Site-Directed Disulfide Mapping of Residues Contributing to the Ca super(2+) and K super(+) Binding Pocket of the NCKX2 Na super(+)/Ca super(2+)-K super(+) Exchanger

The Na super(+)/Ca super(2+)-K super(+) exchanger (NCKX) gene products are polytopic membrane proteins that utilize the existing cellular Na super(2+) and K super(+) gradients to extrude cytoplasmic Ca super(2+). NCKX proteins are made up of two clusters of hydrophobic segments, both thought to cons...

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Bibliographic Details
Published in:Biochemistry (Easton) 2005-05, Vol.44 (21), p.7787-7795
Main Authors: Kinjo, T G, Kang, K J, Szerencsei, R T, Winkfein, R J, Schnetkamp, PPM
Format: Article
Language:English
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Summary:The Na super(+)/Ca super(2+)-K super(+) exchanger (NCKX) gene products are polytopic membrane proteins that utilize the existing cellular Na super(2+) and K super(+) gradients to extrude cytoplasmic Ca super(2+). NCKX proteins are made up of two clusters of hydrophobic segments, both thought to consist of five putative membrane-spanning alpha -helices, and separated by a large cytoplasmic loop. The two most conserved regions within the NCKX sequence are known as the alpha 1 and alpha 2 repeats, and are found within the first and second set of transmembrane domains, respectively. The alpha repeats have previously been shown to contain residues critical for transport function. Here we used site-directed disulfide mapping to report that the alpha repeats are found in close proximity in three-dimensional space, bringing together key functional NCKX residues, e.g., the two critical acidic residues, Glu super(188) and Asp super(548). Glu188Cys in the alpha 1 repeat could form a disulfide cross-link with Asp548Cys in the alpha 2 repeat. Surprisingly, cysteine substitutions of Ser super(185) in the alpha 1 repeat could form disulfide cross-links with cysteine substitutions of three residues in the alpha 2 repeat (Ser super(545), Asp super(548), and Ser super(552)), thought to cover close to two full turns of an alpha helix, implying an area of increased flexibility. Using the same method, Asp super(575), a residue critical for the K super(+) dependence of NCKX, was shown to be in the proximity of Ser super(185) and Glu super(188), consistent with its role in enabling K super(+) to bind to a single Ca super(2+) and K super(+) binding pocket.
ISSN:0006-2960
DOI:10.1021/bi0502442