Or E, Goldshleger R, Karlish S J
Biochemistry Department, Weizmann Institute of Science, Rehovot, Israel.
J Biol Chem. 1996 Feb 2;271(5):2470-7. doi: 10.1074/jbc.271.5.2470.
This work utilizes proteoliposomes reconstituted with renal Na(+)-K(+)-ATPase to study effects of electrical potential (40-80 mV) on activation of pump-mediated fluxes of Na+ or Rb+ (K+) ions and on inhibitory effects of Rb+ ions or organic cations. The latter include guanidinium derivatives that are competitive Na(+)-like antagonists (David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1141-1149). Cytoplasmic side-positive diffusion potentials significantly decreased the K0.5 of Na+ at the cytoplasmic surface for activation of ATP-dependent Na(+)-K+ exchange but did not affect the inhibitory potency of Rb+ (K+) or any Na(+)-like antagonist. Diffusion potentials did not affect activation of Rb(+)-Rb+ exchange by Rb+ ions at the cytoplasmic surface and had only a minor effect on Rb+ activation at the extracellular surface. Previously, we proposed that the cation binding domain consists of two negatively charged sites, to which two K+ or two Na+ ions bind, and one neutral site for the third Na+ (Glynn, I. M., and Karlish, S.J.D. (1990) Annu. Rev. Biochem. 59, 171-205). The present experiments suggest that binding of a Na+ ion in the neutral site at the cytoplasmic surface is sensitive to voltage. By contrast, binding of Rb+ ions at the extracellular surface of renal pumps appears to be only weakly or insignificantly affected by voltage. Inferences on the identity of the charge-carrying steps, based on experiments using proteoliposomes, are discussed in relation to recent evidence that dissociation of Na+ or association of K+ ions, at the extracellular surface, represent the major charge-carrying steps.
本研究利用重组有肾钠钾ATP酶的蛋白脂质体，来研究电势（40 - 80 mV）对泵介导的钠离子或铷离子（钾离子）通量激活的影响，以及铷离子或有机阳离子的抑制作用。后者包括作为竞争性钠样拮抗剂的胍衍生物（David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1141 - 1149）。细胞质侧为正的扩散电位显著降低了细胞质表面激活ATP依赖的钠钾交换时钠离子的半最大激活浓度（K0.5），但不影响铷离子（钾离子）或任何钠样拮抗剂的抑制效力。扩散电位不影响细胞质表面铷离子对铷铷交换的激活，对细胞外表面铷离子的激活仅有轻微影响。此前，我们提出阳离子结合域由两个带负电荷的位点组成，两个钾离子或两个钠离子与之结合，还有一个中性位点用于结合第三个钠离子（Glynn, I. M., and Karlish, S.J.D. (1990) Annu. Rev. Biochem. 59, 171 - 205）。目前的实验表明，细胞质表面中性位点上钠离子的结合对电压敏感。相比之下，肾泵细胞外表面铷离子的结合似乎仅受到电压的微弱影响或无显著影响。基于使用蛋白脂质体的实验对电荷携带步骤的同一性进行的推断，结合最近关于钠离子解离或钾离子在细胞外表面结合代表主要电荷携带步骤的证据进行了讨论。
