The voltage dependence of a cloned mammalian renal type II Na+/Pi cotransporter (NaPi-2)

The voltage dependence of the rat renal type II Na+/P-i cotransporter (NaPi-2) was investigated by expressing NaPi-2 in Xenopus laevis oocytes and applying the two-electrode voltage clamp. In the steady state, superfusion with inorganic phosphate (P-i) induced inward currents (I-p) in the presence of 96 mM Na+ over the potential range -140 less than or equal to V less than or equal to +40 mV. With P-i as the variable substrate, the apparent affinity constant (K-m(Pi)) was strongly dependent on Na+, increasing sixfold for a twofold reduction in external Na+. K-m(Pi) increased with depolarizing voltage and was more sensitive to voltage at reduced Na+. The Hill coefficient was close to unity and the predicted maximum I-p (I-pmax) was 40% smaller at 50 mM Na+. With Na+ as the variable substrate, K-m(Na) was weakly dependent on both P-i and voltage, the Hill coefficient was close to 3 and I-pmax was independent of P-i at -50 mV. The competitive inhibitor phosphonoformic acid suppressed the steady state holding current in a Na+-dependent manner, indicating the existence of uncoupled Naf slippage. Voltage steps induced pre-steady state relaxations typical for Na+-coupled cotransporters. NaPi-2-dependent relaxations were quantitated by a single, voltage-dependent exponential. At 96 mM Na+, a Boltzmann function was fit to the steady state charge distribution (Q-V) to give a midpoint voltage (V-0.5) in the range -20 to -50 mV and an apparent valency of similar to 0.5 e(-). V-0.5 became more negative as Na+ was reduced. P-i suppressed relaxations in a dose-dependent manner, but had little effect on their voltage age dependence. Reducing external pH shifted V-0.5 to depolarizing potentials and suppressed relaxations in the absence of Na+, suggesting that protons interact with the unloaded carrier. These findings were incorporated into an ordered kinetic model whereby Na+ is the first and last substrate to bind, and the observed voltage dependence arises from the unloaded carrier and first Na+ binding step.