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(The FASEB Journal. 2007;21:916.3)
© 2007 FASEB
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916.3

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

Pakan Sassani1, Alexander Pushkin1, Natalia Abuladze1, Rustam Azimov1, Liyo Kao1, Janos Peti-Peterdi2, Weixin Liu1, Debra Newman1 and Ira Kurtz1

1 Medicine, UCLA, 7-155 Factor Bldg, Los Angeles, CA, 90095,
2 Physiology and Biophysics and Medicine, USC, 1501 San Pablo St, Los Angeles, CA, 90089-2821

ABSTRACT

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.

Supported by NIH.





This Article
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Right arrow Articles by Sassani, P.
Right arrow Articles by Kurtz, I.
PubMed
Right arrow Articles by Sassani, P.
Right arrow Articles by Kurtz, I.