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

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 ([sup.32][P.sub.i], [sup.22]Na) 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.sub.i] species is HP[O.sup.2-.sub.4]. 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 HP[O.sup.2-.sub.4] affinity is voltage dependent and that this voltage dependency is abrogated by lowering the pH or the [Na.sup.+] concentration. In contrast, the voltage dependency of the apparent [Na.sup.+] affinity increased when pH was lowered. De-steady-state current analysis shows that [Na.sup.+] ions bind first and influence the preferred orientation of the transporter in the absence of [P.sub.i]. Pre-steady-state charge movement was partially suppressed by complete removal of [Na.sup.+] from the bath, by reducing extracellular pH (both in the presence and absence of [Na.sup.+]), or by adding [P.sub.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 HP[O.sup.2-.sub.4] binding and proton modulation of the first [Na.sup.+] binding step. Xenopus laevis oocyte; electrophysiology; two-electrode voltage clamp; stoichiometry; structure-function