Study of vesicular ATP release is complicated by Golgi-sequestered VNUT-pHluorins and clondronate inhibiting ATP release mediated by cell lysis in Neuro2A cells

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.