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The Functional Roles of Cysteine Residues in the KCC4 K‐Cl Cotransporter
Author(s) -
Mercado Adriana,
Babilonia Elisa,
Mount David
Publication year - 2008
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.22.1_supplement.936.1
Subject(s) - cysteine , mutant , cotransporter , chemistry , xenopus , alanine , serine , transmembrane protein , function (biology) , biochemistry , mutation , amino acid , transmembrane domain , intracellular , biophysics , microbiology and biotechnology , biology , gene , enzyme , receptor , organic chemistry , sodium
K + ‐Cl − cotransporters (KCCs) are modulated by the cysteine‐reactive compound NEM. Reducing agents also inhibit oligomerization of KCC2, implicating cysteine‐dependent disulfide bridges. KCC4 contains a total of 23 cysteines, 18 conserved; we have examined the functional role of all 23 cysteines, using expression in Xenopus oocytes. Mutation to alanine (transmembrane) or serine (extra/intracellular orientation) yielded several non‐functional mutants. However, converting these to leucine and/or other residues we found that no single cysteine was essential for function. Single mutants of conserved cysteines within TM2 were only modestly functional, suggesting a critical functional role for these residues. Sequential mutation to generate a cysteine‐less protein has been partially successful, preserving function when up to 11 cysteines were modified. The apparent dimerization of KCC4 in immunoblots was not affected in compound mutants, suggesting that cysteines are not required for dimerization of this KCC paralog. In summary, no single cysteine in KCC4 is required for K + ‐Cl − cotransport, nor are cysteines evidently required for dimerization. Progress has also been made in generating functional compound mutants that are profoundly cysteine‐depleted, suitable for defining cysteines targeted by activating/inhibiting stimuli and/or for other structure‐function studies.