Lithium interactions with the type II electrogenic sodium-coupled phosphate cotransporter
Ian C. Forster, Olga Andrini and Anne-Kristine Meinild
Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Switzerland
Introduction: Type IIa or IIb Na+-coupled inorganic phosphate cotransporters (NaPi-IIa/b) are generally considered to be exclusively Na+ dependent. We now show that Li+ can substitute for Na+ as a driving cation and that one Li+ ion can occupy the 1stcation interaction site, which is used to couple the transmembrane electric field to energize thecotransportcycle.
Methods: We expressed NaPi-IIa/b in X. laevisoocytes and performed two-electrode voltage-clamp electrophysiology and uptake assays to investigate the effect of external Li+ on their transport kinetics.
Results: Substitution of 50% external Na+ with Li+ reduced the maximum transport rate but increased the apparent affinity for Pi. Moreover, the steady-state voltage dependence of the Pi-induced current was significantly weaker in the presence of external Li+. By means of simultaneous electrophysiology and 22Na uptake on single oocytes, we established that Li+ ions can substitute for at least one of the 3 Na+ ions that participate incotransport.We performed presteady-state assays to identify which partial reactions in the transport cycle involved Li+ interactions. Our data provide compelling evidence that Li+ ions, in the absence of external Na+ can interact with the empty carrier. However, the total charge displaced with Li+ alone was 70% of that in the presence of Na+, or when 50% of the Na+ was replaced by Li+. If Na+ and Li+ were both present, the voltage dependence of the mobile charge movement was shifted in the depolarizing direction, which was also consistent with the altered steady-state voltage dependence of the phosphate induced current. Our presteady-state data suggested that the charge movements in the presence of Li+ alone reflect the interaction of a single Li+ ion with the transporter, in contrast to 2 Na+ ions when only Na+ is present.
Intepretation: We propose an ordered binding scheme for cotransport in which either a Na+or Li+ ion can bind at the putative 1stcation interaction site. This is followed by the cooperative binding of a Na+ ion, one divalent Pi anion and another Na+ ion completes the loading of the carrier before translocation. With Li+ bound, the 2nd Na+ ion is more tightly bound to the protein, however the translocation rate of the fully loaded carrier is significantly reduced. Model simulations confirm our experimental findings.
Support: Swiss National Science Foundation (to ICF) and Benzon Foundation (to A-KM)