Study of vesicular ATP release is complicated by Golgi-sequestered VNUT-pHluorins and clondronate inhibiting ATP release mediated by cell lysis in Neuro2A cells
Notice bibliographique
Résumé
Introduction: Vesicular ATP release mediates neurotransmission and paracrine signalling in diverse tissues systems, and the Vesicular Nucleotide Transporter (VNUT / SLC17A9) was identified as the transporter that loads ATP into vesicles. In many tissues, like sympathetic nerves and adrenal glands, ATP acts as a co-transmitter with other neurotransmitters like norepinephrine. In this context, we assumed that the identification of VNUT would provide an essential marker protein to study the trafficking, release and recycling of purinergic (ATP-secreting) vesicles. We hypothesized that fusing a pH-sensitive GFP to a lumen facing domain of VNUT would provide a novel tool to image the release of purinergic vesicles, complementing analyses of ATP release directly imaged using GRAB-ATP sensors which are P2Y1 receptors that are uncoupled from their G-proteins and are fused with a cytosolic, circularly permuted GFP that fluoresces when the receptor is bound by ATP. Methods: We generated VNUT-pHluorin fusion constructs using CCTOP, later validated by AlphaFold, to predict lumen facing domains. Constructs were expressed in Neuro2A cells stimulated with a variety of stimuli (e.g. high KCl solutions, hypotonic stress). In parallel, GRAB-ATP sensors were expressed to directly detect and calibrate ATP release. Results: Unlike other SLC17A and SLC18A family proteins that have a long lumen facing loop between the first two transmembrane domains, VNUT has only short lumen facing loops. We inserted pHluorin in the S1-S2, S3-S4, S7-S8 and S11-12 linkers, with the VNUTs1-2-pHluorin giving the best expression. The expression construct localized largely to a slightly acidic Golgi, and did not localize to acidic organelles. 55-105 mM KCl solutions, hypertonic 450 mM sucrose, 10 µM ionomycin and 0.5x hypotonic solution all failed to evoke externalization of the VNUT-pHluorins. GRAB-ATP confirmed that 0.5x solution induces robust ATP release, that was significantly reduced by the putative VNUT inhibitor clodronate (1 µM). Notably, ATP was often released as a large plume from one or two cells, with numerous smaller flickers of ATP released shortly before. Quantitative modelling indicated that the large release events were likely due to hypotonic cell lysis with “flickers” being consistent with vesicle release. The presumed lysis events were not sensitive to extracellular Ca2+ or depletion of ER Ca2+ stores with cyclopiazonic acid. Notably, inhibition of the GRAB-ATP signal by clodronate was mirrored in luciferase assays. Conclusion: All assessed VNUT-pHluorin constructs appeared to be trapped in Golgi-like organelles and fail to externalize in cells that mediate vesicular ATP release. Inhibition of the hypotonically-stimulated ATP release by clondronate appeared to be due to suppression of a cell-lysis like event, rather than inhibition of vesicular ATP release. Collectively, these results provide a cautionary tail when using clodronate to VNUT physiology and highlight the possibility that VNUT may support the filling of vesicles at a point upstream of the formation of fusion-competent vesicles. This work was supported by grant RGPIN-2019-04925 from the Natural Sciences and Engineering Research Council (NSERC) of Canada. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
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Comment cette classification a été obtenuedéplier
Prédiction machine sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Le volet Gemma est une étiquette directe du modèle pour chaque travail de la base, lue sur la notice réduite au titre. Le volet Codex est un classifieur appris des 10 348 étiquettes directes de Codex et calibré sur les taux pondérés de l'échantillon; les champs sans appui suffisant ne portent aucun appel Codex. Le mode candidate est l'union des deux volets; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont pas des étiquettes humaines.
Scores du classifieur distillé par catégorie (deux têtes)
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,001 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,001 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule source (Gemma direct ou Codex distillé), pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».