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

Abstract

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 Pi-induced current began at less hyperpolarizing potentials. Simultaneous electrophysiology and22Na 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 Pianion, 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.