Dimeric interaction between the cytoplasmic domains of the Na+/H+ exchanger NHE1 revealed by symmetrical intermolecular cross-linking and selective co-immunoprecipitation.
To investigate the oligomeric structure of Na(+)/H(+) exchanger 1 (NHE1), permeabilized cells and membranes from cells expressing NHE1 variants were treated with the oxidizing agent Cu(2+)/o-phenanthroline or the bifunctional sulfhydryl reagent methanethiosulfonate. These treatments resulted in symmetrical intermolecular cross-linking at intrinsic (Cys(794) and Cys(561)) or 15 exogenous cysteine residues introduced into the distal carboxyl- (C-) terminal cytoplasmic domain (after aa 600) but not at intrinsic Cys(538) because of masking by its tight association with calcineurin B-homologous protein. Cross-linking was abolished in membranes solubilized with sodium dodecyl sulfate, which dissociates oligomeric NHE1, while it was preserved in those treated with Triton X-100. In addition, treatment with cross-linkers did not produce the tetrameric forms of NHE1 mutants with two cysteine residues. Thus, cross-linking presumably occurs between adjacent C-termini of the NHE1 dimer but not by a stochastic process via random collision of NHE1 molecules. The observations suggest that at least the distal C-termini of the NHE1 dimer are flexible or mobile and are thereby capable of easily making contact with each other over the large cytoplasmic portion of the molecule. Furthermore, co-immunoprecipitation experiments showed that the proximal C-termini (aa 503-580) have a strong propensity to interact directly with each other in parallel. Deletion of aa 562-579 resulted in disruption of disulfide cross-linking between the C-termini and markedly reduced the intracellular pH sensitivity of Na(+)/H(+) exchange, suggesting that the dimeric interaction in this region may control the pH-dependent regulation of NHE1.
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
Takashi
T
Hisamitsu
Tianxiang
T
Pang
Munekazu
M
Shigekawa
Shigeo
S
Wakabayashi
D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010618:Phenanthrolines / Q000737:chemistry; D011233:Precipitin Tests / Q000379:methods; D017434:Protein Structure, Tertiary; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D003432:Cross-Linking Reagents; D004220:Disulfides; D010446:Peptide Fragments; D010618:Phenanthrolines; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D003545:Cysteine; C025205:1,10-phenanthroline