The temperature dependence of the transport kinetics of flounder Na+-coupled inorganic phosphate (Pi) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. 32Pi 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 Pi and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the Pi- 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 Pi 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.
The Journal of Membrane Biology – Springer Journals
Published: Apr 19, 2007
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