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Substrate interactions in the human type IIa sodium-phosphate cotransporter (NaPi-IIa)

Institute of Physiology, University of Zurich, Zurich, Switzerland

Submitted 6 August 2004 ; accepted in final form 14 December 2004

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 (³²P_i, 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 P_i 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 P_i. 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 P_i (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.

Xenopus laevis oocyte; electrophysiology; two-electrode voltage clamp; stoichiometry; structure-function

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