Temperature Dependence of Steady-State and Presteady-State Kinetics of a Type IIb Na⁺/P_i Cotransporter

Abstract

The temperature dependence of the transport kinetics of flounder Na⁺-coupled inorganic phosphate (P_i) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. ³²P_i uptake was strongly temperature-dependent and decreased by ∼80% at a temperature change from 25°C to 5°C. The corresponding activation energy (E _a) was ∼14 kcal mol⁻¹ for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM P_i and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the P_i- and PFA-induced changes in holding current decreased with temperature. E _a at −100 mV for the cotransport and leak modes was ∼16 kcal mol⁻¹ and ∼11 kcal mol⁻¹, respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E _a for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM P_i over a range of external Na⁺. The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na⁺-debinding rate compared with the other voltage-dependent rate constants.