Intermolecular cross-linking of Na+-Ca2+ exchanger proteins: evidence for dimer formation.
The cardiac Na (+)-Ca (2+) exchanger (NCX1) is modeled to contain nine transmembrane segments (TMS) with a pair of oppositely oriented, conserved sequences called the alpha-repeats that are important in ion transport. Residue 122 in the alpha-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 degrees 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 alpha-1 and alpha-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 (+)-Ca (2+) exchanger.
Ren
Xiaoyan
X
Departments of Physiology and Medicine and the Cardiovascular Research Laboratories, David Geffen School of Medicine at UCLA, Los Angeles, California 90095-1760, USA. KPhilipson@mednet.ucla.edu
Nicoll
Debora A
DA
Galang
Giselle
G
Philipson
Kenneth D
KD
D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D008954:Models, Biological; D010618:Phenanthrolines / Q000737:chemistry / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics
D003432:Cross-Linking Reagents; D010618:Phenanthrolines; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D003545:Cysteine; C025205:1,10-phenanthroline