In presynaptic nerve terminals, complex molecular interactions control synaptic vesicles fusion with the plasma membrane and the release of their content in neurotransmitter
Classically the V-ATPase is a proton pump that ensures the acidification of intracellular compartments, including those destined for secretion and notably synaptic vesicles. Our project is based on accumulating, evidence that the membrane sector of the V-ATPase also acts at a late step in membrane fusion. This implies that the V-ATPase plays two roles in neurotransmitter release at different stages in the synaptic vesicle cycle, (i) establishing the proton gradient that transporters use to load vesicles with transmitter; (ii) co-operating with SNAREs to promote synaptic vesicle fusion at the plasma membrane. At the molecular level we will dissect interactions between the V-ATPase membrane sector and the SNARE membrane fusion machinery and design probes to assess their functional relevance. At the functional level we will assess the activity of these probes in Ca2+-dependent neurotransmitter release. The c-subunit of the membrane sector of the V-ATPase forms a transmembrane hexamer suspected to have pore-forming properties, one hypothesis is that this protein forms the fusion pore by which neurotransmitter initially escapes into the synaptic cleft. This project allows a better understanding of the molecular mechanisms of neurotransmitter release and might help developing new molecular strategies that allow controlling excess neurotransmitter release (epilepsy, wrinkles…)
A combination of expertise in biochemical and molecular techniques as well as the electrophysiological recordings of neuronal activity allow analyzing the effects of precise molecular perturbations on synaptic communication
Results are expected at a more advanced state of the project
The project allows uncovering different molecular levers that control neurotransmitter release
A scientific production is expected at a more advanced state of the project
Our project is based on accumulating evidence that the V0 membrane sector of the V-ATPase acts at a late step in membrane fusion by co-operating with SNAREs to promote synaptic vesicle fusion at the plasma membrane. We recently discovered a direct interaction between the v-SNARE VAMP2 and the c-subunit, a major component of the V-ATPase membrane sector. This protein forms very hydrophobic hexamers and has been suggested to mediate calcium regulated acetylcholine release upon reconstitution in proteoliposomes. Our project aims at understanding how and to what extent c-subunit regulates SNARE-mediated neurotransmitter release. In order to answer very specific mechanistic questions about vesicular transport, it is crucial to start by a detailed dissection of the different molecular interactions. We will use this information to confirm and extend our preliminary results; showing that upon reconstitution in proteoliposomes, purified recombinant c-subunit can mediate Ca2+/calmodulin induced choline release. In addition to the pore-forming properties of c-subunit, we will study its influence on SNARE-mediated proteoliposome fusion. These results should provide sufficient molecular details in order to design mutants or probes to perturb these interactions in living cells. Based on these results, electrophysiological recording using caged peptides, laser-mediated protein inactivation, a combination of shRNA and viral transfection of mutant proteins will be used to examine the influence of very specific perturbations of protein complexes on neurotransmitter release in dissociated neuronal cultures. We will also combine patch clamp recording and FM 1-43 fluorescence analysis in order to investigate whether the action of c-subunit in SNARE-mediated neurotransmitter release correlates with a specific release mode: full fusion or Kiss-and Run.
Monsieur Oussama EL FAR (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION PACA) – oussama.el-far@univ-amu.fr
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
UMR641 INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION PACA
UMR641 INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION PACA
Help of the ANR 500,000 euros
Beginning and duration of the scientific project:
October 2011
- 48 Months
