Proton-Sensitive Transitions of Renal Type II Na +-Coupled Phosphate Cotransporter Kinetics

Abstract

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.