Molecular insight into coordination sites for substrates and their coupling kinetics in Na + /HCO 3 - 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 transport coupling of Na + and HCO 3 - in NBCe1. In Xenopus oocytes, decreasing extracellular [HCO 3 - ] 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 HCO 3 - affinity. The residues responsible for the coordination of Na + and HCO 3 - in the substrate pocket of NBCe1 were respectively determined by mutational and molecular simulation studies. Mutation to the residues for HCO 3 - coordination decreased the affinities of NBCe1 for both Na + and HCO 3 - . However, mutation to the residues for Na + coordination decreased the affinity for Na + but had little effect on the affinity for HCO 3 - . Molecular simulation showed that NBCe1 has the capacity to coordinate only two ions of HCO 3 - or CO 3 2- . We propose that (1) NBCe1 has an ordered substrate-binding kinetics with the binding of HCO 3 - 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 HCO 3 - 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 HCO 3 - or CO 3 2- . 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- .