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

The general phosphate need in mammalian cells is accommodated by members of the Pi transport (PiT) family (SLC20), which use either Na+ or H+ to mediate inorganic phosphate (P-i) symport. The mammalian PiT para-logs PiT1 and PiT2 are Na+-dependent Pi ( NaPi) 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 P-32(i) as a traceable Pi source. For PiT1, the Michaelis-Menten constant for Pi was determined as 322.5 +/- 124.5 mu M. PiT2 was analyzed for the first time and showed positive cooperativity in Pi uptake with a half-maximal activity constant for Pi of 163.5 +/- 39.8 mu M. PiT1- and PiT2-mediated Na+-dependent Pi 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 Pi transport at acidic pH. Study of the impact of divalent cations Ca2+ and Mg2+ revealed that Ca2+ was important, but not critical, for NaPi transport function of PiT proteins. To gain insight into the NaPi cotransport function, we analyzed PiT2 and a PiT2 Pi transport knockout mutant using Na-22(+) as a traceable Na+ source. Na+ was transported by PiT2 even without Pi in the uptake medium and also when Pi transport function was knocked out. This is the first time decoupling of Pi 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 Pi import to Na+ transport in PiT2.