title: Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2.
abstract: The general phosphate need in mammalian cells is accommodated by members of the P(i) transport (PiT) family (SLC20), which use either Na(+) or H(+) to mediate inorganic phosphate (P(i)) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na(+)-dependent P(i) (NaP(i)) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with (32)P(i) as a traceable P(i) source. For PiT1, the Michaelis-Menten constant for P(i) was determined as 322.5 +/- 124.5 microM. PiT2 was analyzed for the first time and showed positive cooperativity in P(i) uptake with a half-maximal activity constant for P(i) of 163.5 +/- 39.8 microM. PiT1- and PiT2-mediated Na(+)-dependent P(i) uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na(+) dependency patterns. However, only PiT2 was capable of Na(+)-independent P(i) transport at acidic pH. Study of the impact of divalent cations Ca(2+) and Mg(2+) revealed that Ca(2+) was important, but not critical, for NaP(i) transport function of PiT proteins. To gain insight into the NaP(i) cotransport function, we analyzed PiT2 and a PiT2 P(i) transport knockout mutant using (22)Na(+) as a traceable Na(+) source. Na(+) was transported by PiT2 even without P(i) in the uptake medium and also when P(i) transport function was knocked out. This is the first time decoupling of P(i) from Na(+) transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E(55) and E(575) are responsible for linking P(i) import to Na(+) transport in PiT2.
authors.affiliation: Department of Molecular Biology, Institute of Clinical Medicine, University of Aarhus, Aarhus, Denmark.
authors.forename: Pernille
authors.initials: P
authors.lastname: Bøttger
authors.forename: Susanne E
authors.initials: SE
authors.lastname: Hede
authors.forename: Morten
authors.initials: M
authors.lastname: Grunnet
authors.forename: Boy
authors.initials: B
authors.lastname: Høyer
authors.forename: Dan A
authors.initials: DA
authors.lastname: Klaerke
authors.forename: Lene
authors.initials: L
authors.lastname: Pedersen
mesh_terms: D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D018075:RNA, Complementary / Q000378:metabolism; D012964:Sodium / Q000737:chemistry / Q000378:metabolism*; D012979:Sodium Radioisotopes / Q000378:metabolism; D050611:Sodium-Phosphate Cotransporter Proteins, Type III / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D010710:Phosphates; D010761:Phosphorus Radioisotopes; D020033:Protein Isoforms; D018075:RNA, Complementary; D012979:Sodium Radioisotopes; D050611:Sodium-Phosphate Cotransporter Proteins, Type III; D012964:Sodium; D004262:DNA Restriction Enzymes; D008274:Magnesium; D002118:Calcium