Institute of Chemistry, Technical University of Berlin, Berlin, Germany.
PLoS One. 2012;7(3):e33645. doi: 10.1371/journal.pone.0033645. Epub 2012 Mar 20.
Whereas electrogenic partial reactions of the Na,K-ATPase have been studied in depth, much less is known about the influence of the membrane potential on the electroneutrally operating gastric H,K-ATPase. In this work, we investigated site-specifically fluorescence-labeled H,K-ATPase expressed in Xenopus oocytes by voltage clamp fluorometry to monitor the voltage-dependent distribution between E(1)P and E(2)P states and measured Rb(+) uptake under various ionic and pH conditions. The steady-state E(1)P/E(2)P distribution, as indicated by the voltage-dependent fluorescence amplitudes and the Rb(+) uptake activity were highly sensitive to small changes in intracellular pH, whereas even large extracellular pH changes affected neither the E(1)P/E(2)P distribution nor transport activity. Notably, intracellular acidification by approximately 0.5 pH units shifted V(0.5), the voltage, at which the E(1)P/E(2)P ratio is 50∶50, by -100 mV. This was paralleled by an approximately two-fold acceleration of the forward rate constant of the E(1)P→E(2)P transition and a similar increase in the rate of steady-state cation transport. The temperature dependence of Rb(+) uptake yielded an activation energy of ∼90 kJ/mol, suggesting that ion transport is rate-limited by a major conformational transition. The pronounced sensitivity towards intracellular pH suggests that proton uptake from the cytoplasmic side controls the level of phosphoenzyme entering the E(1)P→E(2)P conformational transition, thus limiting ion transport of the gastric H,K-ATPase. These findings highlight the significance of cellular mechanisms contributing to increased proton availability in the cytoplasm of gastric parietal cells. Furthermore, we show that extracellular Na(+) profoundly alters the voltage-dependent E(1)P/E(2)P distribution indicating that Na(+) ions can act as surrogates for protons regarding the E(2)P→E(1)P transition. The complexity of the intra- and extracellular cation effects can be rationalized by a kinetic model suggesting that cations reach the binding sites through a rather high-field intra- and a rather low-field extracellular access channel, with fractional electrical distances of ∼0.5 and ∼0.2, respectively.
虽然 Na,K-ATP 酶的电致部分反应已经得到了深入研究，但对于膜电位对中性运行的胃 H,K-ATP 酶的影响知之甚少。在这项工作中，我们通过电压钳荧光法特异性地研究了在非洲爪蟾卵母细胞中表达的荧光标记的 H,K-ATP 酶，以监测 E(1)P 和 E(2)P 状态之间的电压依赖性分布，并在各种离子和 pH 条件下测量 Rb(+)摄取。稳态 E(1)P/E(2)P 分布，如电压依赖性荧光幅度和 Rb(+)摄取活性所示，对细胞内 pH 的微小变化非常敏感，而即使是大的细胞外 pH 变化也不会影响 E(1)P/E(2)P 分布或转运活性。值得注意的是，细胞内酸化约 0.5 pH 单位会使 V(0.5)（E(1)P/E(2)P 比为 50∶50 的电压）向负方向移动 100 mV。这与 E(1)P→E(2)P 跃迁的正向速率常数大约两倍的加速和稳态阳离子转运速率的类似增加相平行。Rb(+)摄取的温度依赖性产生约 90 kJ/mol 的活化能，表明离子转运受到主要构象转变的限制。对细胞内 pH 的显著敏感性表明，质子从细胞质侧摄取控制进入 E(1)P→E(2)P 构象转变的磷酸酶水平，从而限制胃 H,K-ATP 酶的离子转运。这些发现强调了细胞质中质子可用性增加的细胞机制的重要性。此外，我们表明，细胞外 Na(+) 会深刻改变电压依赖性 E(1)P/E(2)P 分布，表明 Na(+) 离子可以作为质子的替代物，影响 E(2)P→E(1)P 转变。内外阳离子效应的复杂性可以通过一个动力学模型来合理化，该模型表明阳离子通过一个相当高的电场内通道和一个相当低的电场外通道到达结合位点，其分数电距离分别约为 0.5 和 0.2。
