Localization of Na^+ and K^+ pathways in Na^+, K^+-ATPase

我用寡霉素研究了Na^+， K^+- atp酶的离子转运机制，寡霉素抑制Na^+转运和atp酶活性。我得到了以下发现。1)寡霉素仅抑制Na^+在典型的p型atp酶（Na^+、K^+- atp酶、H^+、K^+- atp酶和Ca^<2+>- atp酶）之间的转运。2)寡霉素在Na^+， K^+- atp酶α亚基的n端200个氨基酸上有一个结合域。该结构域包括第一和第二跨膜段。疏水寡霉素，因此，应该结合在这些片段的某处，虽然我不能确定其精细的结合位点。根据这些结果和其他发现，这是Na^+， K^+- atp酶和离子运输所需氨基酸定位的假设模型，我提出以下假设；第1 - 6个跨膜段的构象变化伴随着Na^+输运。在Na^+转运过程中，寡霉素干扰第一和第二节段的变化，使转运受到抑制。当构象回到初始状态时，K^+被转移。寡霉素不抑制K^+转运。根据这一假设，我研究了寡霉素和Na^+对K^+依赖的对硝基苯基磷酸酶（pNPPase）的活性，这是Na^+， K^+- atp酶的部分反应。当Na^+: K^+比值大于10:1时，寡霉素刺激pNPPase活性。这一结果是出乎意料的，因为预计寡霉素会增加Na^+对pNPPase活性的抑制作用。在本实验条件下，寡霉素增加了K^+对Na^+， K^+- atp酶的亲和力。这一发现提示寡霉素影响离子与细胞外K +结合（和/或Na^+释放位点）的相互作用。

I have investigated the ion transport mechanism of Na^+, K^+-ATPase using oligomycin, which inhibits the Na^+ transport and the ATPase activity. I obtained the following findings. 1) Oligomycin inhibited only the Na^+ transport among the typical P-type-ATPases, i.e., Na^+, K^+-ATPase, H^+, K^+-ATPase and Ca^<2+>-ATPase. 2) Oligomycin had a binding domain in the N-terminal 200 amino acids of Na^+, K^+-ATPase α subunit, . This domain included the first and second transmembrane segments. The hydrophobic oligomycin, therefore, should bind somewhere in these segments, though I cannot identify its fine binding sites. From these results and other findings, which are an assumptive model of Na^+, K^+-ATPase and localization of the amino acids required for ion transport, I propose the following hypothesis ; the conformational change of the first-sixth transmembrane segments is accompanied with Na^+ transport. When Na^+ is transported, oligomycin disturb the change of the first and second segments, so that the transport is inhibited. When the conformation comes back to the original state, K^+ is transported. Oligomycin does not inhibit K^+ transport. According to this hypothesis, I have investigated oligomycin and Na^+ on K^+-dependent p-nitrophenyl phosphatase (pNPPase) activity, which is a partial reaction of Na^+, K^+-ATPase. When Na^+ : K^+ ratio is over10 : 1, oligomycin stimulated pNPPase activity. This result is unexpected, because it is expected that oligomycin increases the inhibitory effect of Na^+ on pNPPase activity. Under this experimental conditions, oligomycin increased the affinity for K^+ to Na^+, K^+-ATPase. This finding suggested that oligomycin affected the interaction between ions and K^+ binding (and/or Na^+ releasing site) at extracellular side.