Molecular mechanisms of synaptic neurotransmitter release

Synaptic transmission between pre- and postsynaptic neurons occurs when the presynaptic neuron terminal is
temporarily depolarized upon an action potential, opening Ca2+ channels near the active zones of synapses.
Because the extracellular Ca2+ concentration is much higher than the cytoplasmic concentration, Ca2+ flows
into the cytoplasm, triggering fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane
in less than a millisecond. Upon fusion, neurotransmitter molecules are released into the synaptic cleft, and
then bind to receptors located in the postsynaptic membrane. Finally, the fusion machinery is recycled for
further rounds of fusion in the presynaptic cell. Major questions about the molecular mechanisms of membrane
fusion and protein recycling remain. The architecture of the fusion machinery between the synaptic vesicles
and plasma membranes is unknown, and the molecular steps after Ca2+-triggering are unknown. Furthermore,
our understanding of the molecular mechanisms governing synaptic release probability and presynaptic
plasticity is incomplete. Obtaining three-dimensional images of synaptic proteins within their natural membrane
environment will be an essential step towards answering these questions. We propose a stepwise, bottom-up
approach starting with simpler systems and moving to increasingly more complex systems. First, we will
employ a hybrid (ex vivo / in vitro) approach where synaptic vesicles are isolated from mouse brain
homogenates and combined with synthetic acceptor vesicles. Functional tests of this hybrid system will be
performed using a new single vesicle fusion assay that discriminates between different stages of membrane
fusion and includes many presynaptic proteins, including but not limited to SNAREs, synaptotagmin, and
complexin. The contact sites between isolated synaptic vesicles and synthetic vesicles will be imaged by cryo-electron tomography (cryo-ET) followed by subtomogram averaging to reveal the architecture of these
presynaptic complexes in their membrane environment. Next, we plan to image the equivalent membrane
contact sites in both isolated synaptosomes and in synapses of neuronal cultures grown on EM grids. We
anticipate that reconstructions of presynaptic complexes obtained with the hybrid approach are the starting
point for locating such complexes in synaptosomes and in synapses in neuronal cultures. These in vivo
reconstructions might reveal new molecular interactions or associations. After fusion, the AAA+ protein NSF
and associated SNAP co-factors are essential for disassembly of SNARE complexes and for quality control of
newly formed SNARE complexes (in conjunction with Munc18 and Munc13). Previously, we determined single-particle cryo-electron microscopy (cryo-EM) structures of the complex of NSF, αSNAP, and the neuronal
SNARE complex under non-hydrolyzing conditions. We now aim to investigate the molecular details of
disassembly, what conformational changes are involved, and how ATP hydrolysis is coupled to these
conformational changes by cryo-EM.

Grant Number: 5R01MH063105-25
NIH Institute/Center: NIH
Principal Investigator: AXEL BRUNGER