Abstract
Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Animals, Newborn
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Calcium / metabolism
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Cell Membrane / metabolism
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Cerebral Cortex / cytology
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Enzyme Inhibitors / pharmacology
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Enzyme-Linked Immunosorbent Assay / methods
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Excitatory Postsynaptic Potentials / drug effects
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In Vitro Techniques
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Liposomes / metabolism
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Macrolides / pharmacology
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Mutation / genetics
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Neurons / drug effects
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Neurons / metabolism*
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Neurons / ultrastructure
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Neurotransmitter Agents / metabolism*
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Neurotransmitter Agents / pharmacology
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Peptides / metabolism
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Peptides / pharmacology
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Protein Binding / drug effects
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Protein Binding / physiology
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Protein Subunits / genetics
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Protein Subunits / metabolism
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Proteolipids / metabolism
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Rats
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Rats, Wistar
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SNARE Proteins / metabolism
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Sequence Alignment / methods
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Synapses / physiology*
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Synaptic Vesicles / metabolism*
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Two-Hybrid System Techniques
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Vacuolar Proton-Translocating ATPases / chemistry
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Vacuolar Proton-Translocating ATPases / metabolism*
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Vesicle-Associated Membrane Protein 2 / genetics
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Vesicle-Associated Membrane Protein 2 / metabolism
Substances
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Enzyme Inhibitors
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Liposomes
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Macrolides
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Neurotransmitter Agents
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Peptides
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Protein Subunits
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Proteolipids
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SNARE Proteins
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Vamp2 protein, rat
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Vesicle-Associated Membrane Protein 2
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proteoliposomes
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bafilomycin A
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Vacuolar Proton-Translocating ATPases
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Calcium