UCSF

The Na-H exchanger NHE1 contributes to intracellular pH (pH_i) homeostasis in normal cells and the constitutively increased pH_i in cancer. NHE1 activity is allosterically regulated by intracellular protons, with greater activity at lower pH_i However, 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)P₂ binding and regulates NHE1 activity. A GST fusion of the wild type C-terminal cytoplasmic domain of NHE1 showed increased maximum PI(4,5)P₂ binding 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)P₂ binding than AXXA3. When expressed in cells, NHE1 activity and pH_i were 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 pH_i enabling cytoskeletal remodeling. These data identify a molecular mechanism for pH-sensitive PI(4,5)P₂ binding 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 pH_i contributes to cell behaviors, most notably the biology of cancer cells.