Temperature dependence of steady-state and presteady-state kinetics of a type IIb Na+/Pi cotransporter

The temperature dependence of the transport kinetics of flounder Na+ stop-coupled inorganic phosphate (P-i) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. (32) P-i uptake was strongly temperature- dependent and decreased by similar to 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was 14 kcal mol(1) stop 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 similar to 16 kcal mol(1) stop and similar to 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+ stop. 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+ stop-debinding rate compared with the other voltage-dependent rate constants.