流和预稳态电流的电压依赖性，分析了这12个突变体的生电阳离子转运特性。虽然突变体D804N、D804A和D808A几乎没有显示出任何钠/钾泵电流，但其他构建体可以根据其稳态电流的[钾离子]和电压依赖性进行分类；突变体N776A和E779Q的行为与野生型酶相似。突变体E779D、E779A、D808E和D808N的共同特点是对细胞外钾离子的表观亲和力降低。突变体N776Q、N776D和D804E与野生型行为有很大偏差；突变体N776D产生的电流显示出较弱的电压依赖性，而突变体N776Q和D804E的电流 - 电压曲线呈现负斜率。从瞬时钠/钠交换电流确定的表观速率常数与电压关系不大，并且在高于 - 60 mV的电位下比野生型更快。因此，在这些突变体中几乎不存在超极化电位下速率常数的特征性电压依赖性增加。相应地，分别使天冬酰胺776和天冬氨酸804的羧酰胺或羧基移位会降低细胞外钠离子亲和力。
