The cornerstone of presynaptic function is the quantal release of transmitter through exocytosis. The quantal release of transmitter must be localized and rapid, repeatable at high frequencies, and amenable to upregulation or downregulation over time. Rapid release is necessary to allow communication between neurons within milliseconds, and temporal precision is necessary to preserve the timing of the electrical information encoded in an action potential. Thus, exocytosis must be precisely coordinated with the influx of Ca2+ induced by depolarization of the nerve terminal. Terminals must be capable of sustained firing and neurotransmitter release because communication between neurons often involves repeated trains of stimuli. Finally, exocytosis must be a highly regulated process to accommodate the neural plasticity that underlies learning and memory. These requirements are satisfied by the following five steps in the cycle of exocytotic release 3–6:
Docking. Synaptic vesicles release neurotransmitter only at the active zone of a nerve terminal, exactly opposite the signal transduction machinery of the postsynaptic cell. This arrangement minimizes the time required for transmitter to reach the postsynaptic cell and also enhances the precision of communication between the two cells. Because the active zone occupies a relatively restricted area along the plasma membrane of the nerve terminal, vesicles must be specifically targeted to this region through a process referred to as docking.
Priming. Morphologic analyses indicate that approximately 10 to 30 vesicles are docked at the active zone of CNS synapses. However, most of these vesicles are not capable of Ca2+-induced release and must undergo an active maturation process known as priming, which requires ATP hydrolysis. Primed vesicles release neurotransmitter within a millisecond of Ca2+ influx into the cell. The speed of this release suggests that priming may initiate a partial fusion of the synaptic vesicle with the plasma membrane. Moreover, the energy obtained from ATP hydrolysis may be used to alter the conformation of proteins involved in exocytosis.
Fusion/Exocytosis. Fusion of the synaptic vesicle with the presynaptic plasma membrane requires deformation of the two apposing membranes. Because this event is tightly linked to the influx of Ca2+, the underlying protein machinery involves a Ca2+ sensor. Fusion of the membranes allows exocytosis: extrusion of vesicle contents into the synaptic cleft.
Endocytosis. After the synaptic vesicle fuses with the plasma membrane and releases its contents, the vesicle membrane is recycled by a process of internalization known as endocytosis. As with other endocytotic processes, the internalization of synaptic vesicles is facilitated by a protein lattice that coats the internal face of the plasma membrane.
Recycling. This mechanism conserves synaptic vesicle membrane and helps to maintain a pool of release-competent vesicles. Two major models of synaptic vesicle recycling have been proposed. In the first, vesicles fuse with larger endosomes after endocytosis; subsequently, synaptic vesicles bud from these endosomes to engage in another round of exocytosis. According to the second model, synaptic vesicles bud directly from deep invaginations of the plasma membrane that are distinct from endosomes. Although synaptic vesicles recycle according to the first model in some neuroendocrine cells in culture, the latter process is believed to predominate in the CNS. A third process, which may occur intermittently at individual presynaptic terminals, is “kiss and run” exocytosis during which vesicles never completely fuse with the plasma membrane; in this model only a portion of a vesicle’s stored transmitter leaks out through a pore that opens between the lumen of the vesicle and the extracellular space.
The exocytotic cycle. The exocytotic cycle consists of several distinct steps: (1) docking of synaptic vesicles at the plasma membrane of the active zone; (2) priming, an ATP-dependent step that prepares each vesicle for release; (3) fusion/exocytosis, the release of neurotransmitter triggered by the influx of Ca2+ through voltage-gated channels in the plasma membrane; (4) endocytosis, retrieval of vesicle membrane facilitated by a protein coat (dashed line) that subsequently dissociates from each vesicle; and (5) recycling, whereby vesicles move through an endosome intermediate (not shown) or invaginations from the plasma membrane.
The entire exocytotic cycle lasts approximately 30 to 60 seconds. Exocytosis occurs within 1 ms and endocytosis occurs within 5 seconds; the other steps of the cycle occupy the remaining time. The speed of exocytosis, which is truly breathtaking, is required for the overall efficiency of information processing in the nervous system.
Proteins Involved in the Exocytotic Cycle
Until the 1990s, the mechanisms underlying vesicle release and recycling remained obscure, partly because the proteins involved in these processes were not known. During the past 20 years many of these proteins have been identified. Thus, the component of synaptic transmission that was for decades represented by a black box, namely, the triggering of presynaptic vesicle exocytosis by Ca2+ entry, has been replaced by a series of precisely defined molecular events. It should be noted that the SNARE proteins that mediate exocytosis are also localized postsynaptically, where they regulate other aspects of membrane function, which are not discussed in this chapter.
The Function of SNARES: Vesicle Docking or Fusion?
The morphologic differences between vesicles docked at the active zone of a nerve terminal and those that are undocked suggest that neurons contain mechanisms for segregating these pools and for targeting vesicles to the active zone. Initial experiments suggested that a group of proteins known as SNAREs might be involved in this process. SNAREs include three synaptic proteins that were first isolated biochemically and have since become integral to our understanding of vesicle fusion and transmitter release: synaptobrevin, also known as vesicle-associated membrane protein (VAMP); syntaxin; and synaptosomal-associated protein of 25 kDa (SNAP-25), each of which have several isoforms. Synaptobrevins span the vesicle membrane, with one transmembrane domain near the C terminus. Syntaxins are also integral membrane proteins. In contrast, SNAP-25 is anchored to the membrane by lipid groups (long-chain fatty acids) attached to central cysteine residues. At the active zone, syntaxin and SNAP-25 localize in the plasma membrane and are often known as t-SNAREs (t for target membranes). In contrast, synaptobrevin is localized primarily in the vesicular membrane and thus has been labeled a v-SNARE, or vesicle SNARE.
Three important findings led to the hypothesis that these proteins are involved in exocytosis at the presynaptic terminal. First, several bacterial toxins known to cause neurologic symptoms were proven to inhibit exocytosis and to cleave specific SNARE proteins 3–2. Likewise, the black widow spider toxin (α-latrotoxin) affects transmitter release by acting on proteins in the presynaptic nerve terminal. Second, mutation of yeast homologs of the SNAREs causes defects in membrane trafficking. These two lines of investigation implicated the SNAREs in synaptic transmission, but did not address the mechanism through which they might promote exocytosis. The third finding, which emerged from in vitro biochemical studies, was that syntaxin, synaptobrevin, and SNAP-25 spontaneously form a thermodynamically stable 1:1:1 complex. Because syntaxin and SNAP-25 are located primarily in the plasma membrane, and synaptobrevin is located primarily in the vesicle membrane, it was proposed that the association of these proteins might serve to target synaptic vesicles to the plasma membrane during the docking step of the exocytotic cycle. This postulate, known as the SNARE hypothesis, has been influential in the study of membrane trafficking in general as well as the molecular mechanisms underlying synaptic transmission.
3–2 Several Neurotoxins Influence Exocytosis
Botulism and tetanus are neurologic disorders caused by toxins from pathogenic bacteria. Botulism, which occurs after the ingestion of contaminated food, is caused by Clostridium botulinum. This strain secretes an armamentarium of seven potent neurotoxins (A, B, C1, D, E, F, and G), each of which affects exocytosis by cleaving several specific synaptic vesicle proteins, including synaptobrevin, syntaxin, and SNAP-25 (see figure and table). The toxins thereby induce an asymmetric descending paralysis due to inactivation of cranial and peripheral nerves. Because of its ability to cause paralysis in muscle, localized injections of botulinum toxin, colloquially known as “Botox,” are used therapeutically to treat blepharospasm, spasms of the muscle surrounding the tear duct, and dystonia, abnormal skeletal muscle tone, as well as for cosmetic purposes.
Tetanus is caused when Clostridium tetanus contaminates a wound. Tetanus toxin is also a protease that targets synaptic vesicle proteins (eg, synaptobrevin) and inhibits exocytosis. The result is a poisoning of both motor neurons and inhibitory neurons, which causes increased muscle tone and spasms; it often strikes the masseter muscles first, resulting in lockjaw.
Black widow spider venom contains α-latrotoxin, a 130-kd protein that induces a profound paralysis by causing a massive release and subsequent depletion of acetylcholine at the neuromuscular junction. The targets of α-latrotoxin are shown in the table.
Each of these toxins functions as a protease that breaks apart its specific protein target and thereby prevents its normal functioning. The discovery of targets for these toxins was a major breakthrough in our understanding of the molecular basis of exocytosis because it led to the identification of several proteins that play key roles in this process.
|Toxin ||Neural Target1 |
|Botulinum neurotoxin (BotT) || |
Tetanus toxin (TetT)
Subsequent evidence has indicated that SNAREs play a direct role in membrane fusion, with other molecules facilitating docking. Genetic deletion of syntaxin in Drosophila decreases exocytosis, but does not decrease the number of docked vesicles in nerve terminals. Similarly, the injection of tetanus toxin (see 3–2) or fragments of SNARE proteins into the presynaptic terminal of the giant axon of the squid leads to an increased number of docked vesicles. A decrease in the number of docked vesicles would be expected if the SNAREs were required for docking. These findings therefore suggest that the SNAREs may function predominantly at a later step in the exocytotic cycle, namely, fusion.
This hypothesis is supported by several lines of evidence. SNAREs that have been reconstituted into lipid vesicles have been assayed for vesicle fusion with the use of fluorescent dyes. Vesicles containing equimolar amounts of syntaxin and SNAP-25 fused with vesicles containing synaptobrevin, indicating that v-SNAREs and t-SNAREs alone can generate fusion between lipid bilayers. The mechanism for fusion has been suggested by in vitro experiments in which SNAREs bind to each other in parallel with the C terminus of each SNARE similarly oriented. Thus, like viral hairpin proteins, which mediate the entry of viruses into cells, the SNAREs (or SNAREpins) may change their conformation or binding properties to allow fusion between opposing bilayers 3–7 and 3–8. The favorable thermodynamics of SNARE complex formation may drive the fusion of two such bilayers over the energy barrier that normally prevents the intermingling of bilayers.
Model of the cycle whereby SNAREs induce membrane fusion and NSF dissociates the SNARE complex to initiate another round of fusion. This cycle includes the following steps: (1) SNAREs form a stable complex. v- and t-SNAREs engage partners on opposite membranes in a parallel fashion with respect to their membrane anchors. (2) SNAREs induce fusion. This fusion of the vesicle with the plasma membrane may be homologous to membrane fusion by viral hairpin proteins that assist the entry of viruses into cells. (3) NSF dissociates the SNARE complex. After binding to the SNAREs with the aid of a SNAP, NSF dissociates the complex with energy derived from adenosine triphosphate (ATP) hydrolysis into adenosine diphosphate (ADP) and free phosphate (P).
Proposed topology of SNAREpins, including the v-SNARE synaptobrevin and the t-SNAREs syntaxin and SNAP-25. Viral hairpin proteins are proposed to form similar structures to induce the fusion of apposing lipid membranes. (Reproduced with permission from Weber T, Zemelman BV, McNew JA, et al. SNAREpins: Minimal machinery for membrane fusion. Cell 1998;92:759.)
Disassembly of the SNAREs by NSF
Because association of the SNAREs appears to drive fusion of the vesicle membrane with the plasma membrane, dissociation of the SNAREs must occur before the vesicle can be reclaimed from the plasma membrane to embark on a new exocytotic cycle. N-Ethylmaleimide sensitive factor (NSF) and the soluble NSF attachment proteins (SNAPs) may perform this function (see 3–7).1 NSF is a soluble tetramer and requires SNAPs to bind to vesicle membranes and participate in exocytosis. A key finding in the search for the molecular composition of the exocytotic apparatus was the discovery that both NSF and the SNAPs also bind with high affinity to the SNAREs, including syntaxin, synaptobrevin, and SNAP-25. Remarkably, the aggregate composed of these proteins can be dissociated into its component proteins by the addition of ATP. This occurs because NSF is an ATPase; by hydrolyzing ATP, NSF generates the energy to dissociate the thermodynamically stable complex of NSF, SNAPs, and SNARES.
Disassembly is likely to occur when SNAREs occupy the same membrane. Indeed, t-SNAREs and v-SNAREs have been found to coexist on synaptic vesicle membranes. These observations suggest the following model: SNAREs induce fusion between a vesicle membrane and a plasma membrane by fastening together in parallel; to allow another round of exocytosis, NSF uses energy obtained from ATP hydrolysis to unzip the stable SNAREs 3–7 and 3–8.
Synaptotagmin: The Ca2+ Sensor for Transmitter Release
Unlike most membrane trafficking events, which appear to proceed constitutively, neurotransmitter release is triggered by Ca2+. Thus, an important goal in the study of synaptic transmission has been to identify the mechanism by which exocytotic machinery senses Ca2+ influx. Several lines of evidence suggest that the 65-kDa protein synaptotagmin is the primary mediator of this process. Investigators first proposed this possibility after the elucidation of synaptotagmin’s primary structure revealed so-called C2 domains similar to those found in protein kinase C, a Ca2+ and phospholipid-dependent protein kinase (Chapter 4). Synaptotagmin contains two C2 domains, C2A and C2B 3–9, each of which binds two Ca2+ atoms. Extensive NMR and crystallographic analyses reveal that these domains form a novel Ca2+-binding motif composed primarily of β sheets. Such binding does not induce a conformational change but is believed to induce a significant shift in electrostatic potential, or charge, in the Ca2+-binding domain, which may function to regulate the interaction of synaptotagmin with other molecules involved in vesicle fusion.
Synaptic vesicle proteins. Proteins involved in transmitter packaging and exocytosis include the proton-ATPase, which generates a proton gradient across the vesicle membrane, and vesicular transporters, which use the proton gradient to actively transport neurotransmitter into the lumen of vesicles. Synaptobrevin is involved in vesicle fusion and possibly targeting, and synaptotagmin functions as a Ca2+ sensor, binding Ca2+ ions with C2 domains encoded in the primary structure of the protein. The small G protein Rab3a, which is bound to vesicles by a lipid anchor, functions to regulate the exocytotic cycle. Synapsins associate peripherally with vesicles and may segregate them into storage pools.
Because there are many different forms of synaptotagmin, a variety of methods have been used to determine whether the predominant form, synaptotagmin I, is a Ca2+ sensor in vivo. Most convincingly, studies of synaptotagmin mutations in Drosophila, C. elegans, and mice have confirmed that synaptotagmin I plays an important role in exocytosis. Knockout and knockin mice have provided particularly useful information. In synaptotagmin I knockouts, fast Ca2+-dependent transmitter release is strongly suppressed, while a second, slower phase of transmitter release, which is also Ca2+-dependent, is not affected. This second phase of release may be mediated by other synaptotagmin isoforms. Furthermore, genetically modifying the affinity of synaptotagmin I for Ca2+ has an exactly parallel effect on the Ca2+ sensitivity of transmitter release. Other parameters of transmitter release remain unchanged in these mice, thus verifying that synaptotagmin I regulates only one step of the exocytotic cycle: fast Ca2+-triggered fusion.
Additional Proteins Involved in Neurotransmitter Release
Thus far we have only mentioned a small number of the 100 or so proteins that are involved in neurotransmitter release and its regulation. The active zone, the site at which vesicles dock and fuse with the presynaptic plasma membrane, is a highly organized macromolecular complex containing so-called “scaffolding” proteins, which, via their multiple protein–protein interaction domains, are critical for the appropriate positioning of the proteins that are key components of the vesicle cycling and fusion machinery. These include families of proteins termed RIMs (the most common variants being RIM1α and RIM2α) and Munc-13s as well as the extraordinarily large proteins bassoon and piccolo. RIMs appear to be particularly important for the localization of Ca2+ channels near the active zone so that the entry of Ca2+ occurs immediately next to the docked synaptic vesicles. Furthermore, individual presynaptic terminals express different complements of these active zone proteins and this likely contributes to significant differences in their functions.
Rab proteins are a large family of small GTP-binding proteins that are crucial in regulating many types of intracellular membrane transport (Chapter 4). Rab3a plays a particularly important role in regulating synaptic vesicle trafficking and exocytosis. It undergoes a cycle of association with and dissociation from synaptic vesicles in parallel with their exocytosis and endocytosis. It appears to function at a late stage in synaptic vesicle exocytosis after docking. Importantly, it directly interacts with RIM1α, and both Rab3a and RIM1α are required for the long-lasting, activity-dependent enhancement of transmitter release (termed presynaptic long-term potentiation [LTP]) that occurs at some presynaptic terminals following their repetitive activation. In addition to Rab3a, presynaptic terminals contain one or more of three other Rab3 isoforms termed Rab3b, c, and d, the exact functions of which are unknown.
Although we have emphasized the central function of the SNAREs, synaptobrevin, syntaxin, and SNAP-25, in mediating vesicle exocytosis, this process is highly regulated due to the participation of several additional proteins. Among the most important of these are complexins, which bind to SNARE complexes and are critical for the function of synaptotagmin I in triggering Ca2+-dependent synaptic vesicle fusion. Another important protein is Munc-18, which binds to syntaxin and thereby regulates the SNARE-dependent fusion of synaptic vesicles.
The number of docked synaptic vesicles at individual presynaptic terminals is small, on the order of 3 to 10. Thus, during prolonged bursts of activity there are mechanisms in place to replenish these vesicles from another pool of vesicles called the “reserve” pool. Synapsins are a family of proteins that bind to synaptic vesicles and tether them to the presynaptic actin cytoskeleton. They are believed to be important for regulating the trafficking of vesicles between the “reserve” pool and the “readily releasable” pool of vesicles that are docked at the presynaptic plasma membrane. When phosphorylated by protein kinases such as protein kinase A, synapsins dissociate from synaptic vesicles, a biochemical change that contributes to short-term, activity-dependent changes in neurotransmitter release.
Endocytosis and Recycling
After fusion and exocytosis, synaptic vesicle proteins are reclaimed from the plasma membrane through clathrin-mediated endocytosis 3–10. Clathrin is composed of two highly conserved subunits that self-assemble into structures called triskelions, each of which consists of three light-chain and three heavy-chain subunits. It forms a proteinaceous coat that helps shape the membrane into a bud that is subsequently pulled away from the inner surface of the plasma membrane. Clathrin is linked to membranes through heteromeric adapter proteins (APs), each of which comprises a large central domain and two projecting ears. At least four types of adapters are known to exist, and each localizes in a different subcellular organelle; the adapter at the plasma membrane is called AP-2. Each type of AP may contain cell type–specific subunits. A neuron-specific subunit known as β3b, or β-NAP, was originally isolated as the antigen responsible for a so-called paraneoplastic syndrome, or tumor-induced autoimmunity syndrome, which results in cerebellar degeneration.
The endocytotic machinery. Clathrin-mediated endocytosis allows synaptic vesicles to be retrieved from the nerve terminal. Because the protein machinery required for this process is complex, only a few of the major components are shown in this figure. Clathrin and the heteromeric AP-2 adaptor complex form the coat around the budding vesicle, which may be linked to the vesicle membrane by synaptotagmin. Dynamin is a pinchase that pinches or severs the neck of the budding vesicle. Amphiphysin, which plays a central role in the coordination of endocytosis, binds to several proteins, including dynamin, AP-2, clathrin, and the phospholipid phosphatase synaptojanin. The locations of both synaptojanin and amphiphysin may differ from those in this figure, which primarily serves to show protein–protein interactions.
To ensure that other types of proteins are not mistakenly recruited into synaptic vesicles, adapters bind to highly specific proteins at the plasma membrane. The only synaptic vesicle protein known to bind AP-2 is synaptotagmin. Thus, this protein may play a dual role: it may serve as a Ca2+ sensor during exocytosis and as a tag during endocytosis. However, different isoforms of synaptotagmin may be involved in these processes.
After endocytosis, the clathrin coat is removed from the membrane to allow further trafficking and recycling to the existing pool of synaptic vesicles. The molecular chaperone heat shock protein 70c (hsp70c) likely is involved in disassembling clathrin, with the assistance of additional tissue-specific cofactors.
An important component of the biochemical machinery underlying endocytosis is the protein dynamin. This protein cycles between phosphorylated and dephosphorylated states during the exocytotic cycle. Biochemical assays have revealed that dynamin encodes a GTPase activity that was linked to endocytosis after the discovery of a mutation in the Drosophila dynamin gene known as shibere. Flies containing this mutation were isolated more than 25 years ago and display defects in cell signaling. Electron micrographs of the nerve terminals of mutant flies revealed hundreds of vesicles attached to each plasma membrane by short ring-coated necks. Discovery of the shibere phenotype suggested that these rings, which are oligomers of shibere protein, may function to pinch off the neck of a vesicle as it buds from the plasma membrane. Recent findings strongly suggest that dynamin is intimately involved in the endocytosis of synaptic vesicles and that GTP hydrolysis helps to drive this process. Thus, as originally suggested by the shibere mutant, dynamin, with the assistance of proteins that are located in the necks of budding vesicles, appears to function as a “pinchase” to sever or pinch off the necks of coated vesicles as they bud from the plasma membrane. Dynamin-mediated endocytosis has also been implicated in the downregulation of G protein–coupled receptors in response to sustained receptor activation (Chapters 4 and 6).
After endocytosis, synaptic vesicles are recycled. As mentioned previously, the biogenesis and recycling of synaptic vesicles may involve budding from both plasma membrane invaginations and endosomal compartments discrete from the plasma membrane. Although the contribution of each pathway to vesicle formation in neurons remains unclear, some of the molecular components of the endosomal pathway have been determined with the use of model neuroendocrine cells. Like endocytosis at the plasma membrane, the budding of synaptic vesicles from endosomes involves protein coats formed from heteromeric APs. However, endosomal budding employs a different AP protein (AP-3) and does not require clathrin. Interestingly, mutations in one of the large subunits of AP-3 (δ3) may disrupt vesicle trafficking in both mice and humans. The phenotypes of mocha, a δ3 mutation in mice, include an increase in baseline electrical activity in the brain and a unique hypersynchronized 6- to 7-Hz EEG pattern. In humans, a similar mutation results in a form of Hermansky–Pudlak syndrome (HPS), which is notable for defects in storage granules. Moreover, tumor-related antibodies to a neuronally expressed β subunit of AP-3 (also known as β3b or β-NAP) induce cerebellar degeneration. Such findings suggest that defects in secretory vesicle biogenesis and its regulation may underlie other neuropsychiatric syndromes.
Complexity and Specialization
During the past two decades, many of the molecules responsible for the quantal release of neurotransmitter have been identified, and some of the protein–protein interactions underlying this complex process and its intricate regulation have been teased apart. Yet, this chapter presents a simplified view. Neurons express multiple isoforms of many of the proteins described here; for example, 16 isoforms of synaptotagmin have been isolated, and each may regulate a different aspect of synaptic transmission. Isoforms of other synaptic proteins exhibit striking cell type–specific distributions in the brain. Furthermore, almost all of the molecular understanding of the process of synaptic vesicle exocyotsis and endocytosis derives from the study of excitatory and inhibitory synapses that utilize the neurotransmitters glutamate and GABA, respectively. Whether the presynaptic release of major modulatory transmitters such as monoamines involves identical molecular machinery remains to be determined.
Given that most, if not all, neuropsychiatric disorders can be conceptualized as being due to dysfunctions in synaptic transmission, elucidating the detailed molecular mechanisms underlying this fundamental signal transduction process is likely to reveal a great deal about the pathophysiology, and perhaps even the pathogenesis, of such brain disorders. Furthermore, it is anticipated that several proteins involved in neurotransmitter release or the termination of transmitter action will be important targets of therapeutic agents. Indeed, the many isoforms of these proteins should facilitate the design of drugs that act on specific neuronal cell types or synaptic processes.