Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na<sup>+</sup>-K<sup>+</sup>-2Cl<sup>−</sup> cotransporter?
Bibliographic record
Abstract
Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the Na+-K+-2Cl- cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLC-PK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by Cl- depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or Cl- depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.001 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
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 teacher head, 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".