PHOSPHATE-TRANSPORT BY THE HUMAN RENAL COTRANSPORTER NAPI-3 EXPRESSEDIN HEK-293 CELLS

The human renal Na-PO4 cotransporter gene NaPi-3 was expressed in huma n embryonic kidney HEK-293 cells, and the transport characteristics we re measured in cells transfected with a vector containing NaPi-3 or wi th the vector alone (sham transfected). The initial rate of (PO4)-P-32 influx had saturation kinetics for external Na and PO4 with K-1/2(Na) of 128 mM (PO4 = 0.1 mM) and K-1/2(PO4) of 0.084 mM (extracellular Na = 143 mM) in sham-and NaPi-3-transfected cells expressing the transpo rter. Transfection had no effect on the Na-independent (PO4)-P-32 infl ux, but transfection increased Na-dependent (PO4)-P-32 influxes 2.5- t o 5-fold. Of the alkali cations, only Na significantly supported PO4 i nflux. Arsenate inhibited flux with an inhibition constant of 0.4 mM. The phosphate transport in sham- and NaPi-3-transfected cells has near ly the same temperature dependence in the absence and presence of extr acellular Na. The Na-dependent phosphate flux decreased with pH in sha m-transfected cells but was pH independent in transfected cells. The N a-dependent (PO4)-P-32 influx was inhibited by p-chloromercuriphenylsu lfonate, phosphonoformate, phloretin, vanadate, and 5-(N-methyl-N-isob utyl)-amiloride but not by amiloride or other amiloride analogs. These functional characteristics are in general agreement with the known be havior of NaPi-3 homologues in the renal tubule of other species and, thus, demonstrate the fidelity of this transfection system for the stu dy of this protein. Commensurate with the increased functional express ion, there was an increase in the amount of NaPi-3 protein by Western analysis.