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°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 ␦ ؊ 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 ␦ ؊ helical dipole at the C-terminal end of TM1 involving Thr-442 that plays a critical role in the function of the cotransporter.

Topological Location and Structural Importance of the NBCe1-A Residues Mutated in Proximal Renal Tubular Acidosis

“…Immunocytochemistry-24 h post-transfection, cells were rinsed with PBS (140 mM NaCl, 3 mM KCl, 6.5 mM Na 2 HPO 4 , 1.5 mM KH 2 PO 4 , pH 7.4) and incubated with a rabbit antihuman NBCe1-A antibody (Ab-162, 1:100 dilutions in PBS) that interacts with an epitope in putative extracellular loop 3 (5). After a 15-min incubation at room temperature, cells were rinsed with PBS and further incubated with goat anti-rabbit IgG conjugated with Cy3 (1:500 dilution in PBS, from Jackson ImmunoResearch) for 30 min at room temperature.…”

“…NBCe1-A exists as a membrane-embedded 140-kDa homodimer, of which each monomer is fully functional (4). The proposed transmembrane segments (TMs) 1 and 8 were recently analyzed by cysteine scanning mutagenesis and several residues were found to be involved in forming the ion translocation pore (5,6). In particular, residue Thr 442 in TM 1 was proposed to form an external gate for the transported substrates (5).…”

“…The proposed transmembrane segments (TMs) 1 and 8 were recently analyzed by cysteine scanning mutagenesis and several residues were found to be involved in forming the ion translocation pore (5,6). In particular, residue Thr 442 in TM 1 was proposed to form an external gate for the transported substrates (5). Interestingly, substitution of Asp 555 to glutamic acid in the proposed TM 5 of NBCe1-A induced an outward rectifying Cl Ϫ current and altered the transport substrate selectivity, indicating it plays an important role in HCO 3 Ϫ selectivity (7).…”

Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis

“…The transmembrane region of NBCe1-A contains 14 transmembrane segments (TM), and the last five TMs may participate in forming a scaffold to accommodate NBCe1-A substrate ion interaction sites (45,46). Three amino acids in NBCe1-A-TM1 (Ala428, Ala435, and Thr442) (44) and one amino acid in TM8 (Leu750) (25) have been determined to line the ion permeation pathway and are potentially involved in ion translocation. Asp555 in TM5 has been implicated in playing an important role in anion selectivity (42), and extracellular loop 4 was reported to play a role in the electrogenicity of NBCe1-A although the mechanism is currently unclear (5).…”

SLC4A11 is an EIPA-sensitive Na+ permeable pHi regulator

“…HEK293 is a good model cell line for studying bicarbonate transport, as expression of bicarbonate transporters is low (1)(2)29) and endogenous expression of SLC4A11 is low (20). In addition, we tested Na ϩ -OH Ϫ (H ϩ ) transport activity in NHE-deficient PS120 cells to differentiate SLC4A11 as a bonafide transporter or activator of NHEs (15).…”