title: Effect of simultaneously replacing putative TM6 and TM12 of human NBCe1-A with those from NBCn1 on surface abundance in Xenopus oocytes.
abstract: HCO₃⁻ translocation across the plasma membrane via the electrogenic Na/HCO₃⁻ cotransporter NBCe1 plays an important role in intracellular pH regulation and transepithelial HCO₃⁻ transport. However, the structural determinants of transporter function remain largely unknown. A previous study showed that the putative fourth extracellular loop (EL4) plays an essential role in determining the electrogenicity of NBCe1. In the present study, we generated eight new chimeras of human NBCe1-A and NBCn1-A. All possess the putative NBCe1 EL4 and are electrogenic. Chimera O, in which the putative sixth transmembrane segment (TM6) and EL5 through the C terminus (Ct) of NBCe1 was replaced by corresponding NBCn1 sequence, produces the smallest hyperpolarization (1-2 mV) when CO₂/HCO₃⁻ is added to the extracellular solution. Biotinylation experiments show that O has a very low abundance at the plasma membrane. However, chimeras in which we simultaneously replaced the putative TM6 and smaller subdomains of the EL5-Ct region for the NBCn1 sequence were strongly electrogenic except for chimera T, in which we replaced TM6 and TM12 of NBCe1 with the corresponding regions of NBCn1. T exhibited greatly reduced transporter surface expression compared to wild-type NBCe1-A, while retaining at least some electrogenic character. We hypothesize that putative TM6 and TM12 are part of a functional unit and that if the two TMs are replaced by those of the same transporter type, high surface expression would require that the surrounding TMs are also from the same transporter type.
authors.affiliation: Department of Biological Sciences, Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science & Technology, Wuhan 430074, Hubei Province, China. liming.chen@mail.hust.edu.cn
authors.forename: Li-Ming
authors.initials: LM
authors.lastname: Chen
authors.forename: Xue
authors.initials: X
authors.lastname: Qin
authors.forename: Fraser J
authors.initials: FJ
authors.lastname: Moss
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Walter F
authors.initials: WF
authors.lastname: Boron
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011048:Polarography; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry* / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: C498898:SLC4A4 protein, human; C422022:SLC4A7 protein, human; D027982:Sodium-Bicarbonate Symporters; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid