Virkki Leila V, Forster Ian C, Biber Jürg, Murer Heini
Institute of Physiology, University of Zurich, Zurich, Switzerland.
Am J Physiol Renal Physiol. 2005 May;288(5):F969-81. doi: 10.1152/ajprenal.00293.2004. Epub 2004 Dec 21.
We have characterized the kinetics of substrate transport in the renal type IIa human sodium-phosphate cotransporter (NaPi-IIa). The transporter was expressed in Xenopus laevis oocytes, and steady-state and pre-steady-state currents and substrate uptakes were characterized by voltage-clamp and isotope flux. First, by measuring simultaneous uptake of a substrate (32Pi, 22Na) and charge in voltage-clamped oocytes, we established that the human NaPi-IIa isoform operates with a Na:Pi:charge stoichiometry of 3:1:1 and that the preferred transported Pi species is HPO4(2-). We then probed the complex interrelationship of substrates, pH, and voltage in the NaPi-IIa transport cycle by analyzing both steady-state and pre-steady-state currents. Steady-state current measurements show that the apparent HPO4(2-) affinity is voltage dependent and that this voltage dependency is abrogated by lowering the pH or the Na+ concentration. In contrast, the voltage dependency of the apparent Na+ affinity increased when pH was lowered. Pre-steady-state current analysis shows that Na+ ions bind first and influence the preferred orientation of the transporter in the absence of Pi. Pre-steady-state charge movement was partially suppressed by complete removal of Na+ from the bath, by reducing extracellular pH (both in the presence and absence of Na+), or by adding Pi (in the presence of 100 mM Na). None of these conditions suppressed charge movement completely. The results allowed us to modify previous models for the transport cycle of NaPi-II transporters by including voltage dependency of HPO4(2-) binding and proton modulation of the first Na+ binding step.
我们已对人肾IIa型钠-磷酸共转运体（NaPi-IIa）中底物转运的动力学进行了表征。该转运体在非洲爪蟾卵母细胞中表达，通过电压钳制和同位素通量对稳态和预稳态电流以及底物摄取进行了表征。首先，通过测量电压钳制卵母细胞中底物（32Pi、22Na）的同时摄取和电荷，我们确定人NaPi-IIa亚型的运行Na:Pi:电荷化学计量比为3:1:1，且优先转运的Pi物种是HPO4(2-)。然后，我们通过分析稳态和预稳态电流，探究了NaPi-IIa转运循环中底物、pH和电压之间复杂的相互关系。稳态电流测量表明，表观HPO4(2-)亲和力依赖于电压，并且通过降低pH或Na+浓度可消除这种电压依赖性。相反，当pH降低时，表观Na+亲和力的电压依赖性增加。预稳态电流分析表明，Na+离子首先结合，并在没有Pi的情况下影响转运体的优先取向。通过从浴液中完全去除Na+、降低细胞外pH（无论有无Na+）或添加Pi（在存在100 mM Na的情况下），预稳态电荷移动被部分抑制。这些条件均未完全抑制电荷移动。这些结果使我们能够通过纳入HPO4(2-)结合的电压依赖性和第一个Na+结合步骤的质子调节，对先前的NaPi-II转运体转运循环模型进行修正。
