title: Role of an extracellular loop in determining the stoichiometry of Na+-HCO₃⁻ cotransporters.
abstract: The Na+–HCO₃⁻ cotransporters (NBCs) of the solute carrier 4 family (SLC4) are critical for regulating pH in cells as well as in fluids such as blood and cerebrospinal fluid. Moreover, mutations and gene disruptions in NBC are linked to a wide range of pathologies. NBCe1 (SLC4A4) is electrogenic because it has an apparent Na+:HCO₃⁻ stoichiometry of 1:2 or 1:3, whereas NBCn1 (SLC4A7) is electroneutral because it has an apparent stoichiometry of 1:1. Because stoichiometry influences the effect of transport on membrane potential and vice versa, a central question is what structural features underlie electrogenicity versus electroneutrality. A previous study on rat NBCe1/n1 chimeras demonstrated that the structural elements determining the electrogenicity of NBCe1-A are located within the transmembrane domain, excluding the large third extracellular loop. In the present study we generated a series of chimeras of human NBCe1-A and human NBCn1-A. We found that replacing merely the predicted fourth extracellular loop (EL4) – containing 32 amino acid residues that include 7 prolines – of human NBCe1-A with EL4 of NBCn1-A creates an electroneutral NBC. The opposite switch converts an electroneutral construct to one with electrogenic properties. The introduction of an N-glycosylation site into EL4 confirms that at least a part of it is exposed to the extracellular fluid. We hypothesize that putative EL4 either contributes to the substrate-binding vestibule or indirectly influences substrate binding by interacting with one or more transmembrane segments, thereby controlling the nature of transport.
authors.affiliation: Department of Biological Science, Key Laboratory of Molecular Biophysics of Ministry of Education, Huazhong University of Science and Technology School of Life Science and Technology, 1037 Luoyu Road, Wuhan, Hubei Province 430074, P.R. China.   liming.chen@mail.hust.edu.cn
authors.forename: Li-Ming
authors.initials: LM
authors.lastname: Chen
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Walter F
authors.initials: WF
authors.lastname: Boron
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002678:Chimera / Q000502:physiology; D045604:Extracellular Fluid / Q000502:physiology; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry* / Q000502:physiology*; D013237:Stereoisomerism; D014982:Xenopus laevis
chemical_list: D020033:Protein Isoforms; C498898:SLC4A4 protein, human; C422022:SLC4A7 protein, human; D027982:Sodium-Bicarbonate Symporters