Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate (5-IP7) are highly energetic inositol metabolites containing phosphoanhydride bonds. Although inositol pyrophosphates are known to regulate various biological events, including growth, survival, and metabolism, the molecular sites of 5-IP7 action in vesicle trafficking have remained largely elusive. We report here that elevated 5-IP7 levels, caused by overexpression of inositol hexakisphosphate (IP6) kinase 1 (IP6K1), suppressed depolarization-induced neurotransmitter release from PC12 cells. Conversely, IP6K1 depletion decreased intracellular 5-IP7 concentrations, leading to increased neurotransmitter release. Consistently, knockdown of IP6K1 in cultured hippocampal neurons augmented action potential-driven synaptic vesicle exocytosis at synapses. Using a FRET-based in vitro vesicle fusion assay, we found that 5-IP7, but not 1-IP7, exhibited significantly higher inhibitory activity toward synaptic vesicle exocytosis than IP6. Synaptotagmin 1 (Syt1), a Ca2+ sensor essential for synaptic membrane fusion, was identified as a molecular target of 5-IP7. Notably, 5-IP7 showed a 45-fold higher binding affinity for Syt1 compared with IP6. In addition, 5-IP7–dependent inhibition of synaptic vesicle fusion was abolished by increasing Ca2+ levels. Thus, 5-IP7 appears to act through Syt1 binding to interfere with the fusogenic activity of Ca2+. These findings reveal a role of 5-IP7 as a potent inhibitor of Syt1 in controlling the synaptic exocytotic pathway and expand our understanding of the signaling mechanisms of inositol pyrophosphates.

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca2+ stimulates fusion-pore formation in a millisecond timescale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca2+. When 10 µM Ca2+ was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.

Intense electric pulses are known to induce several effects on biological cells and membranes. Among the different effects, membrane electropermeabilisation has been the most studied during the past decades. Until today, electropermeabilisation was reported for all tested pulse duration: from nanoseconds to milliseconds. It is well accepted that electropermeabilisation is triggered by a modulation of the transmembrane voltage which is itself induced by the electric field. The high induced transmembrane voltage results in high electric field in the membrane. Under such a physical stress, cell membrane becomes permeable reversibly or irreversibly, depending on the strength of the stress. We propose in this study to measure the transmembrane voltage induced by an external electric field. The measurement of the transmembrane voltage is based on the fluorescence emitted by a voltage sensitive dye, ANNINE-6, which incorporates in cells membrane. We specifically address in this work the impact of one single pulse of 100 ms on the mammalian cell line DC3F. We discuss in this study the importance of the calibration of the fluorescence response.

Anti-allergic effects of dietary polyphenols were extensively studied in numerous allergic disease models, but the molecular mechanisms of anti-allergic effects by polyphenols remain poorly understood. In the present study, we show that the release of granular cargo molecules, contained in distinct subsets of granules of mast cells, is specifically mediated by two sets of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and that various polyphenols differentially inhibit the formation of those SNARE complexes. Expression analysis of RBL-2H3 cells for 11 SNARE genes and a lipid mixing assay of 24 possible combinations of reconstituted SNAREs indicated that the only two active SNARE complexes involved in mast cell degranulation are Syn (syntaxin) 4/SNAP (23 kDa synaptosome-associated protein)-23/VAMP (vesicle-associated membrane protein) 2 and Syn4/SNAP-23/VAMP8. Various polyphenols selectively or commonly interfered with ternary complex formation of these two SNARE complexes, thereby stopping membrane fusion between granules and plasma membrane. This led to the differential effect of polyphenols on degranulation of three distinct subsets of granules. These results suggest the possibility that formation of a variety of SNARE complexes in numerous cell types is controlled by polyphenols which, in turn, might regulate corresponding membrane trafficking.

Membrane-disrupting agents that selectively target virus versus host membranes could potentially inhibit a broad-spectrum of enveloped viruses, but currently such antivirals are lacking. Here, we develop a nanodisc incorporated with a decoy virus receptor that inhibits virus infection. Mechanistically, nanodiscs carrying the viral receptor sialic acid bind to influenza virions and are co-endocytosed into host cells. At low pH in the endosome, the nanodiscs rupture the viral envelope, trapping viral RNAs inside the endolysosome for enzymatic decomposition. In contrast, liposomes containing a decoy receptor show weak antiviral activity due to the lack of membrane disruption. The nanodiscs inhibit influenza virus infection and reduce morbidity and mortality in a mouse model. Our results suggest a new class of antivirals applicable to other enveloped viruses that cause irreversible physical damage specifically to virus envelope by viruses’ own fusion machine. In conclusion, the lipid nanostructure provides another dimension for antiviral activity of decoy molecules.