Identification of Cys140 in helix 4 as an exofacial cysteine residue within the substrate-translocation channel of rat equilibrative nitrobenzylthioinosine (NBMPR)-insensitive nucleoside transporter rENT2
Bibliographic record
Abstract
The human and rat equilibrative nucleoside transporter proteins hENT1, rENT1, hENT2 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs), and are distinguished functionally by differences in transport of nucleobases and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs. In the present study, we have produced recombinant hENT1, rENT1, hENT2 and rENT2 in Xenopus oocytes and investigated uridine transport following exposure to the impermeant thiol-reactive reagent p-chloromercuriphenyl sulphonate (PCMBS). PCMBS caused reversible inhibition of uridine influx by rENT2, but had no effect on hENT1, hENT2 or rENT1. This difference correlated with the presence in rENT2 of a unique Cys residue (Cys140) in the outer half of TM4 that was absent from the other ENTs. Mutation of Cys140 to Ser produced a functional protein (rENT2/C140S) that was insensitive to inhibition by PCMBS, identifying Cys140 as the exofacial Cys residue in rENT2 responsible for PCMBS inhibition. Uridine protected wild-type rENT2 against PCMBS inhibition, suggesting that Cys140 in TM4 lies within or is closely adjacent to the substrate-translocation channel of the transporter. TM4has been shown previously to be within a structural domain (TMs 3Ő6) responsible for interactions with NBMPR, vasoactive drugs and nucleobases.
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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".