Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2

Am J Physiol Cell Physiol. 2006 Dec;291(6):C1377-87. doi: 10.1152/ajpcell.00015.2006. Epub 2006 Jun 21.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Transport
  • Calcium / metabolism
  • DNA Restriction Enzymes / metabolism
  • Humans
  • Hydrogen-Ion Concentration
  • Magnesium / metabolism
  • Oocytes / cytology
  • Oocytes / physiology
  • Phosphates / chemistry
  • Phosphates / metabolism*
  • Phosphorus Radioisotopes / metabolism
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • RNA, Complementary / metabolism
  • Sodium / chemistry
  • Sodium / metabolism*
  • Sodium Radioisotopes / metabolism
  • Sodium-Phosphate Cotransporter Proteins, Type III / genetics
  • Sodium-Phosphate Cotransporter Proteins, Type III / metabolism*
  • Xenopus laevis

Substances

  • Phosphates
  • Phosphorus Radioisotopes
  • Protein Isoforms
  • RNA, Complementary
  • Sodium Radioisotopes
  • Sodium-Phosphate Cotransporter Proteins, Type III
  • Sodium
  • DNA Restriction Enzymes
  • Magnesium
  • Calcium