A Study On The Kinetics And Structural Mechanism For The Transport Of Na⁺ And HCO₃^- By NBCe1

The electrogenic Na⁺-Bicarbonate Cotransporter 1（NBCe1）is a kind of secondary active transporter of solute carrier 4（SLC4）family that mediates the translocation of Na⁺and HCO₃^-across the plasma membrane.NBCe1 plays a very important physiological role in the body.Dysfunction of NBCe1 leads to severe metabolic acidosis,accompanied with a series of syndromes,including migraine headaches,cataracts,high blood pressure,osteoporosis,intellectual disability,etc.Under physiological conditions,NBCe1 usually mediates the absorption or secretion of Na⁺and HCO₃^-with an apparent ion stoichiometric ratio of 1:2 or 1:3,thereby maintaining the acid-base homeostasis of cells and the body.The distribution and function of NBCe1 in various tissues have been thoroughly studied,but the specific molecular mechanism of its substrate transport is still unclear.In this study,we employ molecular biology,structural biology,electrophysiology technology in the Xenopus oocyte system and molecular docking simulation to investigate the molecular mechanism of NBCe1 transport,including the kinetic order of the binding of Na⁺and HCO₃^-,substrate binding site for Na⁺and HCO₃^-,and the actual stoichiometric ratio of NBCe1.The substrate affinity experiments show that the binding of Na⁺to NBCe1 is affected by changes in HCO₃^-concentration,while the binding of HCO₃^-to NBCe1 is not affected by changes of Na⁺concentration.At the same time,all mutations that have a major effect on HCO₃^-affinity also greatly affect the Na⁺affinity.However,mutations that greatly affect Na⁺affinity could have no significant e ffect on the HCO₃^-affinity.These results support a kinetic model for the binding of substrate to NBCe1 as sequential binding that HCO₃^-precedes Na⁺.In addition,the Hill coefficients of Na⁺and HCO₃^-are 1.10 and 1.07respectively,indicating that the binding of Na⁺or HCO₃^-to NBCe1 have no synergistic effect,although it exists as a dimer.Affinity analysis reveal that mutations of residues Thr⁴³⁶,Ile⁸⁰³ and Phe⁸⁴⁰ located in the substrate-binding pocket affect only Na⁺affinity,but not HCO₃^-affinity.We propose that these residues are involved in the coordination of Na⁺.Mutations to residues Asn⁴³⁹,Ser⁴⁸³,Ser⁴⁸⁴,Thr⁷⁵⁸,Val⁷⁹⁸,Ala⁸⁰⁰,Thr⁸⁰¹ and Val⁸⁰² affect both Na⁺and HCO₃^-affinities.We propose that these residues are involved in the coordination of HCO₃^-.In addition,molecular docking simulation is performed to explore the binding of HCO₃^-in NBCe1.The results of simulation are well consistent with the above conclusions obtained based on the mutantional analysis.Thus,the amino acid sites involved in Na⁺and HCO₃^-binding during substrate transport are identified.Molecular docking simulations also show that both HCO₃^-and CO₃^2-can be coordinated in the substrate-binding pocket of NBCe1 by the same set of residues.Due to the steric hindrance effect,NBCe1 can only bind 2 HCO₃^-or CO₃^2-.Based on these observations,we propose that NBCe1 actua lly transports 1 Na⁺and 2 HCO₃^-when operating with an apparent stoichiometric ratio of 1:2;and that NBCe1 actually transports1Na⁺,1HCO₃^-and 1CO₃^2-when operating with an apparent stoichiometric ratio of 1:3.Therefore,the difference in the apparent ion stoichiometric ratio of N BCe1 is not due to the difference in the number of substrate binding sites in NBCe1,but the result of the different types of substrates.Based on the above experiments,in-depth analysis and reference to relevant literature,we propose the following theories in this study:（1）The substrates binding kinetics model of NBCe1 is that HCO₃^-precedes Na⁺,and the binding of HCO₃^-or Na⁺to NBCe1 has no synergistic effect;（2）the respective binding regions of Na⁺and HCO₃^-and the involved amino acid residues are identified;（3）NBCe1 actually transports 1 Na⁺and 2 HCO₃^-in mode of 1:2 and transports 1 Na⁺,1 HCO₃^-and 1 CO₃^2-in mode of 1:3.This study about the ion transport mechanism of NBCe1 provides an important insight into understanding the characteristics and transport mechanism of other proteins in SLC4 family and also provides an important theoretical basis and thought for the development of targeted drugs for related diseases.