Proton-sensitive transitions of renal type IINa+-coupled phosphate cotransporter kinetics

In the kidney proximal tubule, acidification of the glomerular filtrate leads to an inhibition of inorganic phosphate (P-i) reabsorption by type II Na+-coupled cotransporters (NaPi-II). As external pH also alters the divalent/monovalent P-i ratio, it has been difficult to separate putative proton interactions with the cotransporter from direct titration of divalent P-i, the preferred species transported. To distinguish between these possibilities and identify pH-sensitive transitions in the cotransport cycle, the pH-dependent kinetics of two NaPi-II isoforms, expressed in Xenopus laevis oocytes, were investigated electrophysiologically. At -50 mV, both isoforms showed >70% suppression of electrogenic response for an external pH change from 8.0 to 6.2, not attributable to titration of divalent P-i. This was accompanied by a progressive removal of steady-state voltage dependence. The NaPi-II-related uncoupled slippage current was unaffected by a pH change from 7.4 to 6.2, with no shift in the reversal potential, which suggested that protons do not function as substrate. The voltage-dependence of pre-steady-state relaxations was shifted to depolarizing potentials in 100 mM and 0 mM Na-ext(+) and two kinetic components were resolved, the slower of which was pH-dependent The changes in kinetics are predicted by a model in which protons interact with the empty carrier and final Na+ binding step.