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

The secondary active transporter NBCe1 couples the transmembrane movement of Na⁺ and carbonate species with an apparent stoichiometry of 1Na⁺ :2HCO₃^- (the 'influx' mode) or 1Na⁺ :3HCO₃^- (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 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 respectively 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 CO₃^2- . 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⁺  + 2HCO₃^- , whereas NBCe1 in the efflux mode moves 1Na⁺  + 1HCO₃^-  + 1CO₃^2- . 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⁺ :2HCO₃^- or in the 'efflux' mode with an apparent stoichiometry of 1Na⁺ :3HCO₃^- . 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 CO₃^2- . It is proposed that NBCe1 in the influx mode moves 1Na⁺  + 2HCO₃^- across the plasma membrane, whereas NBCe1 in the efflux mode moves 1Na⁺ +1HCO₃^- +1CO₃^2- .