title: NBCe1-A Transmembrane Segment 1 Lines the Ion Translocation Pathway.
abstract: The electrogenic Na(+)/HCO(3)(-) cotransporter (NBCe1-A) transports sodium and bicarbonate across the basolateral membrane of the renal proximal tubule. In this study the structural requirement of transmembrane segment 1 (TM1) residues in mediating NBCe1-A transport was investigated. Twenty-five introduced cysteine mutants at positions Gln-424 to Gly-448 were tested for their sensitivity to the methanethiosulfonate reagents (2-sulfonatoethyl) methanethiosulfonate (MTSES), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and (2-aminoethyl) methanethiosulfonate (MTSEA). Two mutants, T442C and A435C, showed 100 and 70% sensitivity, respectively, to inhibition by all the three methanethiosulfonate (MTS) reagents, I441C had >50% sensitivity to MTSET and MTSEA, and A428C had 50% sensitivity to MTSEA inhibition. A helical wheel plot showed that mutants T442C, A435C, and A428C are clustered on one face of TM1 within a 100 degrees arc. Topology analysis of TM1 with biotin maleimide and 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) revealed Thr-442 marks the C-terminal end of TM1 and that extracellular FGGLLG stretch is in a small aqueous-accessible cavity. Functional studies indicated that Thr-442 resides in a narrow region of the ion translocation pore with strong delta(-) helical dipole influence. Analysis of the corresponding residue of NBCe1-A-Thr-442 in AE1 (Thr-422) shows it is functionally insensitive to MTSES and unlabeled with MTS-TAMRA, indicating that AE1-TM1 is oriented differently from NBCe1-A. In summary, we have identified residues Thr-442, Ala-435, and Ala-428 in TM1 lining the ion translocation pore of NBCe1-A. Our findings are suggestive of a delta(-) helical dipole at the C-terminal end of TM1 involving Thr-442 that plays a critical role in the function of the cotransporter.
authors.affiliation: Division of Nephrology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095-1689, USA.
authors.forename: Quansheng
authors.initials: Q
authors.lastname: Zhu
authors.forename: Rustam
authors.initials: R
authors.lastname: Azimov
authors.forename: Liyo
authors.initials: L
authors.lastname: Kao
authors.forename: Debra
authors.initials: D
authors.lastname: Newman
authors.forename: Weixin
authors.initials: W
authors.lastname: Liu
authors.forename: Natalia
authors.initials: N
authors.lastname: Abuladze
authors.forename: Alexander
authors.initials: A
authors.lastname: Pushkin
authors.forename: Ira
authors.initials: I
authors.lastname: Kurtz
mesh_terms: D019943:Amino Acid Substitution; D001639:Bicarbonates / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D017136:Ion Transport / Q000502:physiology; D008698:Mesylates / Q000737:chemistry; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary / Q000502:physiology; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001639:Bicarbonates; D008698:Mesylates; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters; C093171:methanethiosulfonate ethylammonium; C109153:(2-sulfonatoethyl)methanethiosulfonate; C109154:(2-(trimethylammonium)ethyl)methanethiosulfonate; D005020:Ethyl Methanesulfonate; D012964:Sodium