J Physiol 2022 Jul 01;60013:3083-3111. doi: 10.1113/JP282034.

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

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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- .

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Genes referenced: slc4a4