Effects of N-Ethylmaleimide on Conformational Equilibria in Purified Cardiac Muscarinic Receptors
Bibliographic record
Abstract
Muscarinic receptors purified from porcine atria and devoid of G protein underwent a 9-27-fold decrease in their apparent affinity for the antagonists quinuclidinyl benzilate, N-methylscopolamine, and scopolamine when treated with the thiol-selective reagent N-ethylmaleimide. Their apparent affinity for the agonists carbachol and oxotremorine-M was unchanged. Conversely, the rate of alkylation by N-ethylmaleimide, as monitored by the binding of [(3)H]quinuclidinyl benzilate, was decreased by antagonists while agonists were without effect. The receptor also underwent a time-dependent inactivation that was hastened by N-ethylmaleimide but slowed by quinuclidinyl benzilate and N-methylscopolamine. The destabilizing effect of N-ethylmaleimide was counteracted fully or nearly so at saturating concentrations of each antagonist and the agonist carbachol. Similar effects occurred with human M(2) receptors differentially tagged with the c-Myc and FLAG epitopes, coexpressed in Sf9 cells, and extracted in digitonin/cholate. The degree of coimmunoprecipitation was unchanged by N-ethylmaleimide, which therefore was without discernible effect on oligomeric size. The data are quantitatively consistent with a model in which the purified receptor from porcine atria interconverts spontaneously between two states (i.e. R R*). Antagonists favor the R state; agonists and N-ethylmaleimide favor the comparatively unstable R* state, which predominates after purification. Occupancy by a ligand stabilizes both states, and antagonists impede alkylation by favoring R over R*. Similarities with constitutively active receptors suggest that R and R* are akin to the inactive and active states, respectively. Purified M(2) receptors therefore appear to exist predominantly in their active state.
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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) | 0.001 | 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 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 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".