Role of cystein residues in the normal operation and assembly of K+‐Cl− cotransporter isoform 2 (KCC2)
Bibliographic record
Abstract
K + ‐Cl − cotransporters (KCCs) regulate intracellular K + and Cl − concentration as well as cell volume. Their transport activity is sensitive to N ‐ethylmaleimide (NEM), a thiol reagent that alkylates available sulfhydryl groups, and their C ‐terminus can interact with other CCCs to form oligomers. These features suggest that a number of cystein residues within the KCCs play an important role in carrier activity and/or assembly. To determine whether this is the case, we have exploited a mutagenic approach using rat KCC2 as a model. Substitutions (C8G, C742G, C854G and C982G) were generated by site‐directed mutagenesis and expressed in X. laevis oocytes. Substitution C854G increased while C982G decreased Rb + transport without altering cell surface protein expression. They responded as did wild type (wt) carrier to the inhibitory effect of 250 μM furosemide but were more sensitive to the inhibitory effect of 1 mM NEM. Coimmunoprecipitation studies showed that the quantity of carriers precipitated with wt KCC2 was lower for C854G than for wt KCC2 or C982G. Lastly, FRAP studies in KCC2‐transfected HEK‐293 cells showed that NEM increased post‐bleach fluorescence recovery time in a concentration‐dependent manner. Collectively, the data suggest that C854 and C982 play an important role in KCC2 function, perhaps by supporting homo‐ and/or heterooligomeric structure formation. This work was funded by CIHR and CFI.
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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) | 0.001 | 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 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".