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Cysteines in Extracellular Loop 3 of NBCe1‐A Form Intra‐ and Inter‐Molecular Disulfide Bonds
Author(s) -
Zhu Quansheng,
Azimov Rustam,
Kao Liyo,
Pushkin Alexander,
Kurtz Ira
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.800.10
Subject(s) - cysteine , chemistry , serine , extracellular , residue (chemistry) , hydrogen bond , biochemistry , bicarbonate , stereochemistry , mutant , molecule , enzyme , gene , organic chemistry
NBCe1‐A cotransports sodium and bicarbonate across the basolateral membrane of the proximal tubule. Four cysteines in the putative NBCe1‐A extracellular loop 3 are conserved among all the SLC4 family sodium coupled bicarbonate transporters. To determine the structural importance of these conserved cysteines, we explored the potential disulfide bonding among the 4 cysteine residues. NBCe1‐A constructs bearing various cysteine mutations were expressed in HEK‐293 cells and examined for the presence of disulfide bonds with the cysteine accessibility method (CAM). Initially we analyzed the aqueous accessibility of the 5 endogenous cytoplasmic cysteines. The results revealed that C120 and C399 are not exposed to the aqueous medium, whereas C389, C992 and C1035 are highly exposed, suggesting that the N‐terminal cytoplasmic region of NBCe1‐A is structured. Mutation of the cytoplasmic cysteines to serines eliminated any free aqueous accessible cysteines indicating that cysteines 583, 585, 630 and 642 in extracellular loop 3 partake in either intra‐ or inter‐monomeric disulfide bonding. Mutation of both cysteine 583 and 585 to serine did not free any cysteines, however, upon treatment with 5mM DTT, free cysteines were detected. Further analysis on mutants where 3 of the 4 cysteines were replaced with serine: 583/585/630 or 583/585/642 showed that the remaining cysteine (642 or 630) in each construct could only be freed after DTT treatment. In conclusion, our data suggests that cysteine 583 and 585 form an intra molecular disulfide bond, whereas cysteine 630 and 642 each form disulfide bonds with the same residue on the opposite monomer, and therefore appears to play an important role in the oligomerization of the cotransporter. Supported by NIH