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Functionally Important Residues in the Predicted 3rd Transmembrane Domain of the Type IIa Sodium-phosphate Cotransporter (NaPi-IIa)

Virkki, L.V. ; Forster, I.C. ; Bacconi, A. ; Biber, J. ; Murer, H.

In: The Journal of Membrane Biology, 2005, vol. 206, no. 3, p. 227-238

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    Titre
    • Functionally Important Residues in the Predicted 3rd Transmembrane Domain of the Type IIa Sodium-phosphate Cotransporter (NaPi-IIa)
    Auteur
    • Virkki, L.V.. Institute of Physiology and Center for Integrative Human Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
    • Forster, I.C.. Institute of Physiology and Center for Integrative Human Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
    • Bacconi, A.. Institute of Physiology and Center for Integrative Human Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
    • Biber, J.. Institute of Physiology and Center for Integrative Human Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
    • Murer, H.. Institute of Physiology and Center for Integrative Human Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057, Zürich, Switzerland
    Type de document
    • Postprint
    Langue
    • Anglais
    Publié dans
    • The Journal of Membrane Biology, 2005, vol. 206, no. 3, p. 227-238. Springer-Verlag; www.springer-ny.com
    Autre version électronique
    Identifiant OAI-PMH
    • oai:doc.rero.ch:320830
    Summary
    The type IIa Na+/Pi, cotransporter (NaPi-IIa) mediates electrogenic transport of three Na+ and one divalent Pi ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and Pi transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of Pi transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the Pi-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na+-dependent Pi transport. Since electrogenic NaPi-II transports 3 Na+/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na+ binding sites in NaPi-IIa