title: A Histidine Cluster in the Cytoplasmic Domain of the Na-H Exchanger NHE1 Confers pH-sensitive Phospholipid Binding and Regulates Transporter Activity.
abstract: The Na-H exchanger NHE1 contributes to intracellular pH (pHi) homeostasis in normal cells and the constitutively increased pHiin cancer. NHE1 activity is allosterically regulated by intracellular protons, with greater activity at lower pHiHowever, the molecular mechanism for pH-dependent NHE1 activity remains incompletely resolved. We report that an evolutionarily conserved cluster of histidine residues located in the C-terminal cytoplasmic domain between two phosphatidylinositol 4,5-bisphosphate binding sites (PI(4,5)P2) of NHE1 confers pH-dependent PI(4,5)P2binding and regulates NHE1 activity. A GST fusion of the wild type C-terminal cytoplasmic domain of NHE1 showed increased maximum PI(4,5)P2binding at pH 7.0 compared with pH 7.5. However, pH-sensitive binding is abolished by substitutions of the His-rich cluster to arginine (RXXR3) or alanine (AXXA3), mimicking protonated and neutral histidine residues, respectively, and the RXXR3 mutant had significantly greater PI(4,5)P2binding than AXXA3. When expressed in cells, NHE1 activity and pHiwere significantly increased with NHE1-RXXR3 and decreased with NHE1-AXXA3 compared with wild type NHE1. Additionally, fibroblasts expressing NHE1-RXXR3 had significantly more contractile actin filaments and focal adhesions compared with fibroblasts expressing wild type NHE1, consistent with increased pHienabling cytoskeletal remodeling. These data identify a molecular mechanism for pH-sensitive PI(4,5)P2binding regulating NHE1 activity and suggest that the evolutionarily conserved cluster of four histidines in the proximal cytoplasmic domain of NHE1 may constitute a proton modifier site. Moreover, a constitutively activated NHE1-RXXR3 mutant is a new tool that will be useful for studying how increased pHicontributes to cell behaviors, most notably the biology of cancer cells.
authors.affiliation: From the Department of Cell and Tissue Biology, University of California, San Francisco, California 94143 and.
authors.forename: Bradley A
authors.initials: BA
authors.lastname: Webb
authors.affiliation: From the Department of Cell and Tissue Biology, University of California, San Francisco, California 94143 and.
authors.forename: Katharine A
authors.initials: KA
authors.lastname: White
authors.affiliation: From the Department of Cell and Tissue Biology, University of California, San Francisco, California 94143 and.
authors.forename: Bree K
authors.initials: BK
authors.lastname: Grillo-Hill
authors.affiliation: From the Department of Cell and Tissue Biology, University of California, San Francisco, California 94143 and.
authors.forename: André
authors.initials: A
authors.lastname: Schönichen
authors.affiliation: the Department of Radiation Oncology, Samsung Medical Center, Seoul, South Korea 06351.
authors.forename: Changhoon
authors.initials: C
authors.lastname: Choi
authors.affiliation: From the Department of Cell and Tissue Biology, University of California, San Francisco, California 94143 and diane.barber@ucsf.edu.
authors.forename: Diane L
authors.initials: DL
authors.lastname: Barber
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism*; D022001:Focal Adhesions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010743:Phospholipids; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers
keywords: actin; focal adhesions; pH regulation; phospholipid; sodium-proton exchange