NBCe1-A Transmembrane Segment 1 Lines the Ion Translocation Pathway

• Published in: The Journal of biological chemistry 2009-03, Vol.284 (13), p.8918-8929
• Main Authors: Zhu, Quansheng, Azimov, Rustam, Kao, Liyo, Newman, Debra, Liu, Weixin, Abuladze, Natalia, Pushkin, Alexander, Kurtz, Ira
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Bicarbonates - chemistry; Bicarbonates - metabolism; Cell Line; Ethyl Methanesulfonate - analogs & derivatives; Ethyl Methanesulfonate - chemistry; Humans; Ion Transport - physiology; Membrane Transport, Structure, Function, and Biogenesis; Mesylates - chemistry; Mutation, Missense; Protein Structure, Tertiary - physiology; Sodium - chemistry; Sodium - metabolism; Sodium-Bicarbonate Symporters - antagonists & inhibitors; Sodium-Bicarbonate Symporters - chemistry; Sodium-Bicarbonate Symporters - genetics; Sodium-Bicarbonate Symporters - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M806674200

The electrogenic Na+/ HCO3- 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° 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 δ- 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 δ- helical dipole at the C-terminal end of TM1 involving Thr-442 that plays a critical role in the function of the cotransporter.