Oligomeric Structure and Minimal Functional Unit of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1-A

Abstract

The electrogenic sodium bicarbonate cotransporter NBCe1-A mediates the basolateral absorption of sodium and bicarbonate in the proximal tubule. In this study the oligomeric state and minimal functional unit of NBCe1-A were investigated. Wildtype (wt) NBCe1-A isolated from mouse kidney or heterologously expressed in HEK293 cells was predominantly in a dimeric state as was shown using fluorescence energy transfer, pulldown, immunoprecipitation, cross-linking experiments, and nondenaturing perfluorooctanoate-PAGE. NBCe1-A monomers were found to be covalently linked by S-S bonds. When each of the 15 native cysteine residues were individually removed on a wt-NBCe1-A backbone, dimerization of the cotransporter was not affected. In experiments involving multiple native cysteine residue removal, both Cys 630 and Cys 642 in extracellular loop 3 were shown to mediate S-S bond formation between NBCe1-A monomers. When native NBCe1-A cysteine residues were individually reintroduced into a cysteineless NBCe1-A mutant backbone, the finding that a Cys 992 construct that lacked S-S bonds functioned normally indicated that stable covalent linkage of NBCe1-A monomers was not a necessary requirement for functional activity of the cotransporter. Studies using concatameric constructs of wt-NBCe1-A, whose activity is resistant to methanesulfonate reagents, and an NBCe1-A T442C mutant, whose activity is completely inhibited by methanesulfonate reagents, confirmed that NBCe1-A monomers are functional. Our results demonstrate that wt-NBCe1-A is predominantly a homodimer, dependent on S-S bond formation that is composed of functionally active monomers.

NBCe1A dimer assemble visualized by bimolecular fluorescence complementation

“…Together, these data suggest that NBCe1A form dimers and that the NH 2 termini from the two monomers are in close proximity, and likely pair up, to form a functional unit. A similar result was reported in another study measuring FRET when NBCe1A was tagged on the NH 2 terminus with EYFP or Cerulean and expressed in HEK293 cells (23). However, the fluorescent tags were not fused onto the COOH terminus of NBCe1A in this study.…”

“…Together these data suggest that the NBCe1 exists in a dimeric form. Similar findings were reported where membrane fractions were isolated from mouse renal cortex or HEK293 cells expressing NBCe1A and analyzed by PAGE using mild solubilization in the nondissociative detergent perfluoro-octanoic acid (PFO) (23).This study found that the dimeric form was the most dominant oligomer, but a minor tetramer coexisted with nondenaturing conditions (i.e., no DTT). Biochemical studies of the AE1 (SLC4A1) oligomeric structure have indicated that the AE1 protein forms dimers and tetramers with similar nondenaturing conditions (3,4,22).…”

Interplay between Disulfide Bonding and N-Glycosylation Defines SLC4 Na+-coupled Transporter Extracellular Topography

“…These variants differ in their Nand C-terminal regions but have the identical transmembrane region (8). NBCe1-A exists as a homodimer in the plasma membrane with each monomer functioning independently (10). Each monomer consists of an N-terminal cytoplasmic region, a C-terminal transmembrane region, and a short C-terminal cytoplasmic tail.…”

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

“…The transporter exists as a homodimer with each monomer functioning independently (18). Each NBCe1-A monomer consists of 1,035 amino acids with both of the NH 2 and COOH termini located in the cytosol.…”