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

Bacconi, Andrea ; Ravera, Silvia ; Virkki, Leila ; Murer, Heini ; Forster, Ian

In: Journal of Membrane Biology, 2007, vol. 215, no. 2-3, p. 81-92

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    Titre
    • Temperature Dependence of Steady-State and Presteady-State Kinetics of a Type IIb Na+/Pi Cotransporter
    Auteur
    • Bacconi, Andrea. Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland
    • Ravera, Silvia. Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland
    • Virkki, Leila. Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland
    • Murer, Heini. Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland
    • Forster, Ian. Institute of Physiology and Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland
    Type de document
    • Postprint
    Langue
    • Inglese
    Publié dans
    • Journal of Membrane Biology, 2007, vol. 215, no. 2-3, p. 81-92. Springer-Verlag
    Autre version électronique
    Identifiant OAI-PMH
    • oai:doc.rero.ch:320108
    Summary
    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 kcalmol−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 kcalmol−1 and ∼11 kcalmol−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