Browsing by Subject "Neurotransmitter Agents"
Now showing 1 - 9 of 9
- Results Per Page
- Sort Options
Item Dissection of the Roles of Synaptotagmins in Calcium Dependent Neurotransmitter Release(2020-08-01T05:00:00.000Z) Voleti, Sai Rashmi; Nam, Yunsun; Rizo-Rey, José; Chook, Yuh Min; Ross, Elliott M.Neuronal communication is mediated by neurotransmitter release, which is triggered in response to a calcium influx into the presynaptic cell, caused by the arrival of an action potential. Synaptotagmins (Syts) mediate this calcium-dependent regulation of neurotransmitter release by mechanisms that still remain elusive. Syts have two calcium-binding C2 domains (C2A and C2B) which are crucial for their function. Clarifying how Syt C2 domains interact with the neuronal membrane fusion machinery and the membranes is essential for gaining insights into the mechanisms of calcium-triggered neurotransmitter release. To understand the role of Syts in neurotransmitter release, we explored the origins of functional differences between the C2 domains of two Syt isoforms, Syt1 and Syt7, which mediate synchronous and asynchronous release respectively. Calcium binding to the C2A domain is critical for Syt7 functions whereas Syt1 function depends more on calcium binding ability of its C2B domain. By solving the structures of Syt7 C2A and C2AB fragments and by analyzing their intrinsic calcium binding properties by ITC, we showed that these properties do not give rise to the functional differentiation between Syt1 and Syt7. By characterizing the calcium-dependent phospholipid binding of C2 domains by FRET, we demonstrated that C2A and C2B dominate membrane binding in Syt7 and Syt1, respectively. This suggests that membrane affinity of C2 domains might dictate their functional importance for Syt function. In addition to membrane binding, Syt1 C2B domain is also involved in interactions with the SNARE complex, which is the central component of neuronal membrane fusion machinery. Clarifying the molecular details of these interactions is crucial for understanding how Syt1 cooperates with SNARE complex to trigger calcium-dependent membrane fusion. Previous structural studies have shown three distinct SNARE complex-binding interfaces on Syt1 C2B domain, two of which overlap with membrane binding regions. By using NMR, we showed that only two of these interfaces exist in solution. By using FRET to characterize interactions on membranes, we showed that Syt1-SNARE complex binding primarily occurs via one of the remaining interfaces (primary interface), and this interaction is almost abolished in the presence of calcium. Together, these results suggest a model where, a release of Syt1-SNARE complex interactions by calcium triggers membrane fusion.Item Insights into the Functions of Munc18-1 in Neurotransmitter Release(2013-03-07) Su, Lijing; Rizo-Rey, José; Luo, Xuelian; Kavalali, Ege T.; Liu, YiNeurotransmitter release is an exquisitely regulated process that transmits signals between neurons. The release process includes: docking of synaptic vesicles at the active zone of the pre-synaptic plasma membrane, priming to a release ready state, and then membrane fusion and release of neurotransmitters triggered by Ca2+. Several conserved proteins are involved in regulating the entire process. The central membrane fusion machinery in neurons includes Munc18-1 and the SNARE proteins syntaxin-1, SNAP-25 and synaptobrevin. The SNAREs form tight SNARE complexes that bring the vesicle membrane and plasma membrane into close proximity and provide forces to induce membrane fusion. Munc18-1 is essential because deletion of Munc18-1 in mice leads to a complete loss of neurotransmitter secretion. However, its molecular mechanism of action is still unclear. This work is aimed to unravel the critical roles of Munc18-1 in regulating neurotransmitter release. The functions of Munc18-1 discovered so far are related to the SNAREs. Recently we found that Munc18-1 interacts with synaptobrevin and the SNARE four-helix bundle with week affinity, which have been shown to stimulate in vitro SNARE-dependent liposome fusion. Biophysical characterization of these two interactions could provide important information to uncover the roles of Munc18-1 in membrane fusion. Cross-linking and NMR spectroscopy experiments showed that Munc18-1 interacts with the C-terminus of the synaptobrevin SNARE motif through some positively charged residues located in its domain 3a. NMR spectroscopy and ITC experiments revealed that the Munc18-1 N-terminal domain interacts with the C-terminal part of synaptobrevin and syntaxin-1 on the SNARE four-helix bundle, and that the affinity is higher than full length Munc18-1. In our in vitro reconstitution experiments that try to establish the vital functions of Munc18-1 and Munc13 in neurotransmitter release, I found that Munc18-1 displaces SNAP-25 from syntaxin-1/SNAP-25 complex to form Munc18-1/syntaxin-1 complex on liposomes in the presence of NSF/α-SNAP/ATP. When NSF/α-SNAP were incorporated in the lipid mixing assays between synaptobrevin-liposmes and syntaxin-1/SNAP-25-liposomes, Munc18-1 together with Munc13 activate lipid mixing that is inhibited by NSF/α-SNAP. These results suggest that Munc18-1 functions with Munc13 to promote SNARE complex formation in an NSF/α-SNAP resistant manner and to guide the synaptic vesicle exocytosis through a tightly regulated pathway.Item Mechanistic Insights into the Role of Munc13 in Synaptic Vesicle Docking, Priming, and Fusion(2019-03-06) Quade, Bradley Jackson; Luo, Xuelian; Rizo-Rey, José; Rice, Luke M.; Zhang, XuewuNeurotransmitter release is a fundamental aspect of neuronal communication that relies on the fusion of synaptic vesicles with the presynaptic membrane. These fusion events are tightly regulated by the influx of Ca2+, which is sensed by the complex protein machinery at the axon terminal. In order for these Ca2+-mediated fusion events to occur in the correct time and place, protein machines interact with neurotransmitter filled vesicles to dock and prime them for release. Munc13 is one of the essential components of the docking, priming, and fusion machinery. To understand the role of Munc13 in docking and priming I attempted to structurally characterize the MUN domain and SNARE protein interactions using nuclear magnetic resonance spectroscopy. Paramagnetic relaxation enhancement and pseudocontact shift experiments were performed to identify the binding site of SNAREs or the SNARE complex on the MUN domain and in both cases the data suggested that there may be binding in multiple locations or that the interactions are promiscuous. I also attempted to crystallize the C2C domain of Munc13 alone and in the context of larger fragments. I was able to grow crystals of various fragments of Munc13 containing C2C and adjacent domains, but these crystals were fragile and diffracted poorly. In lieu of a crystal structure, I modeled the C2C domain based on homologous C2 domains and performed sequence conservation analysis to identify functionally important regions of C2C that may bind membranes. Using structural information coupled with reconstitutions, dynamic light scattering, and cryo-electron tomography I explored the functional relevance of membrane binding within the C1, C2B, and C2C domains of Munc13. The C2C domain was identified as a critical component of Munc13 that enables bridging between liposomes with synaptic vesicle and plasma membrane composition in vitro and this bridging ability is integral for synaptic vesicle docking in vivo. The C1C2B area has a large membrane binding interface that changes depending on the ligands present in the system and this enables Munc13 to modulate the distance between membranes. The ability of Munc13 to regulate the distance between membranes in response to ligands may underlie its role in synaptic plasticity.Item Membrane Lipids and Synaptic Vesicle Trafficking in the CNS(2009-01-14) Wasser, Catherine Rebecca; Kavalali, Ege T.Most vesicles within a synapse are dormant. The rest participate in synaptic neurotransmission, with a portion of these preferentially fusing first. Moreover, all synapses experience spontaneous neurotransmitter release which may originate from the random exocytosis of vesicles prepared to fuse immediately upon calcium influx; however, spontaneously fusing vesicles may be independent because they prefer spontaneous fusion. The functional separation argues that the compositions the synaptic vesicle membranes are somehow unique between pools. The first three chapters explore the role of cholesterol in synaptic transmission. We treated hippocampal cultures with methyl-beta-cyclodextrin, which reversibly binds cholesterol, or mevastatin, an inhibitor of cholesterol biosynthesis, to deplete cholesterol. We also used hippocampal cultures from Niemann-Pick type C1-deficient mice defective in intracellular cholesterol trafficking. These conditions revealed augmented spontaneous neurotransmission. In contrast, the same treatments severely impaired responses evoked by action potentials and hypertonicity. These results suggest that synaptic cholesterol balances evoked and spontaneous neurotransmission by hindering spontaneous synaptic vesicle turnover and sustaining evoked exo-endocytosis. Chapter five examines the role of sphingosine on neurotransmitter release. By adding sphingosine to hippocampal cultures, we found that sphingosine enhances neurotransmission in a synaptobrevin-2-dependent manner. Chapter six investigates the stability of actively recycling synaptic vesicles. We employed several approaches (fluorescent and ultrastructural imaging) to monitor not only the fate recycling vesicles, but also the origin and reuse of spontaneously fusing vesicles. We conclude that at rest, the total recycling pool remains active and resists spontaneous fusion up to at least six hours; while spontaneous fusion of spontaneously fusing vesicles is much faster. This argues that vesicles fusing spontaneously do not originate from the recycling pool. In chapter seven, we observe how modifying synaptic vesicle membranes might affect neurotransmitter release. By the uptake of horseradish peroxidase into vesicles followed by hydrogen peroxide perfusion, we induced free radical modification of vesicle membranes and found that modifying recycling pool vesicles increased spontaneous fusion and attenuated evoked release. Taken together, the results of each chapter appear to suggest that the fusion of action potential-dependent and-independent vesicles are regulated by different mechanisms, supporting the theory that some vesicles may be unique within a synapse.Item Molecular and Functional Determinants of Synaptic Vesicle Recycling In CNS Synapses(2007-05-23) Virmani, Tuhin; Kavalali, Ege T.Chemical neurotransmission is the basis for information processing in the brain, and presynaptic terminals respond to a large range of stimulation patterns including baseline rhythms of activity that coordinate neuronal ensembles, to short bursts of activity that encode information. They also release neurotransmitter spontaneously in the absence of any activity. The question then is how can a single subcellular compartment with approximately 100 synaptic vesicles coordinate these complex functions? We used a multifaceted approach to address this question. We first studied the role of synaptotagmin 7 (syt7), a highly alternatively spliced synaptic plasma membrane protein, whose short splice forms inhibit clathrin-mediated endocytosis. We found that in hippocampal synapses, the splice variants formed a bi-directional molecular switch targeting vesicles to kinetically distinct recycling pathways. Additionally, syt7 knockout synapses had less fast endocytosis, while calcium binding site mutant synapses showed increased vesicle endocytosis. We further investigated the slower recycling pathways by exploring rab5 function. A dominant negative rab5 mutation did not alter synaptic function, but constitutively active rab5 or the inhibition of vesicle budding from endosomes by the PI3-kinase inhibitor wortmannin, decreased vesicle pool size and release kinetics. This suggests that central synapses are tuned towards faster modes of recycling. The model of spontaneous neurotransmitter release from this same evoked recycling pool places additional constraints on this system. We explored this hypothesis by directly visualizing presynaptic recycling of spontaneous vesicles using FM dyes, syt1 antibodies and HRP uptake. We found that there are actually two sets of vesicle pools, one for evoked release, and one for spontaneous release that have minimal interaction with one another. Can presynaptic function be a substrate for diseases of the CNS? To test this important question, we studied a mouse model for infantile Batten disease. We found that underlying synaptic deficits in vesicle pool size and mini frequency could produce the neurological phenotypes exhibited by patients well before the onset of neurodegeneration. Taken together, these results show that synaptic vesicle recycling is a very plastic entity and that synapses have the intrinsic ability to modulate their vesicle trafficking pathways in response to the varying demands placed on them.Item [Southwestern News](2002-01-16) Shields, AmyItem Structural Studies of Complexin/SNARE Interactions(2008-09-18) Lee, Daeho; Rizo-Rey, JoséVesicular neurotransmitter release is mediated by exocytosis of synaptic vesicles at the presynaptic active zone of nerve terminals. The Ca2+-triggered release process is extremely fast, lasts less than half a millisecond, and is tightly regulated by Ca2+. Action potentials cause Ca2+ influx through voltage-gated Ca2+ channels, which in turn triggers synaptic vesicle fusion. The typical sub-millisecond latency between an action potential and neurotransmitter release and the precise timing of the release process are crucial for information processing in the nervous system. To achieve this exquisite regulation, many proteins are involved. The goal of our investigations was to delineate interactions between complexin and SNARE components that lead to the formation of a primed minimal fusion machinery. We have generated and used new constructs of short forms of synaptobrevin and complexin, as well as constructs of SNAP-25 and syntaxin that have previously been shown to be part of the minimal fusion machinery. With NMR spectroscopy, the use of the short synaptobrevin constructs has led to experimental results suggesting at least two key intermediates during the docking/priming process that are independent of complexin. We found evidence for a modular assembly of the full SNARE complex. In the absence of Syb2-CT, the N-terminal half of SNARE complex forms a four-helix bundle, while the C-terminal half, starting just after the polar layer, is disordered. In the presence of the Syb2-CT, however, both halves of the SNARE complex form a four-helix bundle. It is interesting to note that Syb2 residues 29-84 are sufficient for formation of the fully assembled SNARE complex. This evidence strongly suggests the existence of at least two intermediates during the docking priming reaction. Furthermore, with NMR spectroscopy, we have found new evidence that complexin can bind to the t-SNARE complex, in contrast to earlier evidence suggesting that complexin regulates the fully assembled SNARE complex. We demonstrated that Cpx-FL binds the t-SNARE complex SN1/SN3/Syx1a(188-259) in solution, as wa suggested for membrane-bound t-SNAREs. Note, however, that the t-SNARE complex does not contain the large complexin-binding interface provided by Syb2. Furthermore, we found that Cpx-FL also binds t-SNARE sub-complexes formed by SN1/SN3, and SN1/Syx1a(188-259), while very little binding was observed between Cpx-FL and Syx1a(188-259) alone. This finding is particularly interesting, because the cryst structure of the fully assembled SNARE complex does not suggest any binding between Cpx26-83 and either SN1 or SN3, whereas the only common component in all of the above experiments was SN1 domain.Item Structure and Function of Proteins Involved in Regulated Secretion: Synaptotagmins and Complexin(2010-01-12) Craig, Timothy Kellogg; Rizo-Rey, JoséThe release of neurotransmitter from neurons is a tightly regulated process. There are a number of proteins required for membrane fusion to occur, and then there are regulatory proteins that allow membrane fusion to proceed at incredible speed with the precise timing necessary for complex functions such as sight, motor control, and conscious thought. This study will explore the role of three such regulators through biophysical and structural methods. There are a number of proteins that are essential for membrane fusion. The SNARE proteins are the plasma membrane protein Syntaxin, the vesicle membrane protein Synaptobrevin, and the plasma membrane associated protein SNAP25. These proteins form a tight complex called the SNARE complex that is required for neurotransmitter release. This complex bridges the vesicle and plasma membranes, bringing them into close proximity. Formation of this complex is thus an important point of regulation for the neurotransmitter release process. This SNARE complex serves not only to bridge two membranes, but also to become an anchoring point for a number of regulators of neurotransmitter release such as Complexin, and Synaptotagmin as well as other required proteins such as Munc13 and Munc18. Complexin is a small soluble protein that binds to the SNARE complex with high affinity and regulates the formation of the SNARE complex. Synaptotagmin is the calcium sensor for fast release of neurotransmitter. Here I present data showing that the N-terminus of Complexin is involved in a critical interaction with the C-terminus of the SNARE complex that is responsible for the excitatory effect of complexin in neurotransmitter release. Synaptotagmins work with Complexins to trigger rapid membrane fusion in response to calcium influx. Synaptotagmin VII is an important protein for the release of glucagon from islets of langerhans. The C2B domain of this protein is nearly 50% identical to the C2B domain of SytI, but when the C2B domains of SytVII and SytI are switched, the protein does not function correctly. In this study the structure of SytVII was determined by x-ray crystallography to 1.44Å resolution in order to determine if the C2B domain of SytVII is structurally different from other C2B domains. Additionally I crystallized and solved the structure of the C2A domain of Synaptotagmin IX in an effort to compare it to the C2 domains of the other members of the synaptotagmin family. This analysis resulted in the surprising conclusion that a high degree of structural similarity does not necessarily relate to interoperability of the domains.Item Unraveling the Functions of Synaptotagmin and Munc13 in Neurotransmitter Release(2015-01-30) Seven, Alpay Burak; Rosen, Michael K.; Rizo-Rey, José; Albanesi, Joseph P.; Jiang, Qiu-XingNeurotransmitter release is a central event in interneuronal communication. The release machinery includes three SNAREs (soluble N-ethylmaleimide sensitive factor adaptor protein receptor) and Munc18-1 as core components. Munc13, synaptotagmin and complexin underlie the exquisitely tight regulation of synaptic exocytosis. The SNAREs form the SNARE complex that brings synaptic vesicles and plasma membrane together. This complex is disassembled by NSF/α-SNAP (soluble N-ethylmaleimide sensitive fusion protein/NSF attachment protein). Munc18 binds to syntaxin, which keeps syntaxin in its closed confirmation and prevents to form the SNARE complex. Interactions between Munc13, Munc18, and syntaxin perform a vital role in regulation of the SNARE complex formation. Tight regulation of the release machinery requires other factors such as Ca2+ sensor Synaptotagmin-1 and negatively charged lipids. Synaptotagmin interacts with the SNARE complex and the negatively charged lipids, and initiates the synchronous fast release by sensing the Ca2+ influx. It is crucial to investigate functions of individual proteins to understand the mechanism of membrane fusion and neurotransmitter release. Therefore, we investigated the mechanism of membrane bridging by synaptotagmin. Our cryo-electron microscopy (cryo-EM) images showed that a majority of synaptotagmin fragment containing both C¬2A and C2B domains (C2AB) molecules bridge membranes directly. Fluorescence spectroscopy demonstrates that the bottom of the C2B domain contacts the membrane in a substantial population of membrane-bound synaptotagmin fragments. NMR analysis of C2AB-nanodiscs shows that a fraction of C2AB molecules binds to membranes with antiparallel orientations of the C2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by synaptotagmin, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release. We have also discovered that Munc13-1 can bridge membranes in a Ca2+-independent manner, which shed light to the docking activity of Munc13-1. We also showed that Munc13-1 can cause efficient lipid mixing and slow content mixing together with Munc18-1 in the absence of synaptotagmin-1. Addition of synaptotagmin facilitates the fusion pore formation in content mixing assays. Recently, we were able to reconstitute key components of the release machinery. We are further investigating our observations with cryo-electron microscopy to understand the mechanism of membrane fusion.