Molecular insight into coordination sites for substrates and their coupling kinetics in Na+/HCO3- cotransporter NBCe1

The secondary active transporter NBCe1 couples the transmembrane movement of Na+ and carbonate species with an apparent stoichiometry of 1Na(+):2HCO(3)(-) (the 'influx' mode) or 1Na(+) :3HCO(3)(-) (the 'efflux' mode). Here, we employed molecular biology, electrophysiology and structural biology approaches to investigate the molecular mechanism for the coupling of Na+ and HCO3- in NBCe1. In Xenopus oocytes, decreasing extracellular [HCO3-] from 66 to 4 mm progressively decreases the Na+ affinity of NBCe1. However, decreasing [Na+] from 96 to 35 mm has little effect on the HCO3- affinity. The residues responsible for the coordination of Na+ and HCO3- in the substrate pocket of NBCe1 were determined by mutational and molecular simulation studies. Mutation to the residues for HCO3- coordination decreased the affinities of NBCe1 for both Na+ and HCO3-. However, mutation to the residues for Na+ coordination decreased the affinity for Na+ but had little effect on the affinity for HCO3-. Molecular simulation showed that NBCe1 has the capacity to coordinate only two ions of HCO3- or CO32-. We propose that (1) NBCe1 has an ordered substrate-binding kinetics with the binding of HCO3- preceding that of Na+; (2) NBCe1 operating in the influx mode moves 1Na(+) + 2HCO(3)(-), whereas NBCe1 in the efflux mode moves 1Na(+) + 1HCO(3)(-) + 1CO(3)(2-). The substrate-binding kinetics of NBCe1 is distinct from the known kinetics models of many other Na+-coupled transporters with Na+ binding preceding the driven solute. Key points Under physiological conditions, the secondary active transporter NBCe1 can operate in the 'influx' mode with an apparent stoichiometry of 1Na(+) :2HCO(3)(-) or in the 'efflux' mode with an apparent stoichiometry of 1Na(+):3HCO(3)(-). NBCe1 has an ordered substrate-binding kinetics with HCO3- preceding the binding of Na+. The kinetics of NBCe1 is distinct from the known kinetics of many other Na+-driven cotransporters for which the binding of Na+ usually precedes the driven substrate. The residues responsible for the coordination of Na+ and those for carbonate species in the substrate-binding pocket of NBCe1 were determined by mutation and molecular simulation studies. The substrate-binding pocket of NBCe1 contains just two coordination sites for HCO3- or CO32-. It is proposed that NBCe1 in the influx mode moves 1Na(+) + 2HCO(3)(-) across the plasma membrane, whereas NBCe1 in the efflux mode moves 1Na(+) +1HCO(3)(-) +1CO(3)(2-).