Intermolecular Cross-Linking of Na+−Ca2+ Exchanger Proteins: Evidence for Dimer Formation

• Published in: Biochemistry (Easton) 2008-06, Vol.47 (22), p.6081-6087
• Main Authors: Ren, Xiaoyan, Nicoll, Debora A, Galang, Giselle, Philipson, Kenneth D
• Format: Article
• Language: English
• Subjects: Cross-Linking Reagents - chemistry; Cross-Linking Reagents - metabolism; Cysteine - genetics; Cysteine - metabolism; Dimerization; Models, Biological; Phenanthrolines - chemistry; Phenanthrolines - metabolism; Sodium-Calcium Exchanger - chemistry; Sodium-Calcium Exchanger - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi800177t

The cardiac Na+−Ca2+ exchanger (NCX1) is modeled to contain nine transmembrane segments (TMS) with a pair of oppositely oriented, conserved sequences called the α-repeats that are important in ion transport. Residue 122 in the α-1 repeat is in proximity to residue 768 in TMS 6, and the two residues can be cross-linked . During studies on the substrate specificity of this intramolecular cross-link, we found evidence that NCX1 can form dimers. At 37 °C in the absence of extracellular Na+, copper phenanthroline catalyzes disulfide bond formation between cysteines at position 122 in adjacent NCX1 proteins. Dimerization was confirmed by histidine tag pull-down experiments that demonstrate the association of untagged NCX1 with histidine-tagged NCX1. Dimerization occurs along a face of the protein that includes parts of the α-1 and α-2 repeats as well as parts of TMS 1 and TMS 2. We do not see cross-linking between residues in TMS 5, TMS 6, or TMS 7. These data provide the first evidence for dimer formation by the Na+−Ca2+ exchanger.