title: Calmodulin-binding autoinhibitory domain controls "pH-sensing" in the Na+/H+ exchanger NHE1 through sequence-specific interaction.
abstract: The calmodulin (CaM)-binding domain reduces the affinity of the Na+/H+ exchanger NHE1 for intracellular H+ by exerting an autoinhibitory function in quiescent cells. We replaced this domain (aa 637-656) with homologous segments from other NHE isoforms (NHE2 and 4) or functionally similar regions from other sources (Na+/Ca2+ exchanger, CaM-dependent protein kinase II, plasma membrane Ca2+-pump, or CaM-binding peptide Trp3). The NHE-1-, NHE2-, and NHE4-segments bound CaM with Kds of 16, 130, and 27 nM, respectively. These chimeric molecules were expressed in the exchanger-deficient cell PS120. NHE1 with incorporated NHE2-segment was activated in response to Ca2+-mobilizing agents ionomycin and thrombin resulting in an alkaline shift of the intracellular pH (pHi)-dependence of 22Na+ uptake, as was the case with the intact rat NHE2. In contrast, incorporation of the NHE4-segment or other CaM-binding segments induced a constitutive alkaline shift of pHi-dependence with concomitant abolishment of Ca2+-dependent activation, indicating that these segments could not function as an autoinhibitory domain in NHE1. Detailed analyses revealed that Leu639, Lys651 and Tyr652, conserved in the NHE1- and NHE2-segments, but not in the NHE4-segment, are important for the autoinhibition. Furthermore, 125I-labeled CaM-binding peptide from NHE1 was efficiently crosslinked to the NHE1 protein, suggesting that the inhibitory domain physically interacts with part(s) of the molecule. Together, these findings support the notion that the reduction of H+ affinity in Na+/H+ exchange occurs through a mechanism involving a highly sequence-specific interaction of the inhibitory domain with its putative acceptor in NHE1.
authors.affiliation: Department of Molecular Physiology, National Cardiovascular Center Research Institute, Suita, Osaka 565, Japan. wak@ri.ncvc.go.jp
authors.forename: S
authors.initials: S
authors.lastname: Wakabayashi
authors.forename: T
authors.initials: T
authors.lastname: Ikeda
authors.forename: T
authors.initials: T
authors.lastname: Iwamoto
authors.forename: J
authors.initials: J
authors.lastname: Pouysségur
authors.forename: M
authors.initials: M
authors.lastname: Shigekawa
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002460:Cell Line; D003432:Cross-Linking Reagents; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
chemical_list: D002147:Calmodulin; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011994:Recombinant Proteins; C513110:Slc9a2 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D015759:Ionomycin