Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements

Members of the SLC20 family or type III [Na.sup.+]-coupled [P.sub.i] cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular [P.sub.i] homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic [P.sub.i] co-transport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-l, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust [Na.sup.+]-dependent [sup.32][P.sub.i] uptake, but only Xenopus PIT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with [Li.sup.+] substituted for [Na.sup.+]. The dependence of the [P.sub.i]-induced current on [P.sub.i] concentration was Michaelian, and the dependence on [Na.sup.+] concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent [P.sub.i] affinity constant showed a minimum in the pH range 6.2-6.8 of ~0.05 mM and increased to ~0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual [sup.22] [Na-.sup.32][P.sub.i] uptake and suggested a 2:1 [Na.sup.+]:[P.sub.i] stoichiometry. A correlation of 32pi uptake and net charge movement indicated one charge translocation per [P.sub.i]. Changes in oocyte surface pH were consistent with transport of monovalent [P.sub.i]. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of [Na.sup.+]: [H.sub.2]P[O.sub.4]:[Na.sup.+]. Significantly, in contrast to type II [Na.sup.+]-[P.sub.i] cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PIT-2 activity. [Na.sup.+]-[P.sub.i] cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor