title: Molecular determinants of transport function in zebrafish Slc34a Na-phosphate transporters.
abstract: The epithelial Na+-coupled phosphate cotransporter family Slc34a (NaPi-II) is well conserved in vertebrates and plays an essential role in maintaining whole body levels of inorganic phosphate (Pi). A three-dimensional model of the transport protein has recently been proposed with defined substrate coordination sites. Zebrafish express two NaPi-II isoforms with high sequence identity but a 10-fold different apparent Kmfor Pi([Formula: see text]). We took advantage of the two zebrafish isoforms to investigate the contribution of specific amino acids to Picoordination and transport. Mutations were introduced to gradually transform the low-affinity isoform into a high-affinity transporter. The constructs were expressed in Xenopus laevis oocytes and functionally characterized. Becaue the cotransport of Piand Na involves multiple steps that could all influence [Formula: see text], we performed a detailed functional analysis to characterize the impact of the mutations on particular steps of the transport cycle. We used varying concentrations of the substrates Piand its slightly larger analog, arsenate, as well as the cosubstrate, Na+Moreover, electrogenic kinetics were performed to assess intramolecular movements of the transporter. All of the mutations were found to affect multiple transport steps, which suggested that the altered amino acids induced subtle structural changes rather than coordinating Pidirectly. The likely positions of the critical residues were mapped to the model of human Slc34a, and their localization in relation to the proposed substrate binding pockets concurs well with the observed functional data.
authors: Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom; andreas.werner@ncl.ac.uk.
authors: Andreas
authors: A
authors: Werner
authors: Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zürich, Switzerland; and.
authors: Monica
authors: M
authors: Patti
authors: Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom.
authors: Hany S
authors: HS
authors: Zinad
authors: Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom.
authors: Amy
authors: A
authors: Fearn
authors: Bio-Imaging Unit, Medical School, University of Newcastle upon Tyne, United Kingdom.
authors: Alex
authors: A
authors: Laude
authors: Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zürich, Switzerland; and.
authors: Ian
authors: I
authors: Forster
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D006801:Humans; D008956:Models, Chemical; D062105:Molecular Docking Simulation; D010710:Phosphates / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D012964:Sodium / Q000737:chemistry*; D050610:Sodium-Phosphate Cotransporter Proteins, Type II / Q000737:chemistry* / Q000648:ultrastructure*; D013045:Species Specificity; D013329:Structure-Activity Relationship; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000737:chemistry* / Q000648:ultrastructure
chemical_list: D000596:Amino Acids; D010710:Phosphates; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D029961:Zebrafish Proteins; D012964:Sodium
keywords: NaPi-II, Slc34a; flounder; membrane transport; phosphate; structure-function; zebrafish