Abstract
The Na-H exchanger NHE1 contributes to intracellular pH (pHi) homeostasis in normal cells and the constitutively increased pHi in cancer. NHE1 activity is allosterically regulated by intracellular protons, with greater activity at lower pHi 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 pHi 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 pHi 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 pHi contributes to cell behaviors, most notably the biology of cancer cells.
Highlights
The Na-H exchanger NHE1 contributes to intracellular pH homeostasis in normal cells and the constitutively increased pHi in cancer
NHE1 activity and pHi were significantly increased with NHE1-RXXR3 and decreased with NHE1AXXA3 compared with wild type NHE1
Histidines flanked by arginine/lysine-rich regions in the proximal C terminus are conserved in other plasma membrane NHE isoforms, the number of histidine residues varies with isoforms having between one and four (Fig. 1C)
Summary
Cluster of Histidine Residues in Proximal C-terminal Cytoplasmic Domain Confer pH-dependent PI[4,5]P2 Binding— Based on previous findings that the proximal C terminus of NHE1 has pHi-dependent PI[4,5]P2 binding [15], we reasoned that this may be mediated by histidine residues, which can titrate within the physiological pH range and function as a pH sensor site [16]. We asked whether the cluster of histidines between the arginine and lysine-rich regions (Fig. 1B) is important for pH-dependent PI[4,5]P2 binding. We generated mutant GST fusions of the C-terminal cytoplasmic domain by substituting the four histidines in the cluster with arginines for a protonated mimetic (NHE1-RXXR3) or with alanines for a neutral mimetic (NHE1AXXA3). Both mutants bound PI[4,5]P2, pH-dependent binding was abolished, with no significant differences in Bmax or kd for each mutant at pH 7.5 compared with pH 7.0 (Fig. 2, B and C; Table 1). 0.97 Ϯ 0.03 0.27 Ϯ 0.01d,e a p Ͻ 0.05 compared to pH 7.5. b p Ͻ 0.05 compared to WT and RXXR3 at pH 7.5. c p Ͻ 0.05 compared to WT and RXXR3 at pH 7.0. d p Ͻ 0.01 compared to WT. e p Ͻ 0.001 compared to pH 7.5
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