Temperature dependence of steady-state and presteady-state kinetics of a type IIb Na+/P i cotransporter.
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. (32)P(i) uptake was strongly temperature-dependent and decreased by approximately 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was approximately 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 approximately 16 kcal mol(-1) and approximately 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.
Bacconi
Andrea
A
Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Ravera
Silvia
S
Virkki
Leila V
LV
Murer
Heini
H
Forster
Ian C
IC
D000818:Animals; D004594:Electrophysiology; D005260:Female; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D005432:Flounder / Q000235:genetics / Q000378:metabolism*; D017245:Foscarnet / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques; D050610:Sodium-Phosphate Cotransporter Proteins, Type II / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013696:Temperature; D014982:Xenopus laevis
D029941:Fish Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D017245:Foscarnet