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

Abstract

Abstract 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 m m progressively decreases the Na + affinity of NBCe1. However, decreasing [Na + ] from 96 to 35 m m 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. image 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− .

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