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Molecular Mechanism of Neurotransmitter Release
Summary: Axel Brunger's goal is to understand the molecular mechanism of synaptic neurotransmitter release. He is particularly interested in the structure, function, and dynamics of proteins involved in Ca2+-triggered synaptic vesicle fusion. Other projects include structural studies of protein complexes that are involved in synaptic formation and maturation. A molecular understanding of these complex protein machineries may ultimately lead to new therapeutics to treat neurological disorders. He is also developing new methodologies for biomolecular imaging at the molecular scale.
Neurotransmitter Release
Neuronal communication is made possible by the release of neurotransmitters, which in turn depends on the fusion of neurotransmitter-laden synaptic vesicles at the ends of nerve cells. Synaptic vesicle fusion is triggered by an influx of Ca2+ ions into the neuron upon depolarization of the neuron, an event that occurs during neurotransmission. Neurotransmitter release is quantized, that is, it involves individual synaptic vesicle fusion events (a few to several tens of such events for each action potential). The process of individual synaptic vesicle fusion is in turn controlled by a handful of proteins, such as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, the Ca2+ sensor synaptotagmin 1, and the modulator complexin. Thus, neurotransmitter release is a biological phenomenon controlled by a few individual molecules. An understanding of the underlying molecular mechanisms requires methods that are capable of observing single vesicles and molecules.
Ideally, observations of single vesicles and molecules would be performed in live neurons. While such studies have been under way (in our laboratory and others), they currently provide limited information, largely because the genetic manipulations and labeling techniques used do not provide the spatial and time resolution required for studying the molecular mechanism of neurotransmitter release. Thus, minimal in vitro systems are needed that have the neurotransmitter release characteristics observed in neurons and that allow manipulations and observations not possible in vivo. Such in vitro systems will set the stage for high-throughput screening of the effect of other factors on the process. They could also become screening tools for the development of therapeutic leads to modulate neurotransmitter release and combat neurological disorders.
Our approach to understanding the molecular basis for neurotransmitter release consists of a combination of structural and biophysical studies of in vitro systems. Structural information about complexes between the individual molecular components is primarily obtained by x-ray crystallography and single-molecule FRET (fluorescence resonance energy transfer) measurements. This structural information provides the framework for investigations targeted at the functional and dynamic aspects of the system, using single-molecule and single-vesicle fluorescence microscopy techniques.
SNARE-Synaptotagmin Interaction
Neurotransmitter release is triggered when Ca2+ binds to synaptotagmin 1, a synaptic vesicle protein that interacts with neuronal SNAREs (consisting of syntaxin, synaptobrevin, and SNAP-25) and membranes. We used single-molecule FRET measurements between membrane-reconstituted SNARE complexes and synaptotagmin 1 to determine an experimentally derived model of the synaptotagmin 1–SNARE complex. Dye-labeled ternary SNARE complex was preassembled in solution and reconstituted into supported lipid bilayers. A dye-labeled, soluble fragment of synaptotagmin 1 was then added in solution above the bilayer, leading to FRET emissions upon binding to SNAREs in the bilayer.
A set of individual pairs of label attachment sites was used to obtain 34 distances for synaptotagmin 1 bound to the SNARE complex in the presence of Ca2+. These distances were used as target restraints for docking calculations. The best model from these docking calculations shows that the "bottom" (a nomenclature commonly used to indicate the face of a C2 fragment opposed to the Ca2+-binding loops) of the C2B domain of synaptotagmin 1 interacts with the middle portion of the SNARE complex on the side that consists of the SNAP-25 helices.
We also characterized the synaptotagmin 3–SNARE complex interaction by single-molecule fluorescence microscopy and crystallography. We measured single-molecule FRET to characterize the conformation of doubly fluorophore-labeled synaptotagmin 3 in solution, in the presence of Ca2+ and SNARE complex. In the absence of SNAREs, the single-molecule FRET efficiency distribution of synaptotagmin 3 exhibited a broad peak and a tail toward higher FRET states, suggesting multiple conformations, as confirmed by molecular dynamics simulations.
Interaction with SNARE complex dramatically affected the synaptotagmin 3 conformations as evidenced by inducing a high FRET state in both the absence and presence of Ca2+, with the effect being more pronounced with Ca2+. This state is represented by the SNARE-induced Ca2+-bound structure of synaptotagmin 3 that we determined by x-ray crystallography. Remarkably, the arrangement of the Ca2+-binding loops of this structure of synaptotagmin 3 matches that of SNARE-bound synaptotagmin 1, suggesting a conserved feature of synaptotagmins. The loops resemble the membrane-interacting loops of certain viral fusion proteins in the postfusion state, suggesting unexpected similarities between both fusion systems.
In Vitro Reconstitution of Ca2+-Triggered Synaptic Vesicle Fusion
We have achieved the long-standing goal to establish an in vitro single-vesicle system with reconstituted synaptic proteins that produces a rapid burst of content release upon injection of Ca2+. Prior to Ca2+ injection, the system is in a state of single interacting pairs of donor and acceptor vesicles, and fusion events are rare. Our system differentiates between single-vesicle interaction, membrane lipid exchange, and complete fusion (i.e., pore formation) upon Ca2+ injection, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Since single vesicles are observed and the time courses of individual fusion events are monitored, our observations are analogous to quantized Ca2+-triggered neurotransmitter release of one or more synaptic vesicles. We limited this study to neuronal SNARE, synaptotagmin, and complexin proteins. However, other synaptic proteins could be added to the system and their functions could be examined.
Since our system includes both lipid- and content-mixing reporting fluorophores, and since we use total internal reflection light microscopy to monitor single vesicles, different events have characteristic fluorescent signals and can be distinguished on this basis. We can distinguish interacting vesicles from just lipid mixing and from full fusion, so we know the nature of the events. In contrast to ensemble measurements in which single vesicles are not monitored—and which is how such reconstituted systems have been typically monitored in the past—this also allows us to distinguish between fusing vesicles and those that burst or leak accidentally. Moreover, most bulk liposome experiments only monitored lipid mixing. Our results demonstrate that the conclusions of these previous studies need revision since the temporal characteristics of content mixing differ from those of lipid mixing for Ca2+-triggered fusion.
Our single-vesicle content-mixing system has shed light into the molecular mechanism of Ca2+-triggered synaptic vesicle fusion. Neuronal SNAREs alone (i.e., without synaptotagmin and complexin) readily promote donor-acceptor vesicle interaction and lipid mixing. Neuronal SNAREs alone do not, however, efficiently promote content mixing, i.e., complete fusion. The combination of SNAREs and synaptotagmin 1 lowers the transition barriers to achieve complete fusion between membranes upon Ca2+ injection. We find that Ca2+ binding to synaptotagmin is essential for this process since mutation of one of the Ca2+-binding sites of synaptotagmin 1 greatly reduces the rapid fusion burst upon Ca2+ injection. Complexin significantly increases the rapid fusion burst at higher Ca2+ concentrations.
The single-vesicle content-mixing system now provides a platform to study the role of the various factors in Ca2+-triggered synaptic vesicle fusion (in addition to the minimal set of proteins used in this initial study).
A Model of Ca2+-Triggered Synaptic Vesicle Fusion
Based on our experiments, we proposed a model (Figure 1) for Ca2+-triggered vesicle fusion that begins with a SNARE-induced stalk state. SNARE complexes then recruit synaptotagmins to the stalk and induce a conformation of synaptotagmin 1 where the Ca2+-binding loops of both C2 domains are close to the membrane, but yet not penetrating the membrane. Upon Ca2+ influx, this conformation of complexed synaptotagmins is enhanced and their Ca2+-binding loops insert into the membrane. Simultaneously, the synaptotagmin-SNARE interaction is strengthened, influencing the interaction with complexin and leading to full zippering of the SNARE complex. The transition from the stalk to the fusion pore could be helped by this additional zippering of the SNARE complex. In concert, the interaction of the synaptotagmin Ca2+-binding loops with the membrane perturbs membrane regions near the stalk. This model will undergo revision as new experimental data become available.
Synapse Development
Synapse formation and maturation are essential for the normal establishment and remodeling of neuronal circuits in the brain. Impairments in synapse formation and maturation are causes of human diseases such as autism spectrum disorders and mental retardation. Among trans-synaptic adhesion factors that play a role in synapse formation are the neurexin and neuroligin family of proteins. Neuroligins and neurexins form a complex in which two neuroligin molecules link to each other, and a neurexin molecule attaches to each side of the pair. Complex formation is downstream from cadherin and SynCAM adhesion protein engagement. As neurons create new synapses during learning, they must form neuroligin-neurexin connections for those synapses to become functionally mature.
To better understand how the two proteins interact, my laboratory determined the structure of the neuroligin-1 and neurexin-1β complex (Figure 2). We then mutated neuroligin-1 at locations that disrupt the observed interface between it and neurexin. These mutations greatly inhibited the complex from forming but did not affect folding of neuroligin-1. In collaboration with
Thomas Südhof (HHMI, Stanford University), we investigated how these neuroligin-1 mutations alter synaptic function, both in cultures of neurons and in mice, following the approaches that his lab had taken previously. We are currently investigating complexes between neuroligins, neurexins, and other ligands.
Developments in Biomolecular Imaging
X-ray diffraction—which reveals atomic structures of proteins, nucleic acids, and their complexes—plays a pivotal role in the understanding of biological systems. Recently there has been much interest in very large assemblies, such as the ribosome. Since crystals of such large assemblies often diffract weakly (resolution worse than ~3.5 Å), there is a need to develop methods that work at such low resolution. In macromolecular assemblies, some of the components may be known at high resolution, while others are unknown. Determining the structure of such complexes, which are often biologically important, should be possible in principle, as the number of independent diffraction intensities at a resolution better than ~5 Å generally exceeds the number of degrees of freedom.
We developed a method (deformable elastic network [DEN] refinement) that adds specific information from known homologous structures but allows global and local deformations of these homology models. Our approach uses the observation that local protein structure tends to be conserved as sequence and function evolve. For test cases at 3.5- to 5-Å resolution, with known structures at high resolution, our method is a significant improvement over conventional refinement, as monitored by coordinate accuracy, the definition of secondary structure, and the quality of electron density maps. Our method was instrumental in the determination of a number of new x-ray crystal structures (carried out by us and by other groups), and it is applicable to the study of weakly diffracting crystals with a high degree of anisotropy or thermal factors, as well as to the study of data from powerful new x-ray light sources that use free-electron lasers. Furthermore, we are developing methods for the determination of low-resolution models based on other sparse information, such as FRET distances obtained from single-molecule experiments.
A grant from the National Institutes of Health provides partial support for the work on neurotransmitter release.
Last updated September 20, 2011
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