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Role of S‐S Bond Formation in the Oligomerization of kNBC1 (NBCe1‐A)

The oligomerization of kNBC1 (NBCe1‐A) which mediates basolateral bicarbonate transport in the renal proximal tubule was studied in HEK293 cells. In cells co‐expressing Cerulean‐kNBC1 and EYFP‐kNBC1, a FRET signal was detected on the plasma membrane indicating interaction between the 2 tagged proteins. His6‐kNBC1 co‐immunoprecipitated with GFP‐kNBC1 from cells co‐expressing both constructs, and GFP‐kNBC1 was pulled down with His6‐kNBC1 coupled to Ni‐beads. These results indicated that kNBC1 is expressed as an oligomer. To estimate the oligomeric structure of kNBC1, plasma membrane proteins were analyzed using non‐denaturing PAGE in the presence of perfluorooctanoate (PFO), and denaturing PAGE in the presence of SDS, with and without DTT. Only monomers were detected using SDS‐PAGE with DTT, whereas without DTT, dimers and to a lesser extent higher oligomers were detected. A dimer was also the predominant kNBC1 oligomic form detected using non‐denaturing PAGE without DTT. Cross‐linking of kNBC1 expressed in HEK293 cells confirmed dimers as the predominant oligomeric form. The results suggested the S‐S‐bond formation is necessary for the oligomerization of kNBC1. To explore this hypothesis we analyzed 2 mutants, a cysteineless kNBC1 mutant, and a cysteineless mutant in which only Cys992 was restored. The cysteineless mutant was retained intracellularly, whereas Cys992 completely restored plasma membrane expression and function. The results indicate that S‐S‐bond formation between kNBC1 monomers is required for the plasma membrane expression of functionally active kNBC1, and that the predominant oligomeric state of the cotransporter is a dimer.