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The t‐soluble NSF‐attachment protein receptor protein Syntaxin‐1a (Stx‐1a) is abundantly expressed at pre‐synaptic terminals where it plays a critical role in the exocytosis of neurotransmitter‐containing synaptic vesicles. Stx‐1a is phosphorylated by Casein kinase 2α (CK2α) at Ser14, which has been proposed to regulate the interaction of Stx‐1a and Munc‐18 to control of synaptic vesicle priming. However, the role of CK2α in synaptic vesicle dynamics remains unclear. Here, we show that CK2α over‐expression reduces evoked synaptic vesicle release. Furthermore, shRNA‐mediated knockdown of CK2α in primary hippocampal neurons strongly enhanced vesicle exocytosis from the reserve pool, with no effect on the readily releasable pool of primed vesicles. In neurons in which endogenous Stx‐1a was knocked down and replaced with a CK2α phosphorylation‐deficient mutant, Stx‐1a(D17A), vesicle exocytosis was also increased. These results reveal a previously unsuspected role of CK2α phosphorylation in specifically regulating the reserve synaptic vesicle pool, without changing the kinetics of release from the readily releasable pool., Syntaxin‐1a (Stx‐1a) is an essential component of the pre‐synaptic release machinery. Stx‐1a is phosphorylated by the kinase CK2α, however, the consequent impact on pre‐synaptic exocytosis is poorly understood. We show that reducing Stx‐1a phosphorylation by knockdown of CK2α enhances pre‐synaptic vesicle exocytosis from the reserve pool, whereas over‐expressing CK2α reduces exocytosis. Replacement of endogenous Stx‐1a with a mutant that cannot be phosphorylated by CK2α enhances exocytosis from the reserve pool. Together, our results support a model whereby phosphorylation of Stx‐1a by CK2α acts to specifically restrict the size of the reserve pool of vesicles, resulting in reduced vesicle exocytosis.

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane through distinct interactions of the C1C2B region with the plasma membrane: (i) one involving a polybasic face that is expected to yield a perpendicular orientation of Munc13-1 and hinder release; and (ii) another involving the DAG-Ca2+-PIP2-binding face that is predicted to result in a slanted orientation and facilitate release. Here, we have tested this model and investigated the role of the C1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays, and synaptic vesicle priming in primary murine hippocampal cultures. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that two distinct faces of this region control neurotransmitter release and short-term presynaptic plasticity.

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane in two different orientations mediated by distinct interactions of the C1C2B region with the plasma membrane: i) one involving a polybasic face that yields a perpendicular orientation of Munc13-1 and hinders release; and ii) another involving the DAG-Ca2+-PIP2-binding face that induces a slanted orientation and facilitates release. Here we have tested this model and investigated the role of the C1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair synaptic vesicle priming in primary murine hippocampal cultures, and Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that re-orientation of Munc13-1 controls neurotransmitter release and short-term presynaptic plasticity.

Neurotransmitter release during synaptic transmission comprises a tightly orchestrated sequence of molecular events, and Munc13-1 is a cornerstone of the fusion machinery. A forward genetic screen for defects in neurotransmitter release in Caenorhabditis elegans identified a mutation in the Munc13-1 ortholog UNC-13 that eliminated its unique and deeply conserved C-terminal module (referred to as HC2M) containing a Ca(2+)-insensitive C2 domain flanked by membrane-binding helices. The HC2M module could be functionally replaced in vivo by protein domains that localize to synaptic vesicles but not to the plasma membrane. HC2M is broadly conserved in other Unc13 family members and is required for efficient synaptic vesicle priming. We propose that the HC2M domain evolved as a vesicle/endosome adaptor and acquired synaptic vesicle specificity in the Unc13ABC protein family.

Recent structural and functional studies of the synaptic vesicle fusion machinery suggest an inhibited tripartite complex consisting of neuronal soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), synaptotagmin, and complexin prior to Ca2+-triggered synaptic vesicle fusion. We speculate that Ca2+-triggered fusion commences with the release of inhibition by Ca2+ binding to synaptotagmin C2 domains. Subsequently, fusion is assisted by SNARE complex zippering and by active membrane remodeling properties of synaptotagmin. This additional, inhibitory role of synaptotagmin may be a general principle since other recent studies suggest that Ca2+ binding to extended synaptotagmin C2 domains enables lipid transport by releasing an inhibited state of the system, and that Munc13 may nominally be in an inhibited state, which is released upon Ca2+ binding to one of its C2 domains.

Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.

RAB3A-interacting molecule (RIM) proteins are important regulators of transmitter release from active zones. At conventional chemical synapses, RIMs contribute substantially to vesicle priming and docking and their loss reduces the readily releasable pool of synaptic vesicles by up to 75%. The priming function of RIMs is mediated via the formation of a tripartite complex with Munc13 and RAB3A, which brings synaptic vesicles in close proximity to Ca(2+) channels and the fusion site and activates Munc13. We reported previously that, at mouse photoreceptor ribbon synapses, vesicle priming is Munc13 independent. In this study, we examined RIM expression, distribution, and function at male and female mouse photoreceptor ribbon synapses. We provide evidence that RIM1α and RIM1β are highly likely absent from mouse photoreceptors and that RIM2α is the major large RIM isoform present at photoreceptor ribbon synapses. We show that mouse photoreceptors predominantly express RIM2 variants that lack the interaction domain for Munc13. Loss of full-length RIM2α in a RIM2α mutant mouse only marginally perturbs photoreceptor synaptic transmission. Our findings therefore strongly argue for a priming mechanism at the photoreceptor ribbon synapse that is independent of the formation of a RIM–Munc13–RAB3A complex and thus provide further evidence for a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses in synaptic vesicle exocytosis. SIGNIFICANCE STATEMENT RAB3A-interacting molecules 1 and 2 (RIM1/2) are essential regulators of exocytosis. At conventional chemical synapses, their function involves Ca(2+) channel clustering and synaptic vesicle priming and docking through interactions with Munc13 and RAB3A, respectively. Examining wild-type and RIM2 mutant mice, we show here that the sensory photoreceptor ribbon synapses most likely lack RIM1 and predominantly express RIM2 variants that lack the interaction domain for Munc13. Our findings demonstrate that the photoreceptor-specific RIM variants are not essential for synaptic vesicle priming at photoreceptor ribbon synapses, which represents a fundamental difference between photoreceptor ribbon synapses and conventional chemical synapses with respect to synaptic vesicle priming mechanisms.

Munc13 proteins are essential regulators of neurotransmitter release at nerve cell synapses. They mediate the priming step that renders synaptic vesicles fusion-competent, and their genetic elimination causes a complete block of synaptic transmission. Here we have described a patient displaying a disorder characterized by a dyskinetic movement disorder, developmental delay, and autism. Using whole-exome sequencing, we have shown that this condition is associated with a rare, de novo Pro814Leu variant in the major human Munc13 paralog UNC13A (also known as Munc13-1). Electrophysiological studies in murine neuronal cultures and functional analyses in Caenorhabditis elegans revealed that the UNC13A variant causes a distinct dominant gain of function that is characterized by increased fusion propensity of synaptic vesicles, which leads to increased initial synaptic vesicle release probability and abnormal short-term synaptic plasticity. Our study underscores the critical importance of fine-tuned presynaptic control in normal brain function. Further, it adds the neuronal Munc13 proteins and the synaptic vesicle priming process that they control to the known etiological mechanisms of psychiatric and neurological synaptopathies.

Neurotransmitter release requires formation of trans-SNARE complexes between the synaptic vesicle and plasma membranes, which likely underlies synaptic vesicle priming to a release-ready state. It is unknown whether Munc18-1, Munc13-1, complexin-1 and synaptotagmin-1 are important for priming because they mediate trans-SNARE complex assembly and/or because they prevent trans-SNARE complex disassembly by NSF-αSNAP, which can lead to de-priming. Here we show that trans-SNARE complex formation in the presence of NSF-αSNAP requires both Munc18-1 and Munc13-1, as proposed previously, and is facilitated by synaptotagmin-1. Our data also show that Munc18-1, Munc13-1, complexin-1 and likely synaptotagmin-1 contribute to maintaining assembled trans-SNARE complexes in the presence of NSF-αSNAP. We propose a model whereby Munc18-1 and Munc13-1 are critical not only for mediating vesicle priming but also for precluding de-priming by preventing trans-SNARE complex disassembly; in this model, complexin-1 also impairs de-priming, while synaptotagmin-1 may assist in priming and hinder de-priming.

RIM1α-deficient synapses show structural defects in presynaptic vesicle distribution and tethering to the active zone that can be reversed by proteasome inhibition., Synaptic vesicles are embedded in a complex filamentous network at the presynaptic terminal. Before fusion, vesicles are linked to the active zone (AZ) by short filaments (tethers). The identity of the molecules that form and regulate tethers remains unknown, but Rab3-interacting molecule (RIM) is a prominent candidate, given its central role in AZ organization. In this paper, we analyzed presynaptic architecture of RIM1α knockout (KO) mice by cryo–electron tomography. In stark contrast to previous work on dehydrated, chemically fixed samples, our data show significant alterations in vesicle distribution and AZ tethering that could provide a structural basis for the functional deficits of RIM1α KO synapses. Proteasome inhibition reversed these structural defects, suggesting a functional recovery confirmed by electrophysiological recordings. Altogether, our results not only point to the ubiquitin–proteasome system as an important regulator of presynaptic architecture and function but also show that the tethering machinery plays a critical role in exocytosis, converging into a structural model of synaptic vesicle priming by RIM1α.

Activity-dependent changes in presynaptic function represent a critical mechanism by which synaptic strength is controlled. However, how changes in synaptic activity couple to presynaptic components to control synaptic vesicle release and recycling are poorly understood. Sphingosine kinase (SphK) is a sphingolipid metabolic enzyme whose activity-dependent recruitment to membrane regions within presynaptic terminals promotes neurotransmitter release. Here, we show that synaptic recruitment of SPHK-1, the SphK ortholog inCaenorhabditis elegans, is mediated by presynaptic calcium influx. Quantitative fluorescence imaging of live presynaptic terminals reveals that blocking presynaptic calcium influx reduces synaptic SPHK-1 abundance whereas increasing calcium influx increases SPHK-1 synaptic abundance. CALM-1, the calcium and integrin binding protein ortholog, colocalizes with SPHK-1 at release sites and regulates muscarinic-mediated synaptic SPHK-1 recruitment. We identify two additional sphingolipid metabolic enzymes that are concentrated at presynaptic terminals, and mutants lacking one of these, HYL-1/ceramide synthase, have defects in synaptic transmission and in synaptic vesicle cycling. Finally, we show that SPHK-1 activity is required for the recruitment of the priming protein UNC-13/Munc13 to presynaptic terminals following activation by muscarinic signaling. These findings suggest that calcium-dependent regulation of local S1P metabolism at synapses may be an important mechanism by which synaptic vesicle priming factors are recruited to release sites to promote synaptic transmission.

In a presynaptic nerve terminal, synaptic strength is determined by the pool of readily releasable vesicles (RRP) and the probability of release (P) of each RRP vesicle. These parameters are controlled at the active zone and vary across synapses, but how such synapse specific control is achieved is not understood. ELKS proteins are enriched at vertebrate active zones and enhance P at inhibitory hippocampal synapses, but ELKS functions at excitatory synapses are not known. Studying conditional knockout mice for ELKS, we find that ELKS enhances the RRP at excitatory synapses without affecting P. Surprisingly, ELKS C-terminal sequences, which interact with RIM, are dispensable for RRP enhancement. Instead, the N-terminal ELKS coiled-coil domains that bind to Liprin-α and Bassoon are necessary to control RRP. Thus, ELKS removal has differential, synapse-specific effects on RRP and P, and our findings establish important roles for ELKS N-terminal domains in synaptic vesicle priming. DOI: http://dx.doi.org/10.7554/eLife.14862.001, eLife digest Nerve cells in the brain communicate with one another at connections known as synapses: one nerve cell releases signaling molecules called neurotransmitters into the synapse, which are then sensed by the second cell. For the brain to work correctly, it is important that the nerve cells control when and how much neurotransmitter they release. Nerve cells package neurotransmitters into small packets called vesicles. These vesicles can be released at the so-called active zones of each synapse, though only a small subset of vesicles at a synapse are releasable. Many proteins at the active zone control the release of vesicles to influence how nerve cells communicate with each another. ELKS is one of the proteins found at the active zones of nerve cells that release either of the two most common neurotransmitters in the brain: glutamate and GABA. Held et al. have now found that the ELKS protein affects the release of these two neurotransmitters in different ways in the two types of nerve cells. The experiments showed that the number of releasable neurotransmitter-filled vesicles was lower in mouse nerve cells that release glutamate when the genes for the ELKS proteins were deleted in these cells. When the ELKS genes were deleted in the nerve cells that release GABA, the number of releasable vesicles remained the same, though the vesicles were less likely to be released. The fact that removing ELKS has different effects at these two types of synapses suggests that the active zone is not the same at all synapses. Furthermore, these results imply that ELKS is capable of fine-tuning the communication between nerve cells. Future experiments will address how glutamate- and GABA-releasing active zones differ at the molecular and structural levels. Ultimately, this will lead to a better understanding of how information is processed in the brain. DOI: http://dx.doi.org/10.7554/eLife.14862.002

Current models of synaptic vesicle trafficking implicate a core complex of proteins comprised of N -ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs in synaptic vesicle fusion and neurotransmitter release. Despite this progress, major challenges remain in establishing the in vivo functions of these proteins and their roles in determining the physiological properties of synapses. The present study employs glutamatergic adult neuromuscular synapses of Drosophila , which exhibit conserved properties of short-term synaptic plasticity with respect to mammalian glutamatergic synapses, to address these issues through genetic analysis. Our findings establish an in vivo role for SNAP-25 in synaptic vesicle priming, and support a zippering model of SNARE function in this process. Moreover, these studies define the contribution of SNAP-25-dependent vesicle priming to the detailed properties of short-term depression elicited by paired-pulse (PP) and train stimulation. In contrast, NSF is shown here not to be required for WT PP depression, but to be critical for maintaining neurotransmitter release during sustained stimulation. In keeping with this role, disruption of NSF function results in activity-dependent redistribution of the t-SNARE proteins, SYNTAXIN and SNAP-25, away from neurotransmitter release sites (active zones). These findings support a role for NSF in replenishing active zone t-SNAREs for subsequent vesicle priming, and provide new insight into the spatial organization of SNARE protein cycling during synaptic activity. Together, the results reported here establish in vivo contributions of SNAP-25 and NSF to synaptic vesicle trafficking and define molecular mechanisms determining conserved functional properties of short-term depression.

At a synapse, presynaptic terminals form a specialized area of the plasma membrane called the active zone that mediates neurotransmitter release. RIM1alpha is a multidomain protein that constitutes a central component of the active zone by binding to other active zone proteins such as Munc13 s, alpha-liprins, and ELKS, and to synaptic vesicle proteins such as Rab3 and synaptotagmin-1. In mice, knockout of RIM1alpha significantly impairs synaptic vesicle priming and presynaptic long-term plasticity, but is not lethal. We now find that the RIM1 gene encodes a second, previously unknown RIM1 isoform called RIM1beta that is upregulated in RIM1alpha knock-out mice. RIM1beta is identical to RIM1alpha except for the N terminus where RIM1beta lacks the N-terminal Rab3-binding sequence of RIM1alpha. Using newly generated knock-out mice lacking both RIM1alpha and RIM1beta, we demonstrate that different from the deletion of only RIM1alpha, deletion of both RIM1alpha and RIM1beta severely impairs mouse survival. Electrophysiological analyses show that the RIM1alphabeta deletion abolishes long-term presynaptic plasticity, as does RIM1alpha deletion alone. In contrast, the impairment in synaptic strength and short-term synaptic plasticity that is caused by the RIM1alpha deletion is aggravated by the deletion of both RIM1alpha and RIM1beta. Thus, our data indicate that the RIM1 gene encodes two different isoforms that perform overlapping but distinct functions in neurotransmitter release.

SummaryBefore transmitter-filled synaptic vesicles can fuse with the plasma membrane upon stimulation they have to be primed to fusion competence. The regulation of this priming process controls the strength and plasticity of synaptic transmission between neurons, which in turn determines many complex brain functions. We show that CAPS-1 and CAPS-2 are essential components of the synaptic vesicle priming machinery. CAPS-deficient neurons contain no or very few fusion competent synaptic vesicles, which causes a selective impairment of fast phasic transmitter release. Increases in the intracellular Ca2+ levels can transiently revert this defect. Our findings demonstrate that CAPS proteins generate and maintain a highly fusion competent synaptic vesicle pool that supports phasic Ca2+ triggered release of transmitters.

Neuronal communication underlies all aspects of brain function, including learning, memory, and consciousness. How neurons communicate is controlled by both the formation of neuronal connections during neural development and the regulation of neuronal activity in the adult brain. Rho GTPases have a well-known role in neuronal development, and recent studies published in Genes & Development (Steven et al. 2005; McMullan et al. 2006) have demonstrated that they also regulate neuronal activity in the adult brain—at least in Caenorhabditis elegans. Rho in C. elegans acts as part of a network of Gαq pathways that increase neuronal activity by regulating both production and destruction of the second messenger diacylglycerol (DAG), which is a regulator of synaptic vesicle release. In this issue of Genes & Development, Williams et al. (2007) demonstrate that Gαq acts via the UNC-73RhoGEF to increase Rho activity in neurons, and thus increase levels of DAG. The targets of DAG are known and, in one case, a pathway stretching from binding of ligand on the cell surface to changes in synaptic vesicle priming has been mapped out.

International audience; V-ATPases are multimeric enzymes made of two sectors, a V1 catalytic domain and a V0 membrane domain. They accumulate protons in various intracellular organelles. Acidification of synaptic vesicles by V-ATPase energizes the accumulation of neurotransmitters in these storage organelles and is therefore required for efficient synaptic transmission. In addition to this well-accepted role, functional studies have unraveled additional hidden roles of V0 in neurotransmitter exocytosis that are independent of the transport of protons. V0 interacts with SNAREs and calmodulin, and perturbing these interactions affects neurotransmitter release. Here, we discuss these data in relation with previous results obtained in reconstituted membranes and on yeast vacuole fusion. We propose that V0 could be a sensor of intra-vesicular pH that controls the exocytotic machinery, probably regulating SNARE complex assembly during the synaptic vesicle priming step, and that, during the membrane fusion step, V0 might favor lipid mixing and fusion pore stability.

SummaryMunc13-1 is a presynaptic protein that is essential for synaptic vesicle priming. Deletion of Munc13-1/unc13 causes total arrest of synaptic transmission due to a complete loss of fusion-competent synaptic vesicles. The requirement of Munc13-1 for large dense-core vesicles (LDCVs), however, has not been established. In the present study, we use Munc13-1 knockout (KO) and diacylglycerol (DAG) binding-deficient Munc13-1H567K mutant knockin (KI) mice to determine the role of Munc13-1 in the secretion of insulin-containing LDCVs from primary cultured pancreatic β cells. We show that Munc13-1 is required for the sustained insulin release upon prolonged stimulation. The sustained release involves signaling of DAG second messenger, since it is also reduced in KI mice. Insulin secretion in response to glucose stimulation is characterized by a biphasic time course. Our data show that Munc13-1 plays an essential role in the development of the second phase of insulin secretion by priming insulin-containing LDCVs.

SummaryNeurotransmitter secretion at synapses is controlled by several processes—morphological docking of vesicles at release sites, priming of docked vesicles to make them fusion competent, and calcium-dependent fusion of vesicles with the plasma membrane [1, 2]. In worms, flies, and mice, mutants lacking UNC-13 have defects in vesicle priming [3–5]. Current models propose that UNC-13 primes vesicles by stabilizing Syntaxin's “open” conformation by directly interacting with its amino-terminal regulatory domain [6–8]. However, the functional significance of the UNC-13/Syntaxin interaction has not been tested directly. A truncated protein containing the Munc homology domains (MHD1 and MHD2) and the carboxy-terminal C2 domain partially rescued both the behavioral and secretion defects of unc-13 mutants in C. elegans. A double mutation in MHD2 (F1000A/K1002A) disrupts the UNC-13/Syntaxin interaction. The rate of endogenous synaptic events and the amplitude of nerve-evoked excitatory post-synaptic currents (EPSCs) were both significantly reduced in UNC-13S(F1000A/K1002A). However, the pool of primed (i.e., fusion-competent) vesicles was normal. These results suggest that the UNC-13/Syntaxin interaction is conserved in C. elegans and that, contrary to current models, the UNC-13/Syntaxin interaction is required for nerve-evoked vesicle fusion rather than synaptic-vesicle priming. Thus, UNC-13 may regulate multiple steps of the synaptic-vesicle cycle.

In cultured hippocampal neurons, synaptogenesis is largely independent of synaptic transmission, while several accounts in the literature indicate that synaptogenesis at cholinergic neuromuscular junctions in mammals appears to partially depend on synaptic activity. To systematically examine the role of synaptic activity in synaptogenesis at the neuromuscular junction, we investigated neuromuscular synaptogenesis and neurotransmitter release of mice lacking all synaptic vesicle priming proteins of the Munc13 family. Munc13-deficient mice are completely paralyzed at birth and die immediately, but form specialized neuromuscular endplates that display typical synaptic features. However, the distribution, number, size, and shape of these synapses, as well as the number of motor neurons they originate from and the maturation state of muscle cells, are profoundly altered. Surprisingly, Munc13-deficient synapses exhibit significantly increased spontaneous quantal acetylcholine release, although fewer fusion-competent synaptic vesicles are present and nerve stimulation-evoked secretion is hardly elicitable and strongly reduced in magnitude. We conclude that the residual transmitter release in Munc13-deficient mice is not sufficient to sustain normal synaptogenesis at the neuromuscular junction, essentially causing morphological aberrations that are also seen upon total blockade of neuromuscular transmission in other genetic models. Our data confirm the importance of Munc13 proteins in synaptic vesicle priming at the neuromuscular junction but indicate also that priming at this synapse may differ from priming at glutamatergic and gamma-aminobutyric acid-ergic synapses and is partly Munc13 independent. Thus, non-Munc13 priming proteins exist at this synapse or vesicle priming occurs in part spontaneously: i.e., without dedicated priming proteins in the release machinery.

▪ Abstract Neurotransmitter release is mediated by exocytosis of synaptic vesicles at the presynaptic active zone of nerve terminals. To support rapid and repeated rounds of release, synaptic vesicles undergo a trafficking cycle. The focal point of the vesicle cycle is Ca2+-triggered exocytosis that is followed by different routes of endocytosis and recycling. Recycling then leads to the docking and priming of the vesicles for another round of exo- and endocytosis. Recent studies have led to a better definition than previously available of how Ca2+ triggers exocytosis and how vesicles recycle. In particular, insight into how Munc18-1 collaborates with SNARE proteins in fusion, how the vesicular Ca2+ sensor synaptotagmin 1 triggers fast release, and how the vesicular Rab3 protein regulates release by binding to the active zone proteins RIM1α and RIM2α has advanced our understanding of neurotransmitter release. The present review attempts to relate these molecular data with physiological results in an emerging view of nerve terminals as macromolecular machines.

Synaptic neurotransmitter release is restricted to active zones, where the processes of synaptic vesicle tethering, priming to fusion competence, and Ca2+-triggered fusion are taking place in a highly coordinated manner. We show that the active zone components Munc13-1, an essential vesicle priming protein, and RIM1, a Rab3 effector with a putative role in vesicle tethering, interact functionally. Disruption of this interaction causes a loss of fusion-competent synaptic vesicles, creating a phenocopy of Munc13-1-deficient neurons. RIM1 binding and vesicle priming are mediated by two distinct structural modules of Munc13-1. The Munc13-1/RIM1 interaction may create a functional link between synaptic vesicle tethering and priming, or it may regulate the priming reaction itself, thereby determining the number of fusion-competent vesicles.

We analyzed the synaptic physiology of unc-13 mutants in the nematode C. elegans. Mutants of unc-13 had normal nervous system architecture, and the densities of synapses and postsynaptic receptors were normal at the neuromuscular junction. However, the number of synaptic vesicles at neuromuscular junctions was two- to threefold greater in unc-13 mutants than in wild-type animals. Most importantly, evoked release at both GABAergic and cholinergic synapses was almost absent in unc-13 null alleles, as determined by whole-cell, voltage-clamp techniques. Although mutant synapses had morphologically docked vesicles, these vesicles were not competent for release as assayed by spontaneous release in calcium-free solution or by the application of hyperosmotic saline. These experiments support models in which UNC-13 mediates either fusion of vesicles during exocytosis or priming of vesicles for fusion.

Rab3 is a neuronal GTP-binding protein that regulates fusion of synaptic vesicles and is essential for long-term potentiation of hippocampal mossy fibre synapses1,2,3,4,5. More than thirty Rab GTP-binding proteins are known to function in diverse membrane transport pathways, although their mechanisms of action are unclear. We have now identified a putative Rab3-effector protein called Rim. Rim is composed of an amino-terminal zinc-finger motif and carboxy-terminal PDZ and C2 domains. It binds only to GTP (but not to GDP)-complexed Rab3, and interacts with no other Rab protein tested. There is enrichment of Rab3 and Rim in neurons, where they have complementary distributions. Rab3 is found only on synaptic vesicles, whereas Rim is localized to presynaptic active zones in conventional synapses, and to presynaptic ribbons in ribbon synapses. Transfection of PC12 cells with the amino-terminal domains of Rim greatly enhances regulated exocytosis in a Rab3-dependent manner. We propose that Rim serves as a Rab3-dependent regulator of synaptic-vesicle fusion by forming a GTP-dependent complex between synaptic plasma membranes and docked synaptic vesicles.

A readily releasable pool of quanta, tentatively identified with docked synaptic vesicles, has been defined by analysis of the neurotransmitter release caused by application of hypertonic solutions. The goal of this work is to determine the relationship of this functionally defined readily releasable pool to the one drawn upon by action potential-evoked release. We find that hypertonic solutions do not act through changes in intracellular calcium. Since the release produced by action potentials and hypertonic solutions varies in parallel as the pool size is changed, we conclude that the same pool is shared by both mechanisms. This conclusion, taken together with other observations in the literature, means that the synaptic release probability depends on the size of the readily releasable pool.

Reconstituting Synaptic Vesicle Fusion Membrane fusion reactions have been reconstituted in vitro, but often the reconstituted reactions have not directly mirrored the requirements for synaptic vesicle fusion in vivo. Previous work generally used only N -ethylmaleimide–sensitive factor (NSF) attachment protein SNAP receptors (SNAREs) and one or two additional components and could not explain why deletion of Munc18-1 or Munc13 abolishes neurotransmitter release completely, yielding the severe disruptions of synaptic vesicle release in knockout mouse. Ma et al. (p. 421 , published online 20 December; see the Perspective by Hughson ) now present a faithful reconstitution of synaptic vesicle fusion. Membrane fusion required Munc18-1 and Munc13 when the reconstitution experiments included all eight key components (three SNAREs, Munc18-1, Munc13, synaptotagmin-1, NSF, and α-SNAP).

The unc-13 gene in Caenorhabditis elegans is essential for normal presynaptic function and encodes a large protein with C1- and C2-domains. In protein kinase C and synaptotagmin, C1- and/or C2-domains are regulatory domains for Ca2+, phospholipids, and diacylglycerol, suggesting a role for unc-13 in regulating neurotransmitter release. To determine if a similar protein is a component of the presynaptic machinery for neurotransmitter release in vertebrates, we studied unc-13 homologues in rat. Molecular cloning revealed that three homologues of unc-13 called Munc13-1, -13-2, and -13-3 are expressed in rat brain. Munc13s are large, brain-specific proteins with divergent N termini but conserved C termini containing C1- and C2-domains. Specific antibodies demonstrated that Munc13-1 is a peripheral membrane protein that is enriched in synaptosomes and localized to plasma membranes but absent from synaptic vesicles. Our data suggest that the function of unc-13 in C. elegans is conserved in mammals and that Munc13s act as plasma membrane proteins in nerve terminals. The presence of C1- and C2-domains in these proteins and the phenotype of the C. elegans mutants raise the possibility that Munc13s may have an essential signaling role during neurotransmitter release.

The synaptic vesicle cycle at the nerve terminal consists of vesicle exocytosis with neurotransmitter release, endocytosis of empty vesicles, and regeneration of fresh vesicles. Of all cellular transport pathways, the synaptic vesicle cycle is the fastest and the most tightly regulated. A convergence of results now allows formulation of molecular models for key steps of the cycle. These developments may form the basis for a mechanistic understanding of higher neural function.

SummaryNeurotransmitter release proceeds by Ca2+-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and βSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and βSNAP expression on synaptic vesicle exocytosis, employing a new Ca2+ uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca2+ sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and βSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.

SummaryAt a synapse, the presynaptic active zone mediates synaptic vesicle exocytosis. RIM proteins are active zone scaffolding molecules that—among others—mediate vesicle priming and directly or indirectly interact with most other essential presynaptic proteins. In particular, the Zn2+ finger domain of RIMs binds to the C2A domain of the priming factor Munc13, which forms a homodimer in the absence of RIM but a heterodimer with it. Here, we show that RIMs mediate vesicle priming not by coupling Munc13 to other active zone proteins as thought but by directly activating Munc13. Specifically, we found that the isolated Zn2+ finger domain of RIMs autonomously promoted vesicle priming by binding to Munc13, thereby relieving Munc13 homodimerization. Strikingly, constitutively monomeric mutants of Munc13 rescued priming in RIM-deficient synapses, whereas wild-type Munc13 did not. Both mutant and wild-type Munc13, however, rescued priming in Munc13-deficient synapses. Thus, homodimerization of Munc13 inhibits its priming function, and RIMs activate priming by disrupting Munc13 homodimerization.

During the priming step that leaves synaptic vesicles ready for neurotransmitter release, the SNARE syntaxin-1 transitions from a closed conformation that binds Munc18-1 tightly to an open conformation within the highly stable SNARE complex. Control of this conformational transition is important for brain function, but the underlying mechanism is unknown. NMR and fluorescence experiments now show that the Munc13-1 MUN domain, which plays a central role in vesicle priming, markedly accelerates the transition from the syntaxin-1-Munc18-1 complex to the SNARE complex. This activity depends on weak interactions of the MUN domain with the syntaxin-1 SNARE motif, and probably with Munc18-1. Together with available physiological data, these results provide a defined molecular basis for synaptic vesicle priming, and they illustrate how weak protein-protein interactions can play crucial biological roles by promoting transitions between high-affinity macromolecular assemblies.

Synaptic vesicles store neurotransmitters that are released during calcium-regulated exocytosis. The specificity of neurotransmitter release requires the localization of both synaptic vesicles and calcium channels to the presynaptic active zone. Two 35-kilodalton proteins (p35 or syntaxins) were identified that interact with the synaptic vesicle protein p65 (synaptotagmin). The p35 proteins are expressed only in the nervous system, are 84 percent identical, include carboxyl-terminal membrane anchors, and are concentrated on the plasma membrane at synaptic sites. An antibody to p35 immunoprecipitated solubilized N-type calcium channels. The p35 proteins may function in docking synaptic vesicles near calcium channels at presynaptic active zones.

Neurons release neurotransmitters by calcium-dependent exocytosis of synaptic vesicles. However, the molecular steps transducing the calcium signal into membrane fusion are still an enigma. It is reported here that synaptotagmin, a highly conserved synaptic vesicle protein, binds calcium at physiological concentrations in a complex with negatively charged phospholipids. This binding is specific for calcium and involves the cytoplasmic domain of synaptotagmin. Calcium binding is dependent on the intact oligomeric structure of synaptotagmin (it is abolished by proteolytic cleavage at a single site). These results suggest that synaptotagmin acts as a cooperative calcium receptor in exocytosis.

UNC-13 and its homologs are large multidomain presynaptic proteins required for the priming of synaptic vesicles at active zones. Priming is mediated by the formation of trans -SNARE complexes of synaptobrevin, SNAP-25, and syntaxin. Here we review evidence implicating UNC-13/Munc13 in vesicle priming via interactions with SNARE proteins. Furthermore, we discuss how UNC-13/Munc13 itself might be regulated through diacglycerol binding, phosphorylation, Ca 2 + /calmodulin, ubiquitination, and diffusion/translocation to set the size of readily releasable pool and probability of release of vesicles at release sites.

Munc13 proteins are essential in neurotransmitter release, controlling the priming of synaptic vesicles to a release-ready state. The sequences responsible for this priming activity are unknown. Here we identify a large alpha-helical domain of mammalian Munc13-1 that is autonomously folded and is sufficient to rescue the total arrest in neurotransmitter release observed in hippocampal neurons lacking Munc13s.

alpha-RIMs and Munc13s are active zone proteins that control priming of synaptic vesicles to a readily releasable state, and interact with each other via their N-terminal sequences. The alpha-RIM N-terminal sequence also binds to Rab3s (small synaptic vesicle GTPases), an interaction that regulates presynaptic plasticity. We now demonstrate that alpha-RIMs contain adjacent but separate Munc13- and Rab3-binding sites, allowing formation of a tripartite Rab3/RIM/Munc13 complex. Munc13 binding is mediated by the alpha-RIM zinc-finger domain. Elucidation of the three-dimensional structure of this domain by NMR spectroscopy facilitated the design of a mutation that abolishes alpha-RIM/Munc13 binding. Selective disruption of this interaction in the calyx of Held synapse decreased the size of the readily releasable vesicle pool. Our data suggest that the ternary Rab3/RIM/Munc13 interaction approximates synaptic vesicles to the priming machinery, providing a substrate for presynaptic plasticity. The modular architecture of alpha-RIMs, with nested binding sites for Rab3 and other targets, may be a general feature of Rab effectors that share homology with the alpha-RIM N-terminal sequence.

The animation shows calcium-stimulated exocytosis of synaptic vesicles followed by clathrin-mediated vesicle recycling. Many of the molecular components that are involved in synaptic vesicle priming, docking, fusion, and endocytosis are shown. Although the process in the animation describes synaptic vesicle cycling, similar cellular processes occur for most calcium-coupled secretory and clathrin-mediated endocytotic events. The animation could be used to help illustrate the sequence of events associated with both exocytosis and endocytosis, as well as aid in understanding the processes involved in neurotransmitter release in response to nerve stimulation. [ Resource Details ]

Second messenger cascades involving G proteins and calcium are known to modulate neurotransmitter release. A prominent effect of such a cascade is the downmodulation of presynaptic calcium influx, which markedly reduces evoked neurotransmitter release. Here we show that G-protein-mediated signalling, such as through GABA (gamma-amino butyric acid) subtype B (GABA(B)) receptors, retards the recruitment of synaptic vesicles during sustained activity and after short-term depression. This retardation occurs through a lowering of cyclic AMP, which blocks the stimulatory effect of increased calcium concentration on vesicle recruitment. In this signalling pathway, cAMP (functioning through the cAMP-dependent guanine nucleotide exchange factor) and calcium/calmodulin cooperate to enhance vesicle priming. The differential modulation of the two forms of synaptic plasticity, presynaptic inhibition and calcium-dependent recovery from synaptic depression, is expected to have interesting consequences for the dynamic behaviour of neural networks.

UNC-13 is a highly conserved plasma membrane-associated synaptic protein implicated in the regulation of neurotransmitter release through the direct modulation of the SNARE exocytosis complex. Previously, we characterized the Drosophila homologue (DUNC-13) and showed it to be essential for neurotransmitter release immediately upstream of vesicular fusion (“priming”) at the neuromuscular junction (NMJ). Here, we show that the abundance of DUNC-13 in NMJ synaptic boutons is regulated downstream of GαS and Gαq pathways, which have inhibitory and facilitatory roles, respectively. Both cAMP modulation and PKA function are required for DUNC-13 synaptic up-regulation, suggesting that the cAMP pathway enhances synaptic efficacy via DUNC-13. Similarly, PLC function and DAG modulation also regulate the synaptic levels of DUNC-13, through a mechanism that appears independent of PKC. Our results suggest that proteasome-mediated protein degradation is the primary mechanism regulating DUNC-13 levels at the synapse. Both PLC- and PKA-mediated pathways appear to regulate synaptic levels of DUNC-13 through controlling the rate of proteasome-dependent DUNC-13 degradation. We conclude that the functional abundance of DUNC-13 at the synapse, a key determinant of synaptic vesicle priming and neurotransmitter release probability, is primarily regulated by the rate of protein degradation, rather than translocation or transport, convergently controlled via both cAMP and DAG signal transduction pathways. © 2003 Wiley Periodicals, Inc. J Neurobiol 54: 417–438, 2003

Synaptic vesicles must be primed to fusion competence before they can fuse with the plasma membrane in response to increased intracellular Ca 2+ levels. The presynaptic active zone protein Munc13-1 is essential for priming of glutamatergic synaptic vesicles in hippocampal neurons. However, a small subpopulation of synapses in any given glutamatergic nerve cell as well as all γ-aminobutyratergic (GABAergic) synapses are largely independent of Munc13-1. We show here that Munc13-2, the only Munc13 isoform coexpressed with Munc13-1 in hippocampus, is responsible for vesicle priming in Munc13-1 independent hippocampal synapses. Neurons lacking both Munc13-1 and Munc13-2 show neither evoked nor spontaneous release events, yet form normal numbers of synapses with typical ultrastructural features. Thus, the two Munc13 isoforms are completely redundant in GABAergic cells whereas glutamatergic neurons form two types of synapses, one of which is solely Munc13-1 dependent and lacks Munc13-2 whereas the other type employs Munc13-2 as priming factor. We conclude that Munc13-mediated vesicle priming is not a transmitter specific phenomenon but rather a general and essential feature of multiple fast neurotransmitter systems, and that synaptogenesis during development is not dependent on synaptic secretory activity.

Neurotransmitters are released by synaptic vesicle fusion at the active zone. The active zone of a synapse mediates Ca2+-triggered neurotransmitter release, and integrates presynaptic signals in regulating this release. Much is known about the structure of active zones and synaptic vesicles, but the functional relation between their components is poorly understood. Here we show that RIM1alpha, an active zone protein that was identified as a putative effector for the synaptic vesicle protein Rab3A, interacts with several active zone molecules, including Munc13-1 (ref. 6) and alpha-liprins, to form a protein scaffold in the presynaptic nerve terminal. Abolishing the expression of RIM1alpha in mice shows that RIM1alpha is essential for maintaining normal probability of neurotransmitter release, and for regulating release during short-term synaptic plasticity. These data indicate that RIM1alpha has a central function in integrating active zone proteins and synaptic vesicles into a molecular scaffold that controls neurotransmitter release.

Rim1 was previously identified as a Rab3 effector localized to the presynaptic active zone in vertebrates. Here we demonstrate that C. elegans unc-10 mutants lacking Rim are viable, but exhibit behavioral and physiological defects that are more severe than those of Rab3 mutants. Rim is localized to synaptic sites in C. elegans, but the ultrastructure of the presynaptic densities is normal in Rim mutants. Moreover, normal levels of docked synaptic vesicles were observed in mutants, suggesting that Rim is not involved in the docking process. The level of fusion competent vesicles at release sites was reduced fivefold in Rim mutants, but calcium sensitivity of release events was unchanged. Furthermore, expression of a constitutively open form of syntaxin suppressed the physiological defects of Rim mutants, suggesting Rim normally acts to regulate conformational changes in syntaxin. These data suggest Rim acts after vesicle docking likely via regulating priming.

Neurotransmitters are released by Ca(2+)-triggered exocytotic fusion of synaptic vesicles. Before fusion, vesicles dock at a specialised presynaptic plasma membrane region, the active zone, where they are primed to a fusion competent state. The nature of this priming reaction has long been enigmatic. Recent evidence demonstrates that priming is an essential and rate-limiting step in secretion from neurons and neuroendocrine cells. Members of the Unc-13 protein family, which are highly conserved during evolution and act as novel targets of the diacylglycerol second-messenger pathway, have been identified to play an essential role in this process.

Brain function requires precisely orchestrated connectivity between neurons. Establishment of these connections is believed to require signals secreted from outgrowing axons, followed by synapse formation between selected neurons. Deletion of a single protein, Munc18-1, in mice leads to a complete loss of neurotransmitter secretion from synaptic vesicles throughout development. However, this does not prevent normal brain assembly, including formation of layered structures, fiber pathways, and morphologically defined synapses. After assembly is completed, neurons undergo apoptosis, leading to widespread neurodegeneration. Thus, synaptic connectivity does not depend on neurotransmitter secretion, but its maintenance does. Neurotransmitter secretion probably functions to validate already established synaptic connections.

The UNC-13 protein family has been suggested to be critical for synaptic vesicle dynamics based on its interactions with Syntaxin, Munc-18 and Doc 2alpha. We cloned the Drosophila homolog (Dunc-13) and characterized its function using a combination of electrophysiology and ultrastructural analyses. Dunc-13 contained a C1 lipid-binding motif and two C2 calcium-binding domains, and its expression was restricted to neurons. Elimination of dunc-13 expression abolished synaptic transmission, an effect comparable only to removal of the core complex proteins Syntaxin and Synaptobrevin. Transmitter release remained impaired under elevated calcium influx or application of hyperosmotic saline. Ultrastructurally, mutant terminals accumulated docked vesicles at presynaptic release sites. We conclude that Dunc-13 is essential for a stage of neurotransmission following vesicle docking and before fusion.

Neurotransmitter release at synapses between nerve cells is mediated by calcium-triggered exocytotic fusion of synaptic vesicles. Before fusion, vesicles dock at the presynaptic release site where they mature to a fusion-competent state. Here we identify Munc13-1, a brain-specific presynaptic phorbol ester receptor, as an essential protein for synaptic vesicle maturation. We show that glutamatergic hippocampal neurons from mice lacking Munc13-1 form ultrastructurally normal synapses whose synaptic-vesicle cycle is arrested at the maturation step. Transmitter release from mutant synapses cannot be triggered by action potentials, calcium-ionophores or hypertonic sucrose solution. In contrast, release evoked by alpha-latrotoxin is indistinguishable from wild-type controls, indicating that the toxin can bypass Munc13-1-mediated vesicle maturation. A small subpopulation of synapses of any given glutamatergic neuron as well as all synapses of GABA (gamma-aminobutyric acid)-containing neurons are unaffected by Munc13-1 loss, demonstrating the existence of multiple and transmitter-specific synaptic vesicle maturation processes in synapses.

Activation of protein kinase C (PKC) by phorbol esters stimulates secretion of neurotransmitters from most presynaptic terminals. This action of PKC may be even more general, because secretion from many non-neuronal cells is also stimulated by these agents. Despite recent advances in our understanding of the molecular mechanisms underlying transmitter release, the mode of action of PKC in nerve terminals remains unclear. PKC activation might enhance the presynaptic Ca2+ signal that triggers release, for example by modulating ion channels to increase Ca2+ influx or by decreasing Ca2+ buffering or removal. Alternatively, PKC could act independently of Ca2+ entry, by increasing the number of release sites or releasable vesicles, or by making individual vesicles more sensitive to entering Ca2+. Distinguishing among these possible mechanisms has proven difficult, in part because the electrophysiological measurements typically used for such studies provide only indirect information about Ca2+ signalling and vesicle trafficking. The report by Yawo in this issue of The Journal of Physiology (Yawo, 1999) provides clear new insight into this long-standing question. Extracellular application of phorbol esters increased transmission at the classical giant synapse of the chick ciliary ganglion. Although this was due to a stimulation of transmitter release, direct optical measurements from the giant presynaptic terminal showed that phorbol ester treatment had no effect on presynaptic Ca2+ influx, or Ca2+ buffering and removal. These observations unambiguously establish the important point that the target of phorbol esters lies downstream of the presynaptic Ca2+ signal that triggers release. The work of Yawo (1999) also provides new clues about the mechanisms by which phorbol esters stimulate neurotransmitter release. Phorbol esters activate PKC by binding to the regulatory C1-domain found in most PKC isoforms. However, at least one additional presynaptic target of phorbol esters, the Munc-13 protein family, has recently been identified (Betz et al. 1998). The drug bisindolylmaleimide (BIS) can be used to distinguish between these two target proteins. BIS is a potent and selective inhibitor of the binding of ATP to PKC, yet should have no effect on Munc-13 (which apparently does not bind ATP). Yawo (1999) found that BIS eliminated the action of phorbol esters on transmitter release, suggesting that phorbol esters act via PKC at the chick synapse. In contrast, at the rat calyx of Held synapse, blockade of presynaptic phorbol ester effects requires both inhibition of PKC and disruption of the binding of Munc-13 to the vesicle protein Doc2 (T. Hori & T. Takahashi, personal communication). It therefore seems likely that both PKC and Munc-13 are targets of phorbol esters in certain presynaptic terminals. A molecular understanding of how phorbol esters modulate transmitter release will require further identification of the physiological substrates and binding interactions of PKC and Munc-13. PKC phosphorylates several cytoskeletal proteins in vitro, suggesting that phorbol esters could enhance release by recruiting additional releasable vesicles. This is consistent with previous indications that PKC activators ‘mobilize’ vesicles in Aplysia presynaptic terminals (Ghirardi et al. 1992) and increase the size of the readily releasable pool of vesicles in hippocampal neurons (Stevens & Sullivan, 1998). However, the work of Yawo (1999) indicates that this is unlikely to be the case at the chick synapse. His quantitative analysis of the kinetics of synaptic depression indicated that phorbol esters did not change the size of a readily releasable pool of synaptic vesicles, the rate of vesicle pool refilling, or the number of release sites. Thus, phorbol esters may have diverse actions in different presynaptic terminals, perhaps due to the differential distribution of PKC and Munc-13 isoforms. Yawo (1999) observed that phorbol esters cause a leftward shift in the relationship between extracellular Ca2+ and transmitter release, suggesting that phorbol esters increase the Ca2+ sensitivity of exocytosis without affecting the number of releasable vesicles or Ca2+-binding sites. This implies that phorbol esters are affecting the priming or fusion of synaptic vesicles, and among the many proteins implicated in these two processes are several possible downstream targets of phorbol esters. The SNARE complex proteins that appear to mediate synaptic vesicle priming and fusion reactions serve as PKC substrates. For example, phosphorylation of SNAP-25 by PKC decreases its interaction with syntaxin (Shimazaki et al. 1996), which might enhance Ca2+-dependent exocytosis by accelerating dissociation of the SNARE complex. Further, PKC-dependent phosphorylation of n-Sec1 (also called Munc-18) inhibits its interaction with syntaxin, which might enhance exocytosis by increasing the amount of syntaxin available to form SNARE complexes (Fujita et al. 1996). Synaptotagmin I, a Ca2+-binding protein thought to serve as a Ca2+ receptor for transmitter release, is a substrate for PKC both in vitro and in vivo (S. Hilfiker, V. A. Pieribone, C. Nordstedt, P. Greengard & A. J. Czernik, unpublished observations). PKC might enhance exocytosis by modulating the Ca2+ sensitivity of synaptotagmin, by enhancing its binding to either Ca2+ or to the SNARE complex. Finally, the phorbol ester-mediated enhancement of exocytosis might be mediated through effects on Munc-13 proteins. These proteins interact with syntaxin, as well as with the vesicle protein Doc2, a putative Ca2+-dependent regulator of neurotransmitter release (Betz et al. 1998); phorbol ester binding to Munc-13 could then affect transmitter release by altering the availability of syntaxin, or by affecting the Ca2+ sensitivity of Doc2. Thus, a phorbol ester-mediated change in the affinity and/or availability of SNARE proteins may make more vesicles available for fusion by increasing the priming of vesicles within the releasable pool, followed by an increase in the efficiency by which Ca2+-binding proteins such as synaptotagmin or Doc2 transmit the Ca2+ signal to the SNARE-based fusion machinery. In summary, the work of Yawo (1999) very elegantly emphasizes that PKC enhances the sensitivity of neurotransmitter release to entering Ca2+. Continued investigation of the molecular mechanism(s) underlying this physiological response should further clarify our understanding of synaptic vesicle fusion and its control by modulatory signalling pathways.

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.

C 2 domains are well characterized as Ca 2+/phospholipid-binding modules, but little is known about how they mediate protein–protein interactions. In neurons, a Munc13–1 C 2A-domain/RIM zinc-finger domain (ZF) heterodimer couples synaptic vesicle priming to presynaptic plasticity. We now show that the Munc13–1 C 2A domain homodimerizes, and that homodimerization competes with Munc13–1/RIM heterodimerization. X-ray diffraction studies guided by nuclear magnetic resonance (NMR) experiments reveal the crystal structures of the Munc13–1 C 2A-domain homodimer and the Munc13–1 C 2A-domain/RIM ZF heterodimer at 1.44 Å and 1.78 Å resolution, respectively. The C 2A domain adopts a β-sandwich structure with a four-stranded concave side that mediates homodimerization, leading to the formation of an eight-stranded β-barrel. In contrast, heterodimerization involves the bottom tip of the C 2A-domain β-sandwich and a C-terminal α-helical extension, which wrap around the RIM ZF domain. Our results describe the structural basis for a Munc13–1 homodimer–Munc13–1/RIM heterodimer switch that may be crucial for vesicle priming and presynaptic plasticity, uncovering at the same time an unexpected versatility of C 2 domains as protein–protein interaction modules, and illustrating the power of combining NMR spectroscopy and X-ray crystallography to study protein complexes., In neurons, a heterodimer between Munc13-1 and RIM couples synaptic vesicle priming to presynaptic plasticity. Here, a Munc13-1 homodimer that may regulate this process is identified.

Presynaptic active zones (AZs) are unique subcellular structures at neuronal synapses, which contain a network of specific proteins that control synaptic vesicle (SV) tethering, priming, and fusion. Munc13s are core AZ proteins with an essential function in SV priming. In hippocampal neurons, two different Munc13s—Munc13-1 and bMunc13-2—mediate opposite forms of presynaptic short-term plasticity and thus differentially affect neuronal network characteristics. We found that most presynapses of cortical and hippocampal neurons contain only Munc13-1, whereas ∼10% contain both Munc13-1 and bMunc13-2. Whereas the presynaptic recruitment and activation of Munc13-1 depends on Rab3-interacting proteins (RIMs), we demonstrate here that bMunc13-2 is recruited to synapses by the AZ protein ELKS1, but not ELKS2, and that this recruitment determines basal SV priming and short-term plasticity. Thus, synapse-specific interactions of different Munc13 isoforms with ELKS1 or RIMs are key determinants of the molecular and functional heterogeneity of presynaptic AZs.