Intravesicular pH Research Articles

H+ flux of the voltage-gated proton channel Hv1 in giant unilamellar vesicle (GUV).

The voltage-sensitive proton selective channel HV1 is ubiquitously present in mammalian tissues. An aqueous pore provides the proton pathway. Intervening amino acid side chains render the channel impermeable to water [1]. Based on the hydrogen bonds formed between the permeating water molecules and the channel wall, HV1 would otherwise exhibit a water conductance comparable to aquaporins [2,3]. The proton wire involves Grotthuss-like proton hopping on top of intraluminal water molecules and titratable amino acids. Conceivably, HV1 exploits one or more of the arginines from the voltage sensor as part of the proton wire. Here we measured the unitary H+ transport rate by reconstituting the purified proton channel into giant unilamellar vesicles (GUV). We used the pHluorin tag to measure intravesicular pH and oxanol to probe the membrane potential. Fluorescence correlation spectroscopy served to measure channel density. The simultaneous assessment of diffusion limitations of H+ transport towards and away from the channel allows for evaluating the role of HV1's putative proton antenna. This project was supported by the Marie Skłodowska-Curie grant 860592 (Horizon 2020).

Measuring the pH of Acidic Vesicles in Live Cells with an Optimized Fluorescence Lifetime Imaging Probe.

Acidification of intracellular vesicles, such as endosomes and lysosomes, is a key pathway for regulating the function of internal proteins. Most conventional methods of measuring pH are not satisfactory for quantifying the pH inside these vesicles. Here, we investigated the molecular requirements for a fluorescence probe to measure the intravesicular acidic pH in living cells by means of fluorescence lifetime imaging microscopy (FLIM). The developed probe, m-DiMeNAF488, exhibits a pH-dependent equilibrium between highly fluorescent and moderately fluorescent forms, which has distinct and detectable fluorescence lifetimes of 4.36 and 0.58 ns, respectively. The pKa(τ) value of m-DiMeNAF488 was determined to be 4.58, which would be favorable for evaluating the pH in the acidic vesicles. We were able to monitor the pH changes in phagosomes during phagocytosis by means of FLIM using m-DiMeNAF488. This probe is expected to be a useful tool for investigating acidic pH-regulated biological phenomena.

Oscillating the local milieu of polymersome interiors via single input-regulated bilayer crosslinking and permeability tuning

The unique permselectivity of cellular membranes is of crucial importance to maintain intracellular homeostasis while adapting to microenvironmental changes. Although liposomes and polymersomes have been widely engineered to mimic microstructures and functions of cells, it still remains a considerable challenge to synergize the stability and permeability of artificial cells and to imitate local milieu fluctuations. Herein, we report concurrent crosslinking and permeabilizing of pH-responsive polymersomes containing Schiff base moieties within bilayer membranes via enzyme-catalyzed acid production. Notably, this synergistic crosslinking and permeabilizing strategy allows tuning of the mesh sizes of the crosslinked bilayers with subnanometer precision, showing discriminative permeability toward maltooligosaccharides with molecular sizes of ~1.4-2.6 nm. The permselectivity of bilayer membranes enables intravesicular pH oscillation, fueled by a single input of glucose. This intravesicular pH oscillation can further drive the dissipative self-assembly of pH-sensitive dipeptides. Moreover, the permeabilization of polymersomes can be regulated by intracellular pH gradient as well, enabling the controlled release of encapsulated payloads.

Computational Investigation of the pH Dependence of Stability of Melanosome Proteins: Implication for Melanosome formation and Disease.

Intravesicular pH plays a crucial role in melanosome maturation and function. Melanosomal pH changes during maturation from very acidic in the early stages to neutral in late stages. Neutral pH is critical for providing optimal conditions for the rate-limiting, pH-sensitive melanin-synthesizing enzyme tyrosinase (TYR). This dramatic change in pH is thought to result from the activity of several proteins that control melanosomal pH. Here, we computationally investigated the pH-dependent stability of several melanosomal membrane proteins and compared them to the pH dependence of the stability of TYR. We confirmed that the pH optimum of TYR is neutral, and we also found that proteins that are negative regulators of melanosomal pH are predicted to function optimally at neutral pH. In contrast, positive pH regulators were predicted to have an acidic pH optimum. We propose a competitive mechanism among positive and negative regulators that results in pH equilibrium. Our findings are consistent with previous work that demonstrated a correlation between the pH optima of stability and activity, and they are consistent with the expected activity of positive and negative regulators of melanosomal pH. Furthermore, our data suggest that disease-causing variants impact the pH dependence of melanosomal proteins; this is particularly prominent for the OCA2 protein. In conclusion, melanosomal pH appears to affect the activity of multiple melanosomal proteins.

Differential Neuropeptidomes of Dense Core Secretory Vesicles (DCSV) Produced at Intravesicular and Extracellular pH Conditions by Proteolytic Processing.

Neuropeptides mediate cell–cell signaling in the nervous and endocrine systems. The neuropeptidome is the spectrum of peptides generated from precursors by proteolysis within dense core secretory vesicles (DCSV). DCSV neuropeptides and contents are released to the extracellular environment where further processing for neuropeptide formation may occur. To assess the DCSV proteolytic capacity for production of neuropeptidomes at intravesicular pH 5.5 and extracellular pH 7.2, neuropeptidomics, proteomics, and protease assays were conducted using chromaffin granules (CG) purified from adrenal medulla. CG are an established model of DCSV. The CG neuropeptidome consisted of 1239 unique peptides derived from 15 proneuropeptides that were colocalized with 64 proteases. Distinct CG neuropeptidomes were generated at the internal DCSV pH of 5.5 compared to the extracellular pH of 7.2. Class-specific protease inhibitors differentially regulated neuropeptidome production involving aspartic, cysteine, serine, and metallo proteases. The substrate cleavage properties of CG proteases were assessed by multiplex substrate profiling by mass spectrometry (MSP-MS) that uses a synthetic peptide library containing diverse cleavage sites for endopeptidases and exopeptidases. Parallel inhibitor-sensitive cleavages for neuropeptidome production and peptide library proteolysis led to elucidation of six CG proteases involved in neuropeptidome production, represented by cathepsins A, B, C, D, and L and carboxypeptidase E (CPE). The MSP-MS profiles of these six enzymes represented the majority of CG proteolytic cleavages utilized for neuropeptidome production. These findings provide new insight into the DCSV proteolytic system for production of distinct neuropeptidomes at the internal CG pH of 5.5 and at the extracellular pH of 7.2.

The intracellular Ca2+ release channel TRPML1 regulates lower urinary tract smooth muscle contractility

TRPML1 (transient receptor potential mucolipin 1) is a Ca2+-permeable, nonselective cation channel that is predominantly localized to the membranes of late endosomes and lysosomes (LELs). Intracellular release of Ca2+ through TRPML1 is thought to be pivotal for maintenance of intravesicular acidic pH as well as the maturation, fusion, and trafficking of LELs. Interestingly, genetic ablation of TRPML1 in mice (Mcoln1-/- ) induces a hyperdistended/hypertrophic bladder phenotype. Here, we investigated this phenomenon further by exploring an unconventional role for TRPML1 channels in the regulation of Ca2+-signaling activity and contractility in bladder and urethral smooth muscle cells (SMCs). Four-dimensional (4D) lattice light-sheet live-cell imaging showed that the majority of LELs in freshly isolated bladder SMCs were essentially immobile. Superresolution microscopy revealed distinct nanoscale colocalization of LEL-expressing TRPML1 channels with ryanodine type 2 receptors (RyR2) in bladder SMCs. Spontaneous intracellular release of Ca2+ from the sarcoplasmic reticulum (SR) through RyR2 generates localized elevations of Ca2+ ("Ca2+ sparks") that activate plasmalemmal large-conductance Ca2+-activated K+ (BK) channels, a critical negative feedback mechanism that regulates smooth muscle contractility. This mechanism was impaired in Mcoln1-/- mice, which showed diminished spontaneous Ca2+ sparks and BK channel activity in bladder and urethra SMCs. Additionally, ex vivo contractility experiments showed that loss of Ca2+ spark-BK channel signaling in Mcoln1-/- mice rendered both bladder and urethra smooth muscle hypercontractile. Voiding activity analyses revealed bladder overactivity in Mcoln1-/- mice. We conclude that TRPML1 is critically important for Ca2+ spark signaling, and thus regulation of contractility and function, in lower urinary tract SMCs.

Molecular Tetrahedrons as Selective and Efficient Ion Transporters via a Two-Station Swing-Relay Mechanism

The traditional approach to utilizing an ion-relay mechanism for ion transport requires three or more ion-relay stations. Herein, we describe a novel strategy, incorporating a swing action to reali...

Cellular bicarbonate accumulation and vesicular proton transport promote calcification in the sea urchin larva.

The sea urchin embryo develops a calcitic endoskeleton through intracellular formation of amorphous calcium carbonate (ACC). Intracellular precipitation of ACC, requires [Formula: see text] concentrating as well as proton export mechanisms to promote calcification. These processes are of fundamental importance in biological mineralization, but remain largely unexplored. Here, we demonstrate that the calcifying primary mesenchyme cells (PMCs) use Na+/H+-exchange (NHE) mechanisms to control cellular pH homeostasis during maintenance of the skeleton. During skeleton re-calcification, pHi of PMCs is increased accompanied by substantial elevation in intracellular [Formula: see text] mediated by the [Formula: see text] cotransporter Sp_Slc4a10. However, PMCs lower their pHi regulatory capacities associated with a reduction in NHE activity. Live-cell imaging using green fluorescent protein reporter constructs in combination with intravesicular pH measurements demonstrated alkaline and acidic populations of vesicles in PMCs and extensive trafficking of large V-type H+-ATPase (VHA)-rich acidic vesicles in blastocoelar filopodial cells. Pharmacological and gene expression analyses underline a central role of the VHA isoforms Sp_ATP6V0a1, Sp_ATP6V01_1 and Sp_ATPa1-4 for the process of skeleton re-calcification. These results highlight novel pH regulatory strategies in calcifying cells of a marine species with important implications for our understanding of the mineralization process in times of rapid changes in oceanic pH.

Intracellular ABCB1 as a Possible Mechanism to Explain the Synergistic Effect of Hydroxychloroquine-Azithromycin Combination in COVID-19 Therapy.

The co-administration of hydroxychloroquine with azithromycin is proposed in COVID-19 therapy. We hypothesize a new mechanism supporting the synergistic interaction between these drugs. Azithromycin is a substrate of ABCB1 (P-glycoprotein) which is localized in endosomes and lysosomes with a polarized substrate transport from the cell cytosol into the vesicle interior. SARS-CoV-2 and drugs meet in these acidic organelles and both basic drugs, which are potent lysosomotropic compounds, will become protonated and trapped within these vesicles. Consequently, their intra-vesicular concentrations can attain low micromolar effective cytotoxic concentrations on SARS-CoV-2 while concomitantly increase the intra-vesicular pH up to around neutrality. This last effect inhibits lysosomal enzyme activities responsible in virus entry and replication cycle. Based on these considerations, we hypothesize that ABCB1 could be a possible enhancer by confining azithromycin more extensively than expected when the trapping is solely dependent on the passive diffusion. This additional mechanism may therefore explain the synergistic effect when azithromycin is added to hydroxychloroquine, leading to apparently more rapid virus clearance and better clinical benefit, when compared to monotherapy with hydroxychloroquine alone.

P579 Randomised open-label controlled trial of ciprofloxacin/doxycycline/hydroxychloroquine combination compared with standard budesonide in active Crohn’s disease (APRICOT)

Abstract Background Mucosal E. coli are increased in Crohn’s disease (CD). They replicate within macrophages where they are inaccessible to penicillins and gentamicin. Hydroxychloroquine is successfully used together with doxycycline, typically for 12 months, to treat Whipple’s disease. It raises macrophage intra-vesicular pH and inhibits replication of bacteria , including E. coli that require an acid pH. Ciprofloxacin and doxycycline are also effective against E. coli replicating inside macrophages. Methods Adult patients with active CD (CDAI>220 plus CRP≥5mg/l and/or faecal calprotectin >250 μg/g) were randomised to receive either standard oral budesonide (Entocort CR 9mg/day for 8 weeks, 6mg/day for 2 weeks, 3mg/day for 2 weeks) or antibiotics/hydroxychloroquine (AB/HCQ) - oral ciprofloxacin 500mg bd, doxycycline 100mg bd, hydroxychloroquine 200 mg tds for 4 weeks, followed by continuation of doxycycline 100 mg bd and hydroxychloroquine 200 mg tds for a further 20 weeks. The use of anti-TNF in the previous 3 months was an exclusion. Primary endpoint parameters were remission at 10 weeks, remission maintained through to 24 weeks, and remission maintained through to 52 weeks. Secondary endpoints included remission and/or response (fall in CDAI of >70) at 10 weeks and remission at 4 weeks. Patients who failed to respond by 10 weeks were invited to cross-over onto the alternative therapy. Results 59 patients were recruited across 8 sites, lower than target (100) because recruitment slowed due to widening access to biologics. Including cross-over, 39 patients received AB/HCQ and 39 received budesonide. No significant differences were seen comparing AB/HCQ with budesonide at 10, 24 or 52 weeks on either intention-to-treat or per-protocol analysis (see table). Withdrawals by 10 weeks due to adverse events were seen in 16 AB/HCQ, - including nausea (3), photosensitivity (2), Achilles pain (2) and activity of CD (4); and 7 budesonide. Although not significant when analysed per protocol, when patients on AB/HCQ who responded at 10 weeks and later remitted were included, 9/24 patients were in remission at 24 weeks and 4/23 at 52 weeks. No correlation was seen between response to AB/HCQ and ASCA/OmpC status. Conclusion The longer-term remissions seen in some patients receiving AB/HCQ are encouraging and a larger phase 3 study is justified.

Differential Release of Exocytosis Marker Dyes Indicates Stimulation-Dependent Regulation of Synaptic Activity.

There is a general consensus that synaptic vesicular release by a full collapse process is the primary machinery of synaptic transmission. However, competing view suggests that synaptic vesicular release operates via a kiss-and-run mechanism. By monitoring the release dynamics of a synaptic vesicular marker, FM1-43 from individual synapses in hippocampal neurons, we found evidence that the release of synaptic vesicle was delayed by several seconds after the start of field stimulation. This phenomenon was associated with modified opening kinetics of fusion pores. Detailed analysis revealed that some synapses were completely inactive for a few seconds after stimulation, despite immediate calcium influx. This delay in vesicular release was modulated by various stimulation protocols and different frequencies, indicating an activity-dependent regulation mechanism for neurotransmitter exocytosis. Staurosporine, a drug known to induce “kiss-and-run” exocytosis, increased the proportion of delayed synapses as well as the delay duration, while fluoxetine acted contrarily. Besides being a serotonin reuptake inhibitor, it directly enhanced vesicle mobilization and reduced synaptic fatigue. Exocytosis was never delayed, when it was monitored with pH-sensitive probes, synaptopHlourin and αSyt-CypHerE5 antibody, indicating an instantaneous formation of a fusion pore that allowed rapid equilibration of vesicular lumenal pH but prevented FM1-43 release because of its slow dissociation from the inner vesicular membrane. Our observations suggest that synapses operate via a sequential “kiss-and-run” and “full-collapse” exocytosis mechanism. The initially narrow vesicular pore allows the equilibration of intravesicular pH which then progresses toward full fusion, causing FM1-43 release.

The Effect of Buffers on Weak Acid Uptake by Vesicles.

The assessment of weak acid membrane permeability (Pm) frequently involves large unilamellar vesicles. It relies on measurements of the intravesicular pH drop, ΔpHin, in response to a sudden augmentation of external acid concentration. However, ΔpHin may be primarily governed by non-instantaneous protonation and deprotonation reactions of (i) the acid itself, (ii) the buffer molecules, and (iii) the fluorescent pH reporter dye. Moreover, buffer concentration and acid gradient also serve as determinants of ΔpHin, as we show here. The uniexponential time constant (τ) of ΔpHin(t) is an invalid measure of Pm as Arrhenius plots of Pm and reveal different activation energies for acid influx. We calculate Pm by fitting a mathematical model to experimental stopped-flow traces. The model takes into account not only the time course of total internal buffer capacity but also (i) water self-dissociation, (ii) volume changes due to acid induced osmotic water flow, and (iii) the spontaneous membrane proton leak. It allows extracting a Pm of 30.8 ± 3.5 μm/s for formic acid for 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles.

Real-Time Recording of the Cellular Effects of the Anion Transporter Prodigiosin

Summary The unraveling of the cellular effects of anion transporters is key to their potential development as apoptosis-inducing or autophagy-disrupting therapeutics. Here, we conducted a systematic study of the cellular responses to the anion transporter prodigiosin by using a pH on/off responsive near-infrared (NIR)-fluorescent probe in HeLa and LAMP1-GFP-transfected HeLa cell lines. The sequence of localized and global cellular acidity changes and the resulting outcomes induced by the anion transporter were visualized with high temporal and spatial resolution. The results show that prodigiosin causes the pH within the lysosomal lumen to rise, after which a non-organelle-specific increase in acidity of the cytosol takes place, which prompts cells to undergo apoptosis. This was confirmed by the quantification of NIR-emissive lysosomes, the intra-cellular fluorescence intensity, and fluorescence volume over time. This NIR probe overcame the limitations of acridine orange, which to date have severely restricted researchers in this field.

Polarized light microscopy reveals physiological and drug-induced changes in surfactant membrane assembly in alveolar type II pneumocytes

In alveolar type II (AT II) cells, pulmonary surfactant (PS) is synthetized, stored and exocytosed from lamellar bodies (LBs), specialized large secretory organelles. By applying polarization microscopy (PM), we confirm a specific optical anisotropy of LBs, which indicates a liquid-crystalline mesophase of the stored surfactant phospholipids (PL) and an unusual case of a radiation-symmetric, spherocrystalline organelle. Evidence is shown that the degree of anisotropy is dependent on the amount of lipid layers and their degree of hydration, but unaffected by acutely modulating vital cell parameters like intravesicular pH or cellular energy supply. In contrast, physiological factors that perturb this structure include osmotic cell volume changes and LB exocytosis. In addition, we found two pharmaceuticals, Amiodarone and Ambroxol, both of which severely affect the liquid-crystalline order. Our study shows that PM is an easy, very sensitive, but foremost non-invasive and label-free method able to collect important structural information of PS assembly in live AT II cells which otherwise would be accessible by destructive or labor intense techniques only. This may open new approaches to dynamically investigate LB biosynthesis - the incorporation, folding and packing of lipid membranes - or the initiation of pathological states that manifest in altered LB structures. Due to the observed drug effects, we further suggest that PM provides an appropriate way to study unspecific drug interactions with alveolar cells and even drug-membrane interactions in general.

ATP is stored in lamellar bodies to activate vesicular P2X4 in an autocrine fashion upon exocytosis.

Vesicular P2X4 receptors are known to facilitate secretion and activation of pulmonary surfactant in the alveoli of the lungs. P2X4 receptors are expressed in the membrane of lamellar bodies (LBs), large secretory lysosomes that store lung surfactant in alveolar type II epithelial cells, and become inserted into the plasma membrane after exocytosis. Subsequent activation of P2X4 receptors by adenosine triphosphate (ATP) results in local fusion-activated cation entry (FACE), facilitating fusion pore dilation, surfactant secretion, and surfactant activation. Despite the importance of ATP in the alveoli, and hence lung function, the origin of ATP in the alveoli is still elusive. In this study, we demonstrate that ATP is stored within LBs themselves at a concentration of ∼1.9 mM. ATP is loaded into LBs by the vesicular nucleotide transporter but does not activate P2X4 receptors because of the low intraluminal pH (5.5). However, the rise in intravesicular pH after opening of the exocytic fusion pore results in immediate activation of vesicular P2X4 by vesicular ATP. Our data suggest a new model in which agonist (ATP) and receptor (P2X4) are located in the same intracellular compartment (LB), protected from premature degradation (ATP) and activation (P2X4), and ideally placed to ensure coordinated and timely receptor activation as soon as fusion occurs to facilitate surfactant secretion.

Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana.

Two-pore channels (TPCs) comprise a subfamily (TPC1-3) of eukaryotic voltage- and ligand-gated cation channels with two non-equivalent tandem pore-forming subunits that dimerize to form quasi-tetramers. Found in vacuolar or endolysosomal membranes, they regulate the conductance of sodium and calcium ions, intravesicular pH, trafficking and excitability. TPCs are activated by a decrease in transmembrane potential and an increase in cytosolic calcium concentrations, are inhibited by low luminal pH and calcium, and are regulated by phosphorylation. Here we report the crystal structure of TPC1 from Arabidopsis thaliana at 2.87 Å resolution as a basis for understanding ion permeation, channel activation, the location of voltage-sensing domains and regulatory ion-binding sites. We determined sites of phosphorylation in the amino-terminal and carboxy-terminal domains that are positioned to allosterically modulate cytoplasmic Ca(2+) activation. One of the two voltage-sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal Ca(2+) and adopts a conformation distinct from the activated state observed in structures of other voltage-gated ion channels. The structure shows that potent pharmacophore trans-Ned-19 (ref. 17) acts allosterically by clamping the pore domains to VSD2. In animals, Ned-19 prevents infection by Ebola virus and other filoviruses, presumably by altering their fusion with the endolysosome and delivery of their contents into the cytoplasm.

Mechanistic model and analysis of doxorubicin release from liposomal formulations

Reliable and predictive models of drug release kinetics in vitro and in vivo are still lacking for liposomal formulations. Developing robust, predictive release models requires systematic, quantitative characterization of these complex drug delivery systems with respect to the physicochemical properties governing the driving force for release. These models must also incorporate changes in release due to the dissolution media and methods employed to monitor release. This paper demonstrates the successful development and application of a mathematical mechanistic model capable of predicting doxorubicin (DXR) release kinetics from liposomal formulations resembling the FDA-approved nanoformulation DOXIL® using dynamic dialysis. The model accounts for DXR equilibria (e.g. self-association, precipitation, ionization), the change in intravesicular pH due to ammonia release, and dialysis membrane transport of DXR. The model was tested using a Box–Behnken experimental design in which release conditions including extravesicular pH, ammonia concentration in the release medium, and the dilution of the formulation (i.e. suspension concentration) were varied. Mechanistic model predictions agreed with observed DXR release up to 19h. The predictions were similar to a computer fit of the release data using an empirical model often employed for analyzing data generated from this type of experimental design. Unlike the empirical model, the mechanistic model was also able to provide reasonable predictions of release outside the tested design space. These results illustrate the usefulness of mechanistic modeling to predict drug release from liposomal formulations in vitro and its potential for future development of in vitro – in vivo correlations for complex nanoformulations.

Bio-Enabled Gold Superstructures with Built-In and Accessible Electromagnetic Hotspots.

The bio-enabled synthesis of a novel class of surface enhanced Raman scattering probes is presented for functional imaging with built-in and accessible electromagnetic hotspots formed between densely packed satellites grown on a plasmonic core. The superstructures serve as nanoscale sensors to spatiotemporally map intravesicular pH changes along endocytic pathways inside live cells.

Nanoprobing the acidification process during intracellular uptake and trafficking

Many nanoparticular drug delivery approaches rely on a detailed knowledge of the acidification process during intracellular trafficking of endocytosed nanoparticles (NPs). Therefore we produced a nanoparticular pH sensor composed of the fluorescent pH-sensitive dual wavelength dye carboxy seminaphthorhodafluor-1 (carboxy SNARF-1) coupled to the surface of amino-functionalized polystyrene NPs (SNARF-1-NP). By applying a calibration fit function to confocal laser scanning microscopy (CLSM) images, local pH values were determined. The acidification and ripening process of endo/lysosomal compartments containing nanoparticles was followed over time and was found to progress up to 6h to reach an equilibrium pH distribution (maximum pH5.2 [±0.2]). The SNARF-1-NP localization in endo/lysosomal compartments was confirmed by transmission electron microscopy (TEM) and quantitative co-localization analysis with fluorescent endolysosomal marker Rab-proteins by confocal laser scanning microscopy (CLSM). The herein described nanoparticular pH-sensor is a versatile tool to monitor dynamic pH processes inside the endolysosomal compartments. From the Clinical EditorIn this interesting article, the authors elegantly designed a nanoparticular pH sensor with fluorescence probe with the capability to measure intracellular and intravesicular pH changes. The application of this method would enable the further understanding of nanoparticle uptake and intracellular physiology.

The membrane domain of vacuolar H(+)ATPase: a crucial player in neurotransmitter exocytotic release.

V-ATPases are multimeric enzymes made of two sectors, a V1 catalytic domain and a V0 membrane domain. They accumulate protons in various intracellular organelles. Acidification of synaptic vesicles by V-ATPase energizes the accumulation of neurotransmitters in these storage organelles and is therefore required for efficient synaptic transmission. In addition to this well-accepted role, functional studies have unraveled additional hidden roles of V0 in neurotransmitter exocytosis that are independent of the transport of protons. V0 interacts with SNAREs and calmodulin, and perturbing these interactions affects neurotransmitter release. Here, we discuss these data in relation with previous results obtained in reconstituted membranes and on yeast vacuole fusion. We propose that V0 could be a sensor of intra-vesicular pH that controls the exocytotic machinery, probably regulating SNARE complex assembly during the synaptic vesicle priming step, and that, during the membrane fusion step, V0 might favor lipid mixing and fusion pore stability.