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

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Abstract
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 Pispecies is HPO42−. 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 HPO42−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 HPO42−binding and proton modulation of the first Na+binding step.