Mutations in the unc-13 gene cause diverse defects in the nervous system of the nematode C. elegans. Molecular cloning of the gene and sequencing of the cDNA reveal that the product encodes a protein, 1734 amino acids in length, with a central domain with sequence similarity to the regulatory region of protein kinase C. The domain was expressed in Escherichia coli and shown to bind specifically to a phorbol ester in the presence of calcium: diacylglycerol inhibited the binding in a competitive manner (Maruyama, 1991).
The C. elegans unc-13 mutant is a member of a class of mutants that exhibit un-coordinated movement. Mutations of the unc-13 gene cause diverse defects in C. elegans, including abnormal neuronal connections and modified synaptic transmission in the nervous system. unc-13 cDNA encodes a protein (UNC-13) of 1734 amino acid residues with a predicted molecular mass of 198 kDa and sequence identity to the C1/C2 regions but not to the catalytic domain of the ubiquitously expressed protein kinase C family. To characterize the phorbol ester binding site of the UNC-13 protein, cDNA encoding the C1/C2-like regions (amino acid residues 546-940) was expressed in Escherichia coli and the 43 kDa recombinant protein was purified. Phorbol ester binding to the 43 kDa protein is zinc- and phospholipid-dependent, stereospecific and of high affinity (Kd 67 nM). UNC-13 specific antisera detects a protein of approx. 190 kDa in wild-type (N2) but not in mutant (e1019) C. elegans cell extracts. It is concluded that UNC-13 represents a novel class of phorbol ester receptor (Ahmed, 1992).
The C. elegans Unc-13 protein is a novel member of the phorbol ester receptor family having a single cysteine-rich region with high homology to those present in protein kinase C (PKC) isozymes and the chimaerins. The cysteine-rich region of Unc-13 was expressed in Escherichia coli and its interactions with phorbol esters and related analogs, its phospholipid requirements, and its inhibitor sensitivity were quantitatively analyzed. [3H]Phorbol 12,13-dibutyrate [3H]PDBu binds with high affinity to the cysteine-rich region of Unc-13. This affinity is similar to that of other single cysteine-rich regions from PKC isozymes as well as n-chimaerin. As also described for PKC isozymes and n-chimaerin, Unc-13 binds diacylglycerol with an affinity about 2 orders of magnitude weaker than [3H]PDBu. Structure-activity analysis reveals significant but modest differences between recombinant cysteine-rich regions of Unc-13 and PKC delta. In addition, Unc-13 requires slightly higher concentrations of phospholipid for reconstitution of [3H]PDBu binding. Calphostin C, a compound described as a selective inhibitor of PKC, is also able to inhibit [3H]PDBu binding to Unc-13, suggesting that this inhibitor is not able to distinguish between different classes of phorbol ester receptors. In conclusion, although these results reveal some differences in ligand and lipid cofactor sensitivities, Unc-13 represents a high affinity cellular target for the phorbol esters as well as for the lipid second messenger diacylglycerol, at least in C. elegans. The use of phorbol esters or some 'specific' antagonists of PKC does not distinguish between cellular pathways involving different PKC isozymes or novel phorbol ester receptors such as n-chimaerin or Unc-13 (Kazanietz, 1995).
The C. elegans unc-13, unc-18, and unc-64 genes are required for normal synaptic transmission. The UNC-18 protein binds to the unc-64 gene product C. elegans syntaxin (Ce syntaxin: see Drosophila Syntaxin). However, it is not clear how this protein complex is regulated. UNC-13 has been shown to transiently interact with the UNC-18-Ce syntaxin complex, resulting in rapid displacement of UNC-18 from the complex. Genetic and biochemical evidence is presented that UNC-13 contributes to the modulation of the interaction between UNC-18 and Ce syntaxin (Sassa, 1999).
Serotonin inhibits synaptic transmission at C. elegans neuromuscular junctions, and a signaling pathway is described that mediates this effect. Exogenous serotonin inhibits acetylcholine release, whereas serotonin antagonists stimulates release. The effects of serotonin on synaptic transmission are mediated by GOA-1 (a Galpha0 subunit) and DGK-1 (a diacylglycerol [DAG] kinase), both of which act in the ventral cord motor neurons. Mutants lacking goa-1 accumulate abnormally high levels of the DAG-binding protein UNC-13 at motor neuron nerve terminals, suggesting that serotonin inhibits synaptic transmission by decreasing the abundance of UNC-13 at release sites (Nurrish, 1999).
Neurotransmitter release at C. elegans neuromuscular junctions is facilitated by a presynaptic pathway composed of a Gqalpha (EGL-30), EGL-8 phospholipase Cbeta (PLCbeta), and the diacylglycerol- (DAG-) binding protein UNC-13. Activation of this pathway increases release of acetylcholine at neuromuscular junctions, whereas inactivation decreases release. Phorbol esters stimulate acetylcholine release, and this effect is blocked by a mutation that eliminates phorbol ester binding to UNC-13. Expression of a constitutively membrane-bound form of UNC-13 restores acetylcholine release to mutants lacking the egl-8 PLCbeta. Activation of this pathway with muscarinic agonists causes UNC-13 to accumulate in punctate structures in the ventral nerve cord. These results suggest that presynaptic DAG facilitates synaptic transmission and that part of this effect is mediated by UNC-13 (Lackner, 1999).
The synaptic physiology of unc-13 mutants was analyzed in the nematode C. elegans. Mutants of unc-13 have 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 is two- to three-fold greater in unc-13 mutants than in wild-type animals. Most importantly, evoked release at both GABAergic and cholinergic synapses is almost absent in unc-13 null alleles, as determined by whole-cell, voltage-clamp techniques. Although mutant synapses have morphologically docked vesicles, these vesicles are 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 (Richmond, 1999).
The priming step of synaptic vesicle exocytosis is thought to require the formation of the SNARE complex, which comprises the proteins synaptobrevin, SNAP-25 and syntaxin. In solution syntaxin adopts a default, closed configuration that is incompatible with formation of the SNARE complex. Specifically, the amino terminus of syntaxin binds the SNARE motif and occludes interactions with the other SNARE proteins. The N terminus of syntaxin also binds the presynaptic protein UNC-13. Studies in mouse, Drosophila (Aravamudan, 1999) and Caenorhabditis elegans suggest that UNC-13 functions at a post-docking step of exocytosis, most likely during synaptic vesicle priming. Therefore, UNC-13 binding to the N terminus of syntaxin may promote the open configuration of syntaxin. To test this model, mutations were engineered into C. elegans syntaxin that cause the protein to adopt the open configuration constitutively. The open form of syntaxin can bypass the requirement for UNC-13 in synaptic vesicle priming. Thus, it is likely that UNC-13 primes synaptic vesicles for fusion by promoting the open configuration of syntaxin (Richmond, 2001).
Syntaxin adopts a closed configuration in solution. However, mutations in two highly conserved amino acids (L165A, E166A) cause syntaxin to adopt a constitutively open configuration in vitro. The corresponding mutations were made in C. elegans syntaxin (L166A, E167A; open syntaxin). Similar to vertebrate open syntaxin, the mutated C. elegans protein can bind synaptobrevin but not UNC-18 in pull-down assays (Richmond, 2001).
Expression of open syntaxin can fully rescue null mutations of syntaxin. unc-64(js115) is a null allele of the gene encoding C. elegans syntaxin. Homozygotes of unc-64(js115) are completely paralysed and arrest development after hatching. This developmental defect is fully rescued by expression of wild-type syntaxin or the open form of syntaxin in null mutants. Expression of either form of syntaxin rescues the behavioural phenotypes associated with unc-64(js115). Furthermore, expression of open syntaxin does not affect neuronal development (Richmond, 2001).
UNC-13 contains several C2 domains that are calcium-binding motifs. The presence of these domains suggest that UNC-13 might be a calcium sensor for synaptic vesicle exocytosis. If UNC-13 were the sole calcium sensor, then in the absence of UNC-13 there should be no calcium-dependent release. Consistent with this hypothesis, unc-13(s69) mutants completely lack calcium-dependent evoked responses, and overexpression of wild-type syntaxin fails to rescue evoked release in the unc-13(s69) mutants. However, overexpression of open syntaxin completely restores evoked responses to wild-type levels. These normal responses to calcium in the absence of UNC-13 are consistent with the observation that overexpression of rat UNC-13 in chromaffin cells does not affect the calcium sensitivity of release. Together, these data demonstrate that UNC-13 is not the calcium sensor that triggers fusion of synaptic vesicles (Richmond, 2001).
Vesicles become fusion competent at the priming step of exocytosis. At the molecular level, priming is thought to be mediated by the formation of the SNARE complex. Overexpression of Munc13-1 in bovine chromaffin cells accelerates the forward rate constant for the priming of morphologically docked, large dense-core vesicles without affecting the rate of fusion or the calcium sensitivity of release. This stage of dense-core vesicle exocytosis coincides with the association of the SNARE proteins. Although Munc13-1 levels are normally very low in chromaffin cells, these observations suggest that UNC-13 can function to promote dense-core vesicle priming, possibly by promoting formation of the SNARE complex. The data confirm and extend these studies by demonstrating that UNC-13 promotes the priming of synaptic vesicles by acting through syntaxin. Specifically, the role of UNC-13 may be to bind the autoinhibitory domain of syntaxin to promote or maintain the open state and thus facilitate formation of the SNARE complex (Richmond, 2001).
The rescue of the Unc-13 phenotype from no evoked responses to wild-type levels of evoked responses by open syntaxin is a dramatic result; however, these animals are not completely wild type. First, body thrashing and locomotory activity of the mutant is greatly reduced compared with the wild type. Second, measures of endogenous release of synaptic vesicles in the presence of calcium is also greatly reduced compared with the wild type. There are several possible explanations for these results. One potential explanation is that the L166A and E167A mutations do not completely mimic the conformation of syntaxin when it is bound to UNC-13. Alternatively, UNC-13 may have an additional role in vesicle exocytosis, possibly to tether synaptic vesicles near calcium channels. Nevertheless, these data suggest that UNC-13 stimulates priming by opening syntaxin either through the direct interaction previously demonstrated or by acting on another protein, such as UNC-18 (Richmond, 2001).
The C. elegans UNC-13 protein and its mammalian homologs are important for normal neurotransmitter release. A set of transcripts identified from the unc-13 locus in C. elegans results from alternative splicing and apparent alternative promoters. These transcripts encode proteins that are identical in their C-terminal regions but vary in their N-terminal regions. The most abundant protein form is localized to most or all synapses. The sequence alterations, immunostaining patterns, and behavioral phenotypes of 31 independent unc-13 have been analyzed alleles. Many of these mutations are transcript-specific; their phenotypes suggest that the different UNC-13 forms have different cellular functions. A deletion allele has been isolated that is predicted to disrupt all UNC-13 protein products; animals homozygous for this null allele are able to complete embryogenesis and hatch, but they die as paralyzed first-stage larvae. Transgenic expression of the entire gene rescues the behavior of mutants fully; transgenic overexpression of one of the transcripts can partially compensate for the genetic loss of another. This finding suggests some degree of functional overlap of the different protein products (Kohn, 2000).
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, unc-13 homologs were studied in rat. Molecular cloning revealed that three homologs 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. These 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 raises the possibility that Munc13s may have an essential signaling role during neurotransmitter release (Brose, 1995).
Munc13 proteins constitute a family of three highly homologous molecules (Munc13-1, Munc13-2 and Munc13-3). With the exception of a ubiquitously expressed Munc13-2 splice variant, Munc13 proteins are brain-specific. Munc13-1 has a central priming function in synaptic vesicle exocytosis from glutamatergic synapses. In order to identify Munc13-like proteins that may regulate secretory processes in non-glutamatergic neurons or non-neuronal cells, protein profiles were developed for two Munc13-homology-domains (MHDs). MHDs are present in a wide variety of proteins, some of which have previously been implicated in membrane trafficking reactions. Taking advantage of partial sequences in the human expressed sequence tag (EST) database, a novel, ubiquitously expressed, rat protein (Munc13-4) was characterized that belongs to a subfamily of Munc13-like molecules, in which the typical Munc13-like domain structure is conserved. Munc13-4 is predominantly expressed in lung where it is localizes to goblet cells of the bronchial epithelium and to alveolar type II cells, both of which are cell types with secretory function. In the present study a group of novel proteins has been identified; some of these proteins may function in a Munc13-like manner to regulate membrane trafficking. The MHD profiles described in the present study are useful tools for the identification of Munc13-like proteins, which would otherwise have remained undetected (Koch, 2000).
Munc13-1 is one of three closely related rat homologs of C. elegans unc-13. Based on the high degree of similarity between unc-13 and Munc13 proteins, it is thought that their essential function has been conserved from C. elegans to mammals. Munc13-1 is a brain-specific peripheral membrane protein with multiple regulatory domains that may mediate diacylglycerol, phospholipid, and calcium binding. The C-terminus of Munc13-1 interacts directly with a putative coiled coil domain in the N-terminal part of syntaxin. Syntaxin is a component of the exocytotic synaptic core complex, a heterotrimeric protein complex with an essential role in transmitter release. Through this interaction, Munc13-1 binds to a subpopulation of the exocytotic core complex containing synaptobrevin, SNAP25 (synaptosomal-associated protein of 25 kDa), and syntaxin, but to no other tested syntaxin-interacting or core complex-interacting protein. The site of interaction in syntaxin is similar to the binding site for the unc-18 homolog Munc18, but different from that of all other known syntaxin interactors. These data indicate that unc-13-related proteins may indeed be involved in the mediation or regulation of synaptic vesicle exocytosis by modulating or regulating core complex formation. The similarity between the unc-13 and unc-18 phenotypes is paralleled by the coincidence of the binding sites for Munc13-1 and Munc18 in syntaxin. It is possible that the phenotype of unc-13 and unc-18 mutations is caused by the inability of the respective mutated gene products to bind to syntaxin (Betz, 1997).
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. 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 (Betz, 2001).
Munc13-1 and DOC2 have been implicated in the regulation of exocytosis. In vivo these two proteins undergo a transient phorbol ester-mediated and protein kinase C-independent interaction, resulting in the translocation of DOC2 from a vesicular localization to the plasma membrane. The translocation of DOC2 is dependent upon the DOC2 Munc interacting domain that binds specifically to Munc13-1, whereas the association of DOC2 with intracellular membranes is dependent on its C2 domains. This is the first direct in vivo demonstration of a protein-protein interaction between two presynaptic proteins and may represent a molecular basis for phorbol ester-dependent enhancement of exocytosis (Duncan, 2000).
Msec7-1, a mammalian homolog of yeast sec7p, is a specific GDP/GTP exchange factor for small G-proteins of the ARF family. Overexpression of msec7-1 in Xenopus neuromuscular junctions leads to an increase in synaptic transmitter release that is most likely caused by an increase in the pool of readily releasable vesicles. However, the molecular mechanisms by which msec7-1 is targeted to presynaptic compartments and enhances neurotransmitter release are not known. Msec7-1 is shown to interact directly with Munc13-1, a phorbol ester-dependent enhancer of neurotransmitter release that is specifically localized to presynaptic transmitter release zones. Given that Munc13-1 and msec7-1 participate in very similar presynaptic processes and because Munc13-1 is specifically targeted to presynaptic active zones, it is suggested that the msec7-1/Munc13-1 interaction serves to colocalize the two proteins at the active zone, a subcellular compartment with extremely high membrane turnover (Neeb, 1999).
Human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, the subcellular localization of hmunc13 was investigated in a transiently transfected renal cell line, opossum kidney cells. It was found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant, in which the diacylglycerol (C1) binding domain is absent, exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. It is concluded that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, these data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia (Song, 1999).
In chromaffin cells the number of large dense-core vesicles (LDCVs) that can be released by brief, intense stimuli represents only a small fraction of the 'morphologically docked' vesicles at the plasma membrane. Recently, it was shown that Munc13-1 is essential for a post-docking step of synaptic vesicle fusion. To investigate the role of Munc13-1 in LDCV exocytosis, Munc13-1 was overexpressed in chromaffin cells and secretion was stimulated by flash photolysis of caged calcium. Both components of the exocytotic burst, which represent the fusion of release-competent vesicles, are increased by a factor of three. The sustained component, which represents vesicle maturation and subsequent fusion, is increased by the same factor. The response to a second flash, however, is greatly reduced, indicating a depletion of release-competent vesicles. Since there is no apparent change in the number of docked vesicles, it is concluded that Munc13-1 acts as a priming factor by accelerating the rate constant of vesicle transfer from a pool of docked, but unprimed vesicles to a pool of release-competent, primed vesicles (Ashery, 2000).
Munc13-1 is a presynaptic phorbol ester receptor that enhances neurotransmitter release. In the present study the regional, cellular and subcellular expression patterns in rat of two novel Munc13 proteins, Munc13-2 and Munc13-3, were examined. Munc13-1 mRNA is expressed throughout the brain, whereas Munc13-2 mRNA is preferentially present in rostral brain regions, and Munc13-3 mRNA in caudal areas. The novel Munc13 proteins are enriched in synapses. Munc13-3, like Munc13-1, is concentrated in presynaptic terminals. Thus Munc13 proteins are members of a family of neuron-specific, synaptic molecules that bind to syntaxin, an essential mediator of neurotransmitter release. Munc13-2 and Munc13-3 are expressed in a complementary fashion and might act in concert with Munc13-1 to modulate neurotransmitter release (Augustin, 1999a).
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. Munc13-1, a brain-specific presynaptic phorbol ester receptor, has been identified as an essential protein for synaptic vesicle maturation. 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-containing neurons are unaffected by Munc13-1 loss, demonstrating the existence of multiple and transmitter-specific synaptic vesicle maturation processes in synapses (Augustin, 1999b).
Munc13 proteins form a family of three, primarily brain-specific phorbol ester receptors (Munc13-1/2/3) in mammals. Munc13-1 is a component of presynaptic active zones in which it acts as an essential synaptic vesicle priming protein. In contrast to Munc13-1, which is present in most neurons throughout the rat and mouse CNS, Munc13-3 is almost exclusively expressed in the cerebellum. Munc13-3 mRNA is present in granule and Purkinje cells but absent from glia cells. Munc13-3 protein is localized to the synaptic neuropil of the cerebellar molecular layer but is not found in Purkinje cell dendrites, suggesting that Munc13-3, like Munc13-1, is a presynaptic protein at parallel fiber-Purkinje cell synapses. To examine the role of Munc13-3 in cerebellar physiology, Munc13-3-deficient mutant mice were generated. Munc13-3 deletion mutants exhibit increased paired-pulse facilitation at parallel fiber-Purkinje cell synapses. In addition, mutant mice display normal spontaneous motor activity but have an impaired ability to learn complex motor tasks. These data demonstrate that Munc13-3 regulates synaptic transmission at parallel fiber-Purkinje cell synapses. It is proposed that Munc13-3 acts at a similar step of the synaptic vesicle cycle as does Munc13-1, albeit with less efficiency. In view of the present data and the well established vesicle priming function of Munc13-1, it is likely that Munc13-3-loss leads to a reduction in release probability at parallel fiber-Purkinje cell synapses by interfering with vesicle priming. This, in turn, would lead to increases in paired-pulse facilitation and could contribute to the observed deficit in motor learning (Augustin, 2001).
Ribbon synapses, for example of the retina, are specialized synapses that differ from conventional, phasically active synapses in several aspects. Ribbon synapses can tonically and yet very rapidly release neurotransmitter via synaptic vesicle exocytosis. This requires an optimization of the synaptic machinery and is at least partly due to the presence of synaptic ribbons that bind large numbers of synaptic vesicles and which are believed to participate in priming synaptic vesicles for exocytosis. This paper analyzes whether ribbon synapses of the retina employ similar priming factors, i.e. Munc13-1, as do conventional, non-ribbon containing phasically active synapses. Though present in conventional synapses of the retina Munc13-1 is completely absent from ribbon-containing synapses of the retina, both in the outer as well as in the inner plexiform layer. This indicates that ribbon synapses of the retina employ other, possibly more potent priming factors than phasically active conventional synapses (Schmitz, 2001).
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