A Histidine Cluster in the Cytoplasmic Domain of the Na-H Exchanger NHE1 Confers pH-sensitive Phospholipid Binding and Regulates Transporter Activity
Bibliographic record
Abstract
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)P 2 ) of NHE1 confers pH-dependent PI(4,5)P 2 binding and regulates NHE1 activity. A GST fusion of the wild type C-terminal cytoplasmic domain of NHE1 showed increased maximum PI(4,5)P 2 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 (R XX R3) or alanine (A XX A3), mimicking protonated and neutral histidine residues, respectively, and the R XX R3 mutant had significantly greater PI(4,5)P 2 binding than A XX A3. When expressed in cells, NHE1 activity and pH i were significantly increased with NHE1-R XX R3 and decreased with NHE1-A XX A3 compared with wild type NHE1. Additionally, fibroblasts expressing NHE1-R XX R3 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)P 2 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-R XX R3 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".