title: Site-directed disulfide mapping of residues contributing to the Ca2+ and K+ binding pocket of the NCKX2 Na+/Ca2+-K+ exchanger.
abstract: The Na+/Ca2+-K+ exchanger (NCKX) gene products are polytopic membrane proteins that utilize the existing cellular Na+ and K+ gradients to extrude cytoplasmic Ca2+. 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 alpha1 and alpha2 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, Glu188 and Asp548. Glu188Cys in the alpha1 repeat could form a disulfide cross-link with Asp548Cys in the alpha2 repeat. Surprisingly, cysteine substitutions of Ser185 in the alpha1 repeat could form disulfide cross-links with cysteine substitutions of three residues in the alpha2 repeat (Ser545, Asp548, and Ser552), thought to cover close to two full turns of an alpha helix, implying an area of increased flexibility. Using the same method, Asp575, a residue critical for the K+ dependence of NCKX, was shown to be in the proximity of Ser185 and Glu188, consistent with its role in enabling K+ to bind to a single Ca2+ and K+ binding pocket.
authors.affiliation: Hotchkiss Brain Institute, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Drive, Calgary, Alberta, T2N 4N1 Canada.
authors.forename: Tashi G
authors.initials: TG
authors.lastname: Kinjo
authors.forename: KyeongJin
authors.initials: K
authors.lastname: Kang
authors.forename: Robert T
authors.initials: RT
authors.lastname: Szerencsei
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Winkfein
authors.forename: Paul P M
authors.initials: PP
authors.lastname: Schnetkamp
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010449:Peptide Mapping / Q000379:methods*; D010618:Phenanthrolines / Q000737:chemistry / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D017433:Protein Structure, Secondary; D020449:Repetitive Sequences, Amino Acid; D012694:Serine / Q000235:genetics; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D010618:Phenanthrolines; D021382:Protein Sorting Signals; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D003545:Cysteine; D011188:Potassium; D002118:Calcium; C025205:1,10-phenanthroline