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.     

A role for V‐ATPase subunits in synaptic vesicle fusion?

SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors)-mediated exocytotic release of neurotransmitters is a key process in neuronal communication, controlled by a number of molecular interactions. A synaptic vesicle v-SNARE protein (VAMP2 or synaptobrevin), in association with two plasma membrane t-SNAREs (syntaxin 1 and SNAP25), assemble to form a protein complex that is largely accepted as the minimal membrane fusion machine. Acidification of the synaptic vesicle lumen by the large multi-subunit vacuolar proton pump (V-ATPase) is required for loading with neurotransmitters. Recent data demonstrate a direct interaction between the c-subunit of the V-ATPase and VAMP2 that appears to play a role at a late step in transmitter release. In this review, we examine evidence suggesting that the V0 membrane sector of the V-ATPase not only participates in proton pumping, but plays a second distinct role in neurosecretion, downstream of filling and close to vesicle fusion.     

The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.     

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.     

Structure, isolation, composition and reconstitution of the neuronal fusion pore

Neuronal communication is dependent on the fusion of 40-50 nm in diameter synaptic vesicles containing neurotransmitters, at the presynaptic membrane. Here we report for the first time at 5-8A resolution, the presence of 8-10 nm in diameter cup-shaped neuronal fusion pores or porosomes at the presynaptic membrane, where synaptic vesicles dock and fuse to release neurotransmitters. The structure, isolation, composition, and functional reconstitution of porosomes present at the nerve terminal are described. These findings reveal the molecular mechanism of neurotransmitter release at the presynaptic membrane of nerve terminals.     

The synaptic vesicle protein SV2 is a novel type of transmembrane transporter.

The primary function of synaptic vesicles is to store and release neurotransmitter. Synaptic vesicles are locally recycled following exocytosis and rapidly refilled with neurotransmitter from the cytoplasm by a process that depends on the electrochemical gradient generated by a proton pump. Little is known about the molecules that import neurotransmitter into synaptic vesicles. We report here that the sequence of the synaptic vesicle protein SV2 identifies this protein as a novel type of transmembrane transporter. The deduced amino acid sequence of SV2 contains two sets of six predicted transmembrane domains: the six most N-terminal transmembrane domains are highly homologous to a subfamily of transporters that includes the human glucose transporter, while the six most C-terminal domains are homologous to the plasma membrane transporters for neurotransmitters. We propose that SV2 mediates transport of neurotransmitters into synaptic vesicles.     

Negative regulation of neurotransmitter release by calpain: a possible involvement of specific SNAP-25 cleavage

Synaptic transmission is conducted by neurotransmitters released from presynaptic nerve terminals by means of Ca2+-dependent exocytosis of synaptic vesicles. Formation of a complex of soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE) proteins, including vesicle-associated membrane protein-2 (VAMP-2) in the synaptic vesicle membrane, and syntaxin 1 and synaptosomal-associated protein of 25 kDa (SNAP-25) in the plasma membrane, is essential for exocytosis. Ionomycin treatment of cultured rat cerebellar granule cells led to cleavage of SNAP-25, but not syntaxin 1 and VAMP-2, that was dependent on extracellular Ca2+. Cleavage was also induced by N-methyl-D-aspartate (NMDA) treatment, but not by depolarization. The use of various site-specific antibodies to SNAP-25, suggested that the cleavage site was in the N-terminal domain of SNAP-25. Calpain inhibitors abolished the Ca2+-dependent cleavage of SNAP-25 and markedly facilitated Ca2+-dependent glutamate (Glu) release from cerebellar granule cells. These results suggest that calpain may play an important role in the long-lasting regulation of synaptic transmission by suppressing neurotransmitter release, possibly through the proteolytic cleavage of SNAP-25.     

An exciting release on TRPM7

Following fusion, synaptic vesicles do not always release all of their neurotransmitter. According to one model, neurotransmitters bind to a charged matrix within secretory vesicles, and release requires entry of counterions. In the current issue of Neuron, Krapivinsky et al. demonstrate that TRPM7 is localized to synaptic vesicles and is required for release of the positively charged neurotransmitter acetylcholine. The results raise the possibility that TRPM7 is the enigmatic channel that supplies counterions for neurotransmitter release.     

Neurotransmitter release: fusion or 'kiss-and-run'?

The clear synaptic vesicles of neurons release their contents at the presynaptic membrane and are then quickly retrieved. However, it is unclear whether a complete cycle of exocytosis and endocytosis is always involved or whether neurotransmitter can be released by a transient interaction. Recent findings in chromaffin and mast cells suggest that exocytosis is preceded by the formation of a pore that has similar conductance properties to ion channels. The content of the secretory organelle partially escapes at this early step, but the pore can close before the vesicle fuses fully. This article looks at the evidence that quantal release of neurotransmitter from clear synaptic vesicles may occur by a similar 'kiss-and-run' mechanism.     

Tag team action at the synapse

Communication between neurons relies on chemical synapses and the release of neurotransmitters into the synaptic cleft. Neurotransmitter release is an exquisitely regulated membrane fusion event that requires the linking of an electrical nerve stimulus to Ca(2+) influx, which leads to the fusion of neurotransmitter-filled vesicles with the cell membrane. The timing of neurotransmitter release is controlled through the regulation of the soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) proteins-the core of the membrane fusion machinery. Assembly of the fusion-competent SNARE complex is regulated by several neuronal proteins, including complexin and the Ca(2+)-sensor synaptotagmin. Both complexin and synaptotagmin bind directly to SNAREs, but their mechanism of action has so far remained unclear. Recent studies revealed that synaptotagmin-Ca(2+) and complexin collaborate to regulate membrane fusion. These compelling new results provide a molecular mechanistic insight into the functions of both proteins: complexin 'clamps' the SNARE complex in a pre-fusion intermediate, which is then released by the action of Ca(2+)-bound synaptotagmin to trigger rapid fusion.     

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.     

The regulation of neurotransmitter secretion by protein kinase C

The effect of protein kinase C (PKC) on the release of neurotransmitters from a number preparations, including sympathetic nerve endings, brain slices, synaptosomes, and neuronally derived cell lines, is considered. A comparison is drawn between effects of activation of PKC on neurotransmitter release from small synaptic vesicles and large dense-cored vesicles. The enhancement of neurotransmitter release is discussed in relation to the effect of PKC on: 1. Rearrangement of the F-actin-based cytoskeleton, including the possible role of MARCKS in this process, to allow access of large dense-cored vesicles to release sites on the plasma membrane. 2. Phosphorylation of key components in the SNAP/SNARE complex associated with the docking and fusion of vesicles at site of secretion. 3. Ion channel activity, particularly Ca2+ channels.     

Synaptic Vesicle Exocytosis

Presynaptic nerve terminals release neurotransmitters by synaptic vesicle exocytosis. Membrane fusion mediating synaptic exocytosis and other intracellular membrane traffic is affected by a universal machinery that includes SNARE (for “soluble NSF-attachment protein receptor”) and SM (for “Sec1/Munc18-like”) proteins. During fusion, vesicular and target SNARE proteins assemble into an α-helical trans-SNARE complex that forces the two membranes tightly together, and SM proteins likely wrap around assembling trans-SNARE complexes to catalyze membrane fusion. After fusion, SNARE complexes are dissociated by the ATPase NSF (for “N-ethylmaleimide sensitive factor”). Fusion-competent conformations of SNARE proteins are maintained by chaperone complexes composed of CSPα, Hsc70, and SGT, and by nonenzymatically acting synuclein chaperones; dysfunction of these chaperones results in neurodegeneration. The synaptic membrane-fusion machinery is controlled by synaptotagmin, and additionally regulated by a presynaptic protein matrix (the “active zone”) that includes Munc13 and RIM proteins as central components.Synaptic vesicles are uniform organelles of ∼40 nm diameter that constitute the central organelle for neurotransmitter release. Each presynaptic nerve terminal contains hundreds of synaptic vesicles that are filled with neurotransmitters. When an action potential depolarizes the presynaptic plasma membrane, Ca²⁺-channels open, and Ca²⁺ flows into the nerve terminal to trigger the exocytosis of synaptic vesicles, thereby releasing their neurotransmitters into the synaptic cleft (Fig. 1). Ca²⁺ triggers exocytosis by binding to synaptotagmin; after exocytosis, vesicles are re-endocytosed, recycled, and refilled with neurotransmitters. Recycling can occur by multiple parallel pathways, either by fast recycling via local reuse of vesicles (“kiss-and-run” and “kiss-and-stay”), or by slower recycling via an endosomal intermediate (Fig. 1).Open in a separate windowFigure 1.The synaptic vesicle cycle. A presynaptic nerve terminal is depicted schematically as it contacts a postsynaptic neuron. The synaptic vesicle cycle consists of exocytosis (red arrows) followed by endocytosis and recycling (yellow arrows). Synaptic vesicles (green circles) are filled with neurotransmitters (NT; red dots) by active transport (neurotransmitter uptake) fueled by an electrochemical gradient established by a proton pump that acidifies the vesicle interior (vesicle acidification; green background). In preparation to synaptic exocytosis, synaptic vesicles are docked at the active zone, and primed by an ATP-dependent process that renders the vesicles competent to respond to a Ca²⁺-signal. When an action potential depolarizes the presynaptic membrane, Ca²⁺-channels open, causing a local increase in intracellular Ca²⁺ at the active zone that triggers completion of the fusion reaction. Released neurotransmitters then bind to receptors associated with the postsynaptic density (PSD). After fusion pore opening, synaptic vesicles probably recycle via three alternative pathways: local refilling with neurotransmitters without undocking (“kiss-and-stay”), local recycling with undocking (“kiss-and-run”), and full recycling of vesicles with passage through an endosomal intermediate. (Adapted from Südhof 2004.)Due to their small size, synaptic vesicles contain a limited complement of proteins that have been described in detail (Südhof 2004; Takamori et al. 2006). Although the functions of several vesicle components remain to be identified, most vesicle components participate in one of three processes: neurotransmitter uptake and storage, vesicle exocytosis, and vesicle endocytosis and recycling. In addition, it is likely that at least some vesicle proteins are involved in the biogenesis of synaptic vesicles and the maintenance of their exquisite uniformity and stability, but little is known about how vesicles are made, and what determines their size.     

Vacuolar H+ Pumping ATPases in Luminal Acidic Organelles and Extracellular Compartments: Common Rotational Mechanism and Diverse Physiological Roles

Cytoplasmic organelles with an acidic luminal pH include vacuoles, coated vesicles, lysosomes, the Golgi apparatus, and synaptic vesicles. Acidic compartments are also known outside specialized cells such as osteoclasts. The unique acidic pH is formed by V-ATPase (Vacuolar type ATPase), other ion transporters, and the buffering action of proteins inside the organelles. V-ATPase hydrolyzes ATP and transports protons inside an organelle or extracellular compartment. We have summarized recent progress on mouse V-ATPases and their varying localizations together with their mechanism emphasizing similarities with F-type ATPases.     

Tetanus toxin action: inhibition of neurotransmitter release linked to synaptobrevin proteolysis.

Tetanus toxin is a potent neurotoxin that inhibits the release of neurotransmitters from presynaptic nerve endings. The mature toxin is composed of a heavy and a light chain that are linked via a disulfide bridge. After entry of tetanus toxin into the cytoplasm, the released light chain causes block of neurotransmitter release. Recent evidence suggests that the L-chain may act as a metalloendoprotease. Here we demonstrate that blockade of neurotransmission by tetanus toxin in isolated nerve terminals is associated with a selective proteolysis of synaptobrevin, an integral membrane protein of synaptic vesicles. No other proteins appear to be affected by tetanus toxin. In addition, recombinant light chain selectively cleaves synaptobrevin when incubated with purified synaptic vesicles. Our data suggest that cleavage of synaptobrevin is the molecular mechanism of tetanus toxin action.     

ATP-dependent fusion of synaptic vesicles and synaptosomal plasma membrane in a cell-free system

The final step in exocytosis is the fusion of synaptic vesicle membrane with the synaptosomal plasma membrane, leading to the release of the neurotransmitters. We have reconstituted this fusion event in vitro, using isolated synaptic vesicles and synaptosomal plasma membranes from the bovine brain. The membranes of synaptic vesicles were loaded with the lipid--soluble fluorescent probe octadecylrhodamine B at the concentration that resulted in self-quenching of its fluorescence. The vesicles were then incubated with synaptosomal plasma membranes at 37 degrees C and fusion was measured through the dilution-dependent de-quenching of the fluorescence of the probe. Synaptic vesicles by themselves did not fused with plasma membrane, only addition of ATP induced the fusion. W-7 and trifluoroperasine, the drugs reported to inhibit calmodulin-dependent events, were effective inhibitors of the ATP-induced fusion synaptic vesicles and synaptosomal plasma membranes. Our results indicate that the membrane fusion in the nerve terminals during exocytosis may be under direct control of calmodulin-dependent protein phosphorylation.     

The Cytoplasmic Domain of Rat Synaptotagmin I Enhances Synaptic Transmission

Synaptotagmin, an integral membrane protein of synaptic vesicles, functions as a calcium sensor in the temporal control of neurotransmitter release. Although synaptotagmin facilitates lipid membrane fusion in biochemical experiments, overexpression of synaptotagmin inhibits neurotransmission. A facilitatory effect of synaptotagmin on synaptic transmission was never observed. To determine whether synaptotagmin may accelerate synaptic transmission in vivo, we injected the cytoplasmic domain of rat synaptotagmin I (CD-syt) into crayfish motor axons and tested the effect of CD-syt on synaptic response. We confirmed that CD-syt accelerates neuromuscular transmission. The injected preparation had larger synaptic potentials with shorter rise time. Experiments with varying calcium concentrations showed that CD-syt increased the maximum synaptic response of the neuromuscular synapses. Further tests on short-term plasticity of neuromuscular synapses revealed that CD-syt increases the release probability of the release-ready vesicles.     

Amino acid neurotransmission: Dynamics of vesicular uptake

Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg²⁺-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, β-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that “quanta” in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events. Special issue dedicated to Dr. Herman Bachelard.     

Vesicle dynamics: how synaptic proteins regulate different modes of neurotransmission

Central synapses operate neurotransmission in several modes: synchronous/fast neurotransmission (neurotransmitters release is tightly coupled to action potentials and fast), asynchronous neurotransmission (neurotransmitter release is slower and longer lasting), and spontaneous neurotransmission (where small amounts of neurotransmitter are released without being evoked by an action potential). A substantial body of evidence from the past two decades suggests that seemingly identical synaptic vesicles possess distinct propensities to fuse, thus selectively serving different modes of neurotransmission. In efforts to better understand the mechanism(s) underlying the different modes of synaptic transmission, many research groups found that synaptic vesicles used in different modes of neurotransmission differ by a number of synaptic proteins. Synchronous transmission with higher temporal fidelity to stimulation seems to require synaptotagmin 1 and complexin for its Ca²⁺ sensitivity, RIM proteins for closer location of synaptic vesicles (SV) to the voltage operated calcium channels (VGCC), and dynamin for SV retrieval. Asynchronous release does not seem to require functional synaptotagmin 1 as a calcium sensor or complexins, but the activity of dynamin is indispensible for its maintenance. On the other hand, the control of spontaneous neurotransmission remains less clear as deleting a number of essential synaptic proteins does not abolish this type of synaptic vesicle fusion. VGCC distance from the SV seems to have little control on spontaneous transmission, while there is an involvement of functional synaptic proteins including synaptotagmins and complexin. Recently, presynaptic deficits have been proposed to contribute to a number of pathological conditions including cognitive and mental disorders. In this review, we evaluate recent advances in understanding the regulatory mechanisms of synaptic vesicle dynamics and in understanding how different molecular substrates maintain selective modes of neurotransmission. We also highlight the implications of these studies in understanding pathological conditions.