The Voltage Dependence of a Cloned Mammalian Renal Type II Na(+)/P(i) Cotransporter (NaP(i)-2)

The voltage dependence of the rat renal type II Na(+)/P(i) cotransporter (NaP(i)-2) was investigated by expressing NaP(i)-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 ≤ V ≤ +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 Na(+) slippage. Voltage steps induced pre–steady state relaxations typical for Na(+)-coupled cotransporters. NaP(i)-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 ∼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 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.