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From the Physiologisches Institut, Universität Zürich, CH-8057 Zürich, Switzerland

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.

Key Words: ion cotransport • kinetics • steady state • pre–steady state relaxations • Xenopus laevis oocytes

Abbreviations: BBMV, brush border membrane vesicles; I_h, oocyte holding current; I_p, P_i-induced inward currents; I_PFA, PFA-sensitive component; I-V, current–voltage; NaP_i-2 rat type II Na⁺/P_i cotransporter, PFA, phosphonoformic acid; P_i, inorganic phosphate; V_h, oocyte holding potential

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