Lithium interactions with Na +-coupled inorganic phosphate cotransporters: Insights into the mechanism of sequential cation binding

摘要

Type IIa/b Na +-coupled inorganic phosphate cotransporters (NaPi-IIa/b) are considered to be exclusively Na + dependent. Here we show that Li + can substitute for Na + as a driving cation. We expressed NaPi-IIa/b in Xenopus laevis oocytes and performed two-electrode voltage-clamp electrophysiology and uptake assays to investigate the effect of external Li + on their kinetics. Replacement of 50% external Na + with Li + reduced the maximum transport rate and the rate-limiting plateau of the P i-induced current began at less hyperpolarizing potentials. Simultaneous electrophysiology and 22Na uptake on single oocytes revealed that Li + ions can substitute for at least one of the three Na + ions necessary for cotransport. Presteady-state assays indicated that Li + ions alone interact with the empty carrier; however, the total charge displaced was 70% of that with Na + alone, or when 50% of the Na + was replaced by Li +. If Na + and Li + were both present, the midpoint potential of the steady-state charge distribution was shifted towards depolarizing potentials. The charge movement in the presence of Li + alone reflected the interaction of one Li + ion, in contrast to 2 Na + ions when only Na was present. We propose an ordered binding scheme for cotransport in which Li + competes with Na + to occupy the putative first cation interaction site, followed by the cooperative binding of one Na + ion, one divalent Pi anion, and a third Na + ion to complete the carrier loading. With Li + bound, the kinetics of subsequent partial reactions were significantly altered. Kinetic simulations of this scheme support our experimental data.