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

Andrini O, Meinild AK, Ghezzi C, Murer H, Forster IC. Lithium interactions with Na+-coupled inorganic phosphate cotransporters: insights into the mechanism of sequential cation binding. Am J Physiol Cell Physiol 302: C539-C554, 2012. First published November 9, 2011; doi: 10.1152/ajpcell. 00364.2011.-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 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.