Stable dimeric assembly of the second membrane-spanning domain of CFTR (cystic fibrosis transmembrane conductance regulator) reconstitutes a chloride-selective pore
Bibliographic record
Abstract
Structural information is required to define the molecular basis for chloride conduction through CFTR (cystic fibrosis transmembrane conductance regulator). Towards this goal, we expressed MSD2, the second of the two MSDs (membrane-spanning domains) of CFTR, encompassing residues 857-1158 in Sf9 cells using the baculovirus system. In Sf9 plasma membranes, MSD2 migrates as expected for a dimer in non-dissociative PAGE, and confers the appearance of an anion permeation pathway suggesting that dimeric MSD2 mediates anion flux. To assess directly the function and quaternary structure of MSD2, we purified it from Sf9 cells by virtue of its polyhistidine tag and nickel affinity. Reconstitution of MSD2 into liposomes conferred a 4,4'-di-isothiocyanostilbene-2,2'-disulphonate-inhibitable, chloride-selective electrodiffusion pathway. Further, this activity is probably mediated directly by MSD2 as reaction of its single cysteine residue (Cys866) with the thiol modifying reagent, N(alpha)(3-maleimidylpropionyl)biocytin, inhibited chloride flux. Only MSD2 dimers were labelled by N(alpha)(3-maleimidylpropionyl)biocytin, supporting the idea that only dimeric MSD2 can mediate anion flux. As a further test of this hypothesis, we conducted a second purification procedure, wherein purified dimeric and monomeric MSD2 proteins were reconstituted separately. Only proteoliposomes containing stable MSD2 dimers mediated chloride electrodiffusion, providing direct evidence that dimeric MSD2 mediates chloride channel function. In summary, we have shown that the second membrane domain of CFTR can be purified and functionally reconstituted as a chloride channel, providing a tool for probing the structural basis of chloride conduction through CFTR.
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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 | 0.002 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.001 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 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".