Koster J C, Blanco G, Mills P B, Mercer R W
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1996 Feb 2;271(5):2413-21. doi: 10.1074/jbc.271.5.2413.
The intramembrane Glu781 residue of the Na,K-ATPase alpha subunit has been postulated to have a role in the binding and/or occlusion of cations. To ascertain the role of Glu781, the residue was substituted with an aspartate, alanine, or lysine residue and the mutant Na,K-ATPases were coexpressed with the native beta 1 subunit in Sf9 insect cells using the baculovirus expression system. All alpha mutants are able to efficiently assemble with the beta 1 subunit and produce catalytically competent Na,K-ATPase molecules with hydrolytic activities comparable to that of the wild-type enzyme. Analysis of the kinetic properties of the mutated enzymes showed a decrease in apparent affinity for K+ compared to wild-type Na,K-ATPase, with the lysine and alanine substitutions displaying the greatest reduction. All Na,K-ATPase mutants demonstrated a significant increase in apparent affinity for ATP compared to wild-type Na,K-ATPase, while the sensitivity to the cardiotonic inhibitor, ouabain, was unchanged. The dependence on Na+, however, differs among the mutant enzymes with both the Glu781-->Asp and Glu781-->Ala mutants displaying a decrease in the apparent affinity for the cation, while the Glu781-->Lys mutant exhibits a modest increase. Furthermore, in the absence of K+, the Glu781-->Ala mutant displays a Na(+)-ATPase activity and a cellular Na+ influx suggesting that Na+ is substituting for K+ at the extracellular binding sites. The observation that trypsin digestion of the Glu781-->Ala mutant in Na+ medium produces a K(+)-stabilized tryptic fragment also intimates a decreased capacity of the mutant to discriminate between Na+ and K+ at the extracellular loading sites. All together, these data implicate Glu781 of the Na,K-ATPase alpha subunit as an important coordinate of cation selectivity and activation, although the modest effect of Glu781-->Lys substitution seemingly precludes direct involvement of the residue in the cation binding process. In addition, the fifth membrane segment is proposed to represent an important communicative link between the extramembraneous ATP binding domain and the cation transport regions of the Na,K-ATPase.
钠钾ATP酶α亚基的膜内Glu781残基被推测在阳离子的结合和/或封闭过程中起作用。为了确定Glu781的作用，将该残基分别替换为天冬氨酸、丙氨酸或赖氨酸残基，并使用杆状病毒表达系统在Sf9昆虫细胞中将突变的钠钾ATP酶与天然β1亚基共表达。所有α突变体都能够与β1亚基有效组装，并产生具有催化活性的钠钾ATP酶分子，其水解活性与野生型酶相当。对突变酶动力学特性的分析表明，与野生型钠钾ATP酶相比，其对K+的表观亲和力降低，其中赖氨酸和丙氨酸替换导致的降低最为明显。与野生型钠钾ATP酶相比，所有钠钾ATP酶突变体对ATP的表观亲和力均显著增加，而对强心苷抑制剂哇巴因的敏感性未变。然而，不同突变酶对Na+的依赖性有所不同，Glu781→Asp和Glu781→Ala突变体对阳离子的表观亲和力均降低，而Glu781→Lys突变体则略有增加。此外，在没有K+的情况下，Glu781→Ala突变体表现出Na(+)-ATP酶活性和细胞内Na+内流，这表明Na+正在细胞外结合位点替代K+。在Na+培养基中对Glu781→Ala突变体进行胰蛋白酶消化会产生K(+)-稳定的胰蛋白酶片段，这一观察结果也表明该突变体在细胞外加载位点区分Na+和K+的能力下降。总之，这些数据表明钠钾ATP酶α亚基的Glu781是阳离子选择性和激活的重要协同因子，尽管Glu781→Lys替换的影响较小，似乎排除了该残基直接参与阳离子结合过程。此外，第五个膜段被认为是钠钾ATP酶膜外ATP结合结构域与阳离子转运区域之间的重要通信连接。
