Oligomeric structure and minimal functional unit of the electrogenic sodium bicarbonate cotransporter NBCe1-A.
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. Wild-type (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.
Kao
Liyo
L
Division of Nephrology, David Geffen School Medicine, UCLA, Los Angeles, California 90095-1689, USA.
Sassani
Pakan
P
Azimov
Rustam
R
Pushkin
Alexander
A
Abuladze
Natalia
N
Peti-Peterdi
Janos
J
Liu
Weixin
W
Newman
Debra
D
Kurtz
Ira
I
D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D019281:Dimerization; D006801:Humans; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; C498899:Slc4a4 protein, mouse; D027982:Sodium-Bicarbonate Symporters