神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白

近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用.     

Protein-protein interactions and protein modules in the control of neurotransmitter release

Information transfer among neurons is operated by neurotransmitters stored in synaptic vesicles and released to the extracellular space by an efficient process of regulated exocytosis. Synaptic vesicles are organized into two distinct functional pools, a large reserve pool in which vesicles are restrained by the actin-based cytoskeleton, and a quantitatively smaller releasable pool in which vesicles approach the presynaptic membrane and eventually fuse with it on stimulation. Both synaptic vesicle trafficking and neurotransmitter release depend on a precise sequence of events that include release from the reserve pool, targeting to the active zone, docking, priming, fusion and endocytotic retrieval of synaptic vesicles. These steps are mediated by a series of specific interactions among cytoskeletal, synaptic vesicle, presynaptic membrane and cytosolic proteins that, by acting in concert, promote the spatial and temporal regulation of the exocytotic machinery. The majority of these interactions are mediated by specific protein modules and domains that are found in many proteins and are involved in numerous intracellular processes. In this paper, the possible physiological role of these multiple protein-protein interactions is analysed, with ensuing updating and clarification of the present molecular model of the process of neurotransmitter release.     

细胞胞吐的分子基础

近年来对神经末梢突触小泡膜蛋白的蛋白质化学,药理学研究及酵母细胞蛋白运输及分泌的遗传学的研究,发现了胞吐蛋白复合体的存在。ＳＮＡＲＥ假说的提出使得对神经递质释放,内外分泌细胞分泌机制的研究有了突飞猛进的进展。     

Functional interactions between presynaptic calcium channels and the neurotransmitter release machinery

In vertebrates, the physical coupling between presynaptic calcium channels and synaptic vesicle release proteins enhances the efficiency of neurotransmission. Recent evidence indicates that these synaptic proteins may feedback directly on synaptic release by negatively regulating calcium entry, and indirectly through pathways involving second messenger molecules. Studies of individual neurons from both vertebrates and invertebrates have provided novel insights into the roles of scaffolding proteins in calcium channel targeting and neurotransmitter release. These studies require us to expand current models of synaptic transmission.     

Synaptic transmission regulated by a presynaptic MALS/Liprin-alpha protein complex

Neurotransmission requires proper organization of synaptic vesicle pools and rapid release of vesicle contents upon presynaptic depolarization. Genetic studies have begun to reveal a critical role for scaffolding proteins in such processes. Mutations in genes encoding components of the highly conserved MALS/CASK/Mint-1 complex cause presynaptic defects. In all three mutants, neurotransmitter release is reduced in a manner consistent with aberrant vesicle cycling to the readily releasable pool. Recently, liprin-alpha proteins, which define active zone size and morphology, were found to associate with MALS/CASK, suggesting that this complex links the presynaptic release machinery to the active zone, thereby regulating neurotransmitter release.     

神经元突触前可塑性的结构及分子基础

突触可塑性是神经元间信息传递的重要生理调控机制,它包括突触前可塑性和突触后可塑性.突触前可塑性是指通过对神经递质释放过程的干预、修饰,调节突触强度的过程.突触强度的变化,是通过影响量子的大小,活动区的个数和囊泡释放概率来实现的.而突触前囊泡活动尤为重要：从转运、搭靠、融合至内吞进入下一轮循环,每一步都是由一群互相作用的蛋白质共同完成的.     

Rabphilin-3A: A Multifunctional Regulator of Synaptic Vesicle Traffic

We have investigated the function of the synaptic vesicle protein Rabphilin-3A in neurotransmitter release at the squid giant synapse. Presynaptic microinjection of recombinant Rabphilin-3A reversibly inhibited the exocytotic release of neurotransmitter. Injection of fragments of Rabphilin-3A indicate that at least two distinct regions of the protein inhibit neurotransmitter release: the NH₂-terminal region that binds Rab3A and is phosphorylated by protein kinases and the two C2 domains that interact with calcium, phospholipid, and β-adducin. Each of the inhibitory fragments and the full-length protein had separate effects on presynaptic morphology, suggesting that individual domains were inhibiting a subset of the reactions in which the full-length protein participates. In addition to inhibiting exocytosis, constructs containing the NH₂ terminus of Rabphilin-3A also perturbed the endocytotic pathway, as indicated by changes in the membrane areas of endosomes, coated vesicles, and the plasma membrane. These results indicate that Rabphilin-3A regulates synaptic vesicle traffic and appears to do so at distinct stages of both the exocytotic and endocytotic pathways.     

Neurotransmitter release at ribbon synapses in the retina

The synapses of photoreceptors and bipolar cells in the retina are easily identified ultrastructurally by the presence of synaptic ribbons, electron-dense bars perpendicular to the plasma membrane at the active zones, extending about 0.5 microm into the cytoplasm. The neurotransmitter, glutamate, is released continuously (tonically) from these 'ribbon synapses' and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven bursts of release at conventional synapses. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but a few differences have been identified that may be important determinants of tonic transmitter release. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.     

Neurotransmitter release mechanisms studied in Caenorhabditis elegans

The process of regulated exocytosis has received considerable interest as a key component of synaptic transmission. Fusion of presynaptic vesicles and the subsequent release of their neurotransmitter contents is driven by a series of interactions between evolutionarily conserved proteins. Key insights into the molecular mechanisms of vesicle fusion have come from research using genetic model systems such as the nematode worm Caenorhabditis elegans. We review here the current knowledge regarding regulated exocytosis at the C. elegans synapse and future research directions involving this model organism.     

Calcium dependent neurotransmitter release and protein phosphorylation in synaptic vesicles.

A highly purified preparation of synaptic vesicles was prepared to study the relationship between calcium-dependent neurotransmitter release and protein phosphorylation. Calcium ions simultaneously produced significant increases in both the endogenous release of norepinephrine from the synaptic vesicles and the endogenous incorporation of [³²p] phosphate into specific synaptic vesicle proteins. The results are compatible with the hypothesis that the action of calcium on the phosphorylation of specific synaptic vesicle proteins is the molecular mechanism mediating some of the effects of calcium on neurotransmitter release and synaptic vesicle function.     

RIM‐binding proteins recruit BK‐channels to presynaptic release sites adjacent to voltage‐gated Ca2+‐channels

The active zone of presynaptic nerve terminals organizes the neurotransmitter release machinery, thereby enabling fast Ca²⁺‐triggered synaptic vesicle exocytosis. BK‐channels are Ca²⁺‐activated large‐conductance K⁺‐channels that require close proximity to Ca²⁺‐channels for activation and control Ca²⁺‐triggered neurotransmitter release by accelerating membrane repolarization during action potential firing. How BK‐channels are recruited to presynaptic Ca²⁺‐channels, however, is unknown. Here, we show that RBPs (for RIM‐binding proteins), which are evolutionarily conserved active zone proteins containing SH3‐ and FN3‐domains, directly bind to BK‐channels. We find that RBPs interact with RIMs and Ca²⁺‐channels via their SH3‐domains, but to BK‐channels via their FN3‐domains. Deletion of RBPs in calyx of Held synapses decreased and decelerated presynaptic BK‐currents and depleted BK‐channels from active zones. Our data suggest that RBPs recruit BK‐channels into a RIM‐based macromolecular active zone complex that includes Ca²⁺‐channels, synaptic vesicles, and the membrane fusion machinery, thereby enabling tight spatio‐temporal coupling of Ca²⁺‐influx to Ca²⁺‐triggered neurotransmitter release in a presynaptic terminal.     

The actin cytoskeleton and neurotransmitter release: an overview

Here we review evidence that actin and its binding partners are involved in the release of neurotransmitters at synapses. The spatial and temporal characteristics of neurotransmitter release are determined by the distribution of synaptic vesicles at the active zones, presynaptic sites of secretion. Synaptic vesicles accumulate near active zones in a readily releasable pool that is docked at the plasma membrane and ready to fuse in response to calcium entry and a secondary, reserve pool that is in the interior of the presynaptic terminal. A network of actin filaments associated with synaptic vesicles might play an important role in maintaining synaptic vesicles within the reserve pool. Actin and myosin also have been implicated in the translocation of vesicles from the reserve pool to the presynaptic plasma membrane. Refilling of the readily releasable vesicle pool during intense stimulation of neurotransmitter release also implicates synapsins as reversible links between synaptic vesicles and actin filaments. The diversity of actin binding partners in nerve terminals suggests that actin might have presynaptic functions beyond synaptic vesicle tethering or movement. Because most of these actin-binding proteins are regulated by calcium, actin might be a pivotal participant in calcium signaling inside presynaptic nerve terminals. However, there is no evidence that actin participates in fusion of synaptic vesicles.     

Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis

The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.     

Tetanus, botulinum and snake presynaptic neurotoxins

Tetanus and botulinum neurotoxins, produced by anaerobic bacteria of the genus Clostridium, are the most toxic proteins known and are solely responsible for the pathogenesis of tetanus and botulism. They are metallo-proteases that enter nerve terminals and cleave proteins of the neuroexocytosis apparatus causing a persistent, but reversible, inhibition of neurotransmitter release. Botulinum neurotoxins are used in the therapy of many human syndromes caused by hyperactive nerve terminals. Snake presynaptic PLA2 neurotoxins block nerve terminals by binding to the nerve membrane and catalyzing phospholipid hydrolysis with production of lysophospholipids and fatty acids. These compounds change the membrane conformation causing enhanced fusion of synaptic vesicle via hemifusion intermediate with release of neurotransmitter and, at the same time, inhibition of vesicle fission and recycling. It is possible to envisage clinical applications of the lysophospholipid/fatty acid mixture to inhibit hyperactive superficial nerve terminals.

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Short-term plasticity of small synaptic vesicle (SSV) and large dense-core vesicle (LDCV) exocytosis

Synaptic plasticity results from changes in the strength of synaptic transmission upon repetitive stimulation. The amount of neurotransmitter released from presynaptic terminals can regulate short-term plasticity that lasts for a few minutes. This review focuses on short-term plasticity of small synaptic vesicle (SSV) and large dense-core vesicle (LDCV) exocytosis. Whereas SSVs contain classical neurotransmitters and activate ion channels, LDCVs contain neuropeptides and hormones which primarily activate G protein-coupled receptors (GPCRs). Thus, LDCV exocytosis is mainly associated with modulation of synaptic activity and cannot induce synaptic activity by itself. As in SSV exocytosis, repetitive stimulation leads to short-term enhancement of LDCV exocytosis: i.e., activity-dependent potentiation (ADP) which represents potentiation of neurotransmitter release. Short-term plasticity of SSV exocytosis results from Ca²⁺ accumulation, but ADP of LDCV exocytosis does not. Here, we review the signaling mechanisms and differences of short-term plasticity in exocytotic processes of SSV and LDCV.     

Inhibition of SNARE-driven neuroexocytosis by plant extracts

Neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins mediate membrane fusion between synaptic vesicle and presynaptic membrane, resulting in neurotransmitter release. SNARE proteins are specific substrates of botulinum neurotoxins (BoNT) which are now widely used for therapeutic and cosmetic purposes. While BoNT blocks neuroexocytosis by cleaving SNAREs, inhibiting SNARE assembly process might exert the same effect on neurotransmission. In the present study, some extracts of 100 plants reduced neurotransmitter release by inhibiting SNARE complex formation in neuronal cells. The extracts effectively paralyzed muscle of rat phrenic nerve-hemidiaphragm preparation. Our results raise the possibility that SNARE folding inhibitors from natural resources might replace some special BoNT application fields. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chang Hwa Jung and Yoo Soo Yang equally contributed to this work.     

Is synaptotagmin the calcium sensor?

After much debate, recent progress indicates that the synaptic vesicle protein synaptotagmin I probably functions as the calcium sensor for synchronous neurotransmitter release. Following calcium influx into presynaptic terminals, synaptotagmin I rapidly triggers the fusion of synaptic vesicles with the plasma membrane and underlies the fourth-order calcium cooperativity of release. Biochemical and genetic studies suggest that lipid and SNARE interactions underlie synaptotagmin's ability to mediate the incredible speed of vesicle fusion that is the hallmark of fast synaptic transmission.     

Building a bilayer model of the neuromuscular synapse

Progress over the past 10 years has made it possible to construct a simple model of neurotransmitter release. Currently, some models use artificially formed vesicles to represent synaptic vesicles and a planar lipid bilayer as a presynaptic membrane. Fusion of vesicles with the bilayer is via channel proteins in the vesicle membrane and an osmotic gradient. In this paper, a framework is presented for the successful construction of a more complete model of synaptic transmission. This model includes real synaptic vesicles that fuse with a planar bilayer. The bilayer contains acetylcholine receptor (AChR) channels which function as autoreceptors in the membrane. Vesicle fusion is initiated following a Ca²⁺ flux through voltage-gated Ca²⁺ channels. Key steps in the plan are validated by mathematical modeling. Specifically, the probability that a reconstituted AChR channel opens following the release of ACh from a fusing vesicle, is calculated as a function of time, quantal content, and number of reconstituted AChRs. Experimentally obtainable parameters for construction of a working synapse are given. The inevitable construction of a full working model will mean that the minimal structures necessary for synaptic transmission are identified. This will open the door in determining regulatory and modulatory factors of transmitter release.     

The proteome of the presynaptic active zone: from docked synaptic vesicles to adhesion molecules and maxi-channels

The presynaptic proteome controls neurotransmitter release and the short and long term structural and functional dynamics of the nerve terminal. Using a monoclonal antibody against synaptic vesicle protein 2 we immunopurified a presynaptic compartment containing the active zone with synaptic vesicles docked to the presynaptic plasma membrane as well as elements of the presynaptic cytomatrix. Individual protein bands separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were subjected to nanoscale-liquid chromatography electrospray ionization-tandem mass spectrometry. Combining this method with 2-dimensional benzyldimethyl- n -hexadecylammonium chloride/sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight and immunodetection we identified 240 proteins comprising synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery, proteins involved in intracellular signal transduction, a large variety of adhesion molecules and proteins potentially involved in regulating the functional and structural dynamics of the pre-synapse. Four maxi-channels, three isoforms of voltage-dependent anion channels and the tweety homolog 1 were co-isolated with the docked synaptic vesicles. As revealed by in situ hybridization, tweety homolog 1 reveals a distinct expression pattern in the rodent brain. Our results add novel information to the proteome of the presynaptic active zone and suggest that in particular proteins potentially involved in the short and long term structural modulation of the mature presynaptic compartment deserve further detailed analysis.     

Syntaphilin: a syntaxin-1 clamp that controls SNARE assembly

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that forms the SNARE complex with VAMP/synaptobrevin and SNAP-25. Identifying proteins that modulate SNARE complex formation is critical for understanding the molecular mechanisms underlying neurotransmitter release and its modulation. We have cloned and characterized a protein called syntaphilin that is selectively expressed in brain. Syntaphilin competes with SNAP-25 for binding to syntaxin-1 and inhibits SNARE complex formation by absorbing free syntaxin-1. Transient overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex, and thereby regulates synaptic vesicle exocytosis.