synaptotagmin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - Synaptotagmin 1

Synonyms - synaptotagmin I

Cytological map position - 23A6--B1

Function - calcium sensor in synaptic vesicle fusion

Keywords - synaptic vesicle exocytosis, neuromuscular junction

Symbol - Syt1

FlyBase ID: FBgn0004242

Genetic map position -

Classification - synaptotagmin homolog

Cellular location - membrane

NCBI links: Precomputed BLAST | Entrez Gene | UniGene |
Recent literature
Lee, J. and Littleton, J. T. (2015). Transmembrane tethering of synaptotagmin to synaptic vesicles controls multiple modes of neurotransmitter release. Proc Natl Acad Sci U S A 112: 3793-3798. PubMed ID: 25775572
Synaptotagmin 1 (Syt1) is a synaptic vesicle integral membrane protein that regulates neurotransmitter release by activating fast synchronous fusion and suppressing slower asynchronous release. The cytoplasmic C2 domains of Syt1 interact with SNAREs and plasma membrane phospholipids in a Ca(2+)-dependent manner and can substitute for full-length Syt1 in in vitro membrane fusion assays. To determine whether synaptic vesicle tethering of Syt1 is required for normal fusion in vivo, this study performed a structure-function study with tethering mutants at the Drosophila larval neuromuscular junction. Transgenic animals expressing only the cytoplasmic C2 domains or full-length Syt1 tethered to the plasma membrane failed to restore synchronous synaptic vesicle fusion, and also failed to clamp spontaneous vesicle release. In addition, transgenic animals with shorter, but not those with longer, linker regions separating the C2 domains from the transmembrane segment abolished Syt1's ability to activate synchronous vesicle fusion. Similar defects were observed when C2 domain alignment was altered to C2B-C2A from the normal C2A-C2B orientation, leaving the tether itself intact. Although cytoplasmic and plasma membrane-tethered Syt1 variants could not restore synchronous release in syt1 null mutants, they were very effective in promoting fusion through the slower asynchronous pathway. As such, the subcellular localization of Syt1 within synaptic terminals is important for the temporal dynamics that underlie synchronous and asynchronous neurotransmitter release.

Synaptotagmin (Syt) is the major Ca2+ sensor for synaptic vesicle exocytosis. To characterize Ca2+-mediated synaptic vesicle fusion, Drosophila syt mutants deficient in specific interactions mediated by its two Ca2+ binding C2 domains were examined. In the absence of syt, synchronous release is abolished and a kinetically distinct delayed asynchronous release pathway is uncovered. Synapses containing only the C2A domain of synaptotagmin partially recover synchronous fusion, but have an abolished Ca2+ cooperativity. Mutants that disrupt Ca2+ sensing by the C2B domain have synchronous release with normal Ca2+ cooperativity, but with reduced release probability. These data suggest the Ca2+ cooperativity of neurotransmitter release is likely mediated through synaptotagmin-SNARE interactions, while phospholipid binding and oligomerization trigger rapid fusion with increased release probability. These results indicate that synaptotagmin is the major Ca2+ sensor for evoked release and functions to trigger synchronous fusion in response to Ca2+, while suppressing asynchronous release (Yoshihara, 2002).

Katz and colleagues established the hypothesis that Ca2+ influx into the presynaptic nerve terminal triggers neurotransmitter release (Katz, 1967). Current models for vesicle exocytosis propose that Ca2+ triggers fusion through activation of the SNARE complex. The SNARE complex (the term SNARE stands for SNAP receptors) is formed from the interaction of the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP-25. Together these proteins assemble into a four-helix bundle that is sufficient to drive vesicle fusion in in vitro reconstitution experiments. However, vesicle fusion mediated through the assembly of reconstituted SNARE proteins is slow and Ca2+ independent. This sharply contrasts with synaptic transmission, where SNARE complex assembly and subsequent vesicle fusion is rapid and triggered by Ca2+ (Yoshihara, 2002 and references therein).

The identity of the Ca2+ sensor(s) that triggers vesicle fusion is still under investigation, but many studies point toward an essential function for synaptotagmin in coupling Ca2+ to SNARE-mediated fusion. Synaptotagmins form a large family of C2 domain-containing proteins with seven members in Drosophila and 19 members in mammals (Adolfsen, 2001; Craxton, 2001). Synaptotagmin is the most abundant Ca2+ binding protein present on synaptic vesicles and accounts for 7% of total vesicle protein (Perin, 1990; Chapman, 1994). Synaptotagmin contains two well-characterized Ca2+ binding motifs known as C2 domains. The C2 domain is an abundant motif present in over 100 human proteins and was initially found to encode a Ca2+-activated lipid binding domain in protein kinase C. Biochemical studies have demonstrated numerous Ca2+-dependent interactions mediated by synaptotagmin that suggest it may couple Ca2+ influx to vesicle fusion. Specifically, synaptotagmin binds the SNARE complex and individual t-SNAREs (syntaxin and SNAP-25) in a Ca2+-stimulated manner (Chapman, 1995, 1996; Davis, 1999; Schiavo, 1997; Gerona, 2000; Kee, 1996). Synaptotagmin also binds phospholipids in a Ca2+-dependent manner through lipid interactions with both C2 domains (Brose, 1992; Chapman, 1994; Davis, 1999; Südhof, 1996; Earles, 2001; Fernandez, 2001). In addition, synaptotagmin undergoes homo-oligomerization (Chapman, 1996; Osborne, 1999; Fukuda, 2000; Sugita, 1996; Littleton, 1999) via Ca2+-dependent activation of its C2B domain (Yoshihara, 2002).

Although synaptotagmin has been demonstrated to bind Ca2+ in vitro, it is still unknown if synaptotagmin is the major Ca2+ sensor for synaptic exocytosis, and if so, how it mediates Ca2+ activation of release. Several studies have demonstrated that synaptotagmin is required for normal synaptic transmission. Knockout mice lacking synaptotagmin have greatly reduced synchronous transmitter release following nerve stimulation (Geppert, 1994). Knockin mice with a mutated synaptotagmin that has a 2-fold reduction in Ca2+-dependent phospholipid binding by the C2A domain display a 50% reduction in evoked release (Fernández-Chacón, 2001). Drosophila synaptotagmin (syt) mutants also show reduced evoked neurotransmitter release (Littleton, 1993b, 1994; DiAntonio, 1994; Broadie, 1994; Mackler, 2002). However, mutants that affect SNARE proteins also disrupt evoked release, suggesting this phenotype alone is an insufficient indicator of a specific loss of Ca2+ sensing versus defects in other stages of vesicle trafficking. The fourth order Ca2+ cooperativity of release provides the steep relationship between Ca2+ and vesicle fusion, and is the best indicator for a specific role in Ca2+ sensing. However, alterations in the Ca2+ cooperativity of release have not been conclusively demonstrated for syt mutants. Finally, the relationship between synaptotagmin's biochemical interactions and its role in secretion is largely unknown. To address these questions and determine if synaptotagmin is the major Ca2+ sensor for synaptic exocytosis, an electrophysiological analysis was undertaken of Drosophila syt mutants that disrupt distinct functions of synaptotagmin (Yoshihara, 2002).

Three syt alleles (AD1, AD3, and AD4) have been biochemically characterized, and each has defects in specific molecular interactions (DiAntonio, 1994; Littleton, 2001; Fukuda, 2000). sytAD4 is a null allele caused by an early stop codon that deletes the transmembrane and cytoplasmic domains of the protein and disrupts all of synaptotagmin's known interactions (DiAntonio, 1994). sytAD1 has a premature stop codon that deletes the C2B domain and reduces Ca2+-dependent binding of synaptotagmin to SNAREs and Ca2+-dependent oligomerization, while preserving phospholipid binding by the C2A domain (Littleton, 2001; Davis, 1999). sytAD3 encodes a Y364N change in C2B that does not abolish SNARE or phospholipid binding, but instead disrupts Ca2+-dependent conformational changes in C2B that are required for oligomerization of synaptotagmin (Littleton, 2001; Fukuda, 2000). These mutations allow the role of Ca2+-dependent phospholipid binding, Ca2+-dependent SNARE binding, and Ca2+-dependent oligomerization of synaptotagmin in vesicle fusion to be analyzed and separated. Each of these mutants alters exocytosis in a specific fashion, indicating multiple functions for synaptotagmin (Yoshihara, 2002).

These results provide convincing evidence that synaptotagmin is the major low-affinity Ca2+ sensor at synapses and integrates Ca2+ signals through several effector interactions. The results are summarized in a proposed model. The C2A domain alone can generate synchronous release with a small release probability, likely via Ca2+-dependent phospholipid binding by the C2A domain. Lipid binding may allow synaptic vesicles to intimately contact the plasma membrane and facilitate SNARE complex formation. In addition to lipid interactions, high-affinity SNARE binding by an intact C2A-C2B domain of synaptotagmin mediates the Ca2+ cooperativity of release, likely by initiating and stabilizing fusion pores formed from SNARE-synaptotagmin complexes. Cooperativity could be accounted for by multiple Ca2+ ions binding to one synaptotagmin monomer and triggering SNARE association (stoichiometric model), or by the requirement of multiple synaptotagmin-SNARE interactions per fusion event (stochastic model). Ca2+ binding by the C2B domain, which can promote oligomerization of synaptotagmin, dramatically increases release probability, but likely does not contribute to Ca2+ cooperativity. Ca2+-triggered oligomerization, which has been measured to occur on a submillisecond timescale (Davis, 1999), may be required to rapidly concentrate multiple synaptotagmin bound SNARE complexes into a single interface at the contact point of two lipid bilayers. The zippering together of multiple SNARE complexes, together with the lipid association mediated by the C2 domains, may be sufficient to account for the incredible speed with which fusion occurs in vivo in response to Ca2+ (Yoshihara, 2002).

In addition to providing strong evidence that synaptotagmin is the Ca2+ sensor for the rapid phase of synaptic vesicle fusion, these data support two kinetically and mechanistically distinct phases of release, consistent with reports from mammalian CNS synapses (Goda, 1994). Recordings from paired hippocampal neurons in culture (Goda, 1994) have revealed a fast component of release with a time constant of 5–10 ms, and a second asynchronous component with a time constant of 100–200 ms. Both of these phases of release have fourth order cooperativity and support the two-Ca2+ sensor model of exocytosis: a low-affinity Ca2+ sensor that activates the fast synchronous phase of release and a second distinct high-affinity Ca2+ sensor that triggers the slow asynchronous release mechanism. The similarities between mammalian CNS synapses and Drosophila NMJs argue that the mechanisms mediating Ca2+ cooperativity of release have been conserved across evolution. This work provides evidence that synaptotagmin is the only low-affinity Ca2+ sensor at the Drosophila NMJ and that it normally suppresses the second asynchronous phase of release, generating the high fidelity of normal synaptic transmission. In the complete absence of synaptotagmin (sytAD4), the fast component of release is completely abolished and the second asynchronous phase of release is fully uncovered. The cooperativity of the residual delayed release in the null mutant is unchanged, but reflects the cooperativity of the asynchronous release mechanism. Although an increased variability in release latency was previously observed (Broadie, 1994), the presence of two distinct release mechanisms was not previously realized and the distinction between the cooperativity of fast release versus asynchronous release was not measured. The intact cooperativity of residual release previously reported in the null mutant (Broadie, 1994) reflects the cooperativity of the asynchronous release mechanism, since no synchronous release component remains in the null. In sytAD1, both the synchronous and asynchronous phases coexist and can be kinetically separated, suggesting the AD1 mutant protein can trigger the fast phase of release (with a cooperativity of 1 instead of 4), but cannot fully suppress delayed release. The partial suppression of the asynchronous phase results in a decrease in the absolute number of delayed release events, but the population time constant for the remaining asynchronous events is unchanged. Both the high-affinity and low-affinity Ca2+ sensors that underlie these two phases of release likely impinge upon the basic SNARE fusion machinery, since mutations in syntaxin eliminate both components of release (Yoshihara, 2002).

Although this model is consistent with the data and the previous biochemical characterization of the synaptotagmin mutant proteins used in this study, the fact that other interactions also contribute to the defects observed in syt mutants cannot be ruled out. However, the finding that the cooperativity of neurotransmitter release (n) is abolished in sytAD1 mutants (from 4 to 1) provides the best evidence to date that synaptotagmin functions as the Ca2+ sensor for fast exocytosis. Several other manipulations have also been reported to mildly reduce n, but none have been shown to have as severe an effect on Ca2+ cooperativity as syt mutants. Drosophila dunce mutants elevate cAMP levels and show a mild reduction in n to 2.4. It is possible that PKA phosphorylation patterns are altered in the dunce mutant and directly affect synaptotagmin-SNARE interactions. Alternatively, cAMP-dependent facilitation, which is also mediated by Ca2+, may alter additional Ca2+ effectors of release. Experimental decreases in the levels of the v-SNARE synaptobrevin and the t-SNARE syntaxin also cause a slight reduction in the Ca2+ cooperativity of release to 2.6. The reduction in SNARE proteins may cause abnormal cooperation between SNAREs and synaptotagmin during Ca2+ influx, thus leading to subtle reductions in cooperativity (Yoshihara, 2002 and references therein).

Given that synaptotagmin has been shown to stabilize the fusion pore during dense core vesicle fusion (Wang, 2001), it is proposed that synaptotagmin-SNARE interactions rapidly trigger the opening and stabilization of the fusion pore, while preventing fusion pore openings induced by lower Ca2+ concentration via the high-affinity sensor. A role for synaptotagmin-SNARE interactions in vesicle fusion is supported by voltammetry measurements from cracked PC12 cells using dominant-negative synaptotagmin probes that prevent synaptotagmin-SNARE binding (Earles, 2001). In addition, the Ca2+-dependent interaction of synaptotagmin with SNAP-25 has been shown to be essential for the triggering of vesicle fusion in PC12 cells (Zhang, 2002). Recent work on Drosophila synaptotagmin transgenic strains harboring mutants that alter the Ca2+ binding properties of the C2A and C2B domains have been reported. Disruptions of C2A Ca2+ binding cause a 50% decrease in evoked release from heat shock-induced mutant transgenics, but do not result in a null phenotype (Robinson, 2002). However, the mutant transgenes contain intact C2B Ca2+ binding ligands, suggesting C2B phospholipid binding may substitute for defects in C2A. Fernández-Chacón (2002) showed that C2A Ca2+ ligand mutations engineered into the intact C2A-C2B recombinant protein do not cause defective Ca2+-dependent phospholipid binding of the complete protein, confirming functional redundancy for lipid binding between the two C2 domains. Similar redundant lipid binding activities in synaptotagmin have been previously reported (Earles, 2001). Mutations in C2A that prevent Ca2+-dependent phospholipid binding by the intact C2A-C2B domain cause a 50% decrease in release probability, confirming an important role for C2A in lipid binding (Fernández-Chacón, 2001). Robinson (2002) also demonstrates that synaptotagmin IV, which has defective C2A phospholipid binding, can functionally substitute for synaptotagmin I. Interestingly, synaptotagmin IV maintains normal Ca2+-dependent interactions with the t-SNARE syntaxin (Littleton, 1999), suggesting Ca2+-dependent SNARE interactions may be important for synaptotagmin IV's ability to substitute for synaptotagmin I. Disruptions of the Ca2+ binding properties of the C2B domain result in a more severe defect in synaptic transmission, but with phenotypes that are more severe than the null mutant, indicating these transgenes function as dominant-negative inhibitors of vesicle fusion (Mackler, 2002). Although it is still unclear exactly how these dominant-negative phenotypes are manifested biochemically, TEM studies indicate an abundance of docked vesicles in these mutants, suggesting that Ca2+ binding to the C2B domain has a post-docking role in release. Although both the results of this study and those of Mackler (2002) support an essential post-docking role for synaptotagmin as the fast Ca2+ sensor, it cannot be ruled out that there may be subtle defects in endocytosis and vesicle docking. TEM analysis has revealed an overall decrease in the absolute numbers of synaptic vesicles in nerve terminals of null mutants, suggesting synaptotagmin may be required to maintain a completely wild-type pool of recycling vesicles (Reist, 1998). However, the TEM phenotype was similar between sytAD3 and null mutants. Given the striking differences in physiology between the null and sytAD3, the TEM phenotype might reflect a general decrease in overall nerve terminal function that leads to less recycling, but that does not reflect a specific role for synaptotagmin in the process. Indeed, physiological measurements to explore the asynchronous release component remaining in the syt nulls suggests that robust docking and endocytosis mechanisms still exist in the absence of synaptotagmin. Release via the high-affinity Ca2+ sensor is robust in syt nulls under conditions of sustained elevated Ca2+ levels and can continue at high frequencies for hours. This indicates the pool of vesicles that are cycling in the syt null can undergo endocytosis, docking, etc. at high frequency and high fidelity, and does not support the conclusion that this population of vesicles has major defects in docking or endocytosis (Yoshihara, 2002).

In addition to a role for synaptotagmin in triggering fast vesicle fusion, it is also observed that synaptotagmin suppresses delayed release during sustained Ca2+ elevation. It is hypothesized that the positive role of synaptotagmin as the Ca2+ sensor promoting the fast release phase is mechanistically distinct from its role in clamping asynchronous fusion. The best evidence for this model is that the AD3 mutant has a completely intact suppression of delayed release, yet is still defective in synaptotagmin's positive role in promoting fusion. sytAD3 mutants also have less release evoked by salines containing high K+ or Ca2+ ionophores than the null mutant sytAD4. The data indicate that in sytAD4, where there is no promoting or clamping function of synaptotagmin remaining, there is increased release due to the unrestricted activity of the high-affinity Ca2+ sensor. In sytAD3, the suppression of asynchronous release is still intact and thus there is less release promoted through the high-affinity sensor than in sytAD4. The remaining release events in sytAD3 can be attributed to synaptotagmin's triggering of synchronous fusion, which is greatly reduced in this mutant. The data do not allow the differentiation of whether synaptotagmin's suppression of asynchronous release is a Ca2+-dependent or -independent function. Both asynchronous release and facilitation are postulated to be mediated by the high-affinity Ca2+ sensor since they share a similar Ca2+ dependence (Kamiya, 1994). The identity of the high-affinity Ca2+ sensor responsible for delayed release is unknown, but may be another member of the large synaptotagmin family or a distinct class of Ca2+ binding proteins. Though facilitation is important for short-term synaptic plasticity, asynchronous release is extremely deleterious for normal synaptic transmission. The suppression of asynchronous release by synaptotagmin is critical for providing the temporal resolution of synaptic transmission. These properties make Synaptotagmin an efficient Ca2+ sensor that generates the high signal-to-noise ratio of neurotransmitter release that is required for brain function (Yoshihara, 2002).

A synaptotagmin suppressor screen indicates SNARE binding controls the timing and Ca2+ cooperativity of vesicle fusion

The synaptic vesicle Ca2+ sensor Synaptotagmin binds Ca2+ through its two C2 domains to trigger membrane interactions. Beyond membrane insertion by the C2 domains, other requirements for Synaptotagmin activity are still being elucidated. To identify key residues within Synaptotagmin required for vesicle cycling, advantage was taken of observations that mutations in the C2B domain Ca2+-binding pocket dominantly disrupt release from invertebrates to humans. An intragenic screen was performed for suppressors of lethality induced by expression of Synaptotagmin C2B Ca2+-binding mutants in Drosophila. This screen uncovered essential residues within Synaptotagmin that suggest a structural basis for several activities required for fusion, including a C2B surface implicated in SNARE complex interaction that is required for rapid synchronization and Ca2+ cooperativity of vesicle release. Using electrophysiological, morphological and computational characterization of these mutants, a sequence is proposed of molecular interactions mediated by Synaptotagmin that promote Ca2+ activation of the synaptic vesicle fusion machinery (Guan, 2017).

The role of Syt1 role as the Ca2+ sensor for fast synaptic vesicle exocytosis has been well established, although how it triggers fusion is still being elucidated. A large number of studies have established a role for membrane penetration by the Ca2+ binding loops of the C2A and C2B domains of the protein. These interactions with negatively charged lipid bilayers are a hallmark of C2 domain function, and are thought to generate local perturbations in membrane structure that facilitate rapid vesicle fusion. Genetic approaches to specifically mutate the key Ca2+-binding residues within the C2 domains have validated the important nature of this interaction in vivo. These studies demonstrated essential roles for the C2B Ca2+ lipid binding loops, and important, though less essential roles for the C2A Ca2+ binding loops. These approaches relied on targeted mutagenesis of key residues that were first found to be important in vitro based on predicted properties derived from the structure of the protein (Guan, 2017).

Beyond Ca2+-dependent lipid binding, there is still debate about other required interactions mediated through Syt1. The unbiased genetic approach described in this study identified a set of residues that regulate Syt1 function and suggest functional requirements beyond the Ca2+ binding loops. Several key residues were identified that decorate a surface of the Syt1 C2B domain that had been poorly characterized, but recently emerged as a SNARE complex binding surface on Syt1. Remarkably, this study identified mutations in residues S332 (S279 in mammalian Syt1), R334 (R281), Y391 (Y338), E348 (E295) and A455 (A402). All five amino acids form the essential surface interaction residues that dock the C2B domain of Syt1 onto the SNARE complex, based on the recently elucidated primary Syt1-SNARE complex structure (Zhou, 2015). Mutations in the S332 and the R334 residues largely abolished the function of Syt1 in driving synchronous fusion. The R334H mutation, which is predicted to be the most essential residue for SNARE binding, failed to rescue synchronous synaptic vesicle release and prevent the increased asynchronous release that is observed in syt1 null mutants. These observations match well with mutations in SNAP-25 and Syt1 designed to disrupt this interaction at mammalian synapses. In addition, it was observed that the R334 mutation abolished the Ca2+ cooperativity of release, even though R334 does not reside near the Ca2+ binding loops of the protein. Although it is unknown what key features contribute to the Ca2+ cooperativity (n = 3 to 5) normally seen for release, the number of Ca2+ ions that bind to an individual Syt1 protein or the number of Syt1 proteins on a synaptic vesicle that contribute to fusion have all been considered as possible determinants. In addition, prior studies found defects in Ca2+ cooperativity in hypomorphic mutants of the SNARE proteins. Together with observations of the R334H mutation, these findings argue that the number of Syt1 molecules bound to SNARE complexes likely contributes to the higher-order Ca2+ cooperativity value observed for release (Guan, 2017).

Although these data indicate the SNARE binding surface of Syt1 is a critical determinant of neurotransmitter release, when this interaction might occur during the fusion process remains to be elucidated. No morphological docking defect in the R334H or S332L mutants were observed by electron microscopy, suggesting the Syt1-SNARE interaction is functionally important downstream of synaptic vesicle docking. It is unclear when full assembly of SNARE complexes occurs during the synaptic vesicle cycle. One model suggests full SNARE assembly would trigger bilayer fusion, such that docked vesicles likely contain a partially zippered SNARE complex stabilized by the SNARE-binding protein Complexin. Based on the structure of the Syt1-SNARE complex, it is possible that Syt1 could interact with a partially zippered SNARE complex that contains Complexin. This would allow the Syt1-SNARE interaction to play a role in orienting the Syt1 C2 domains near the site of future membrane interactions, serving as a scaffold for the fusion process. Beyond the defect in synchronous release, the R334H mutant also fails to clamp asynchronous release and the enhanced spontaneous release observed in syt1 null mutants. As such, a Syt1-SNARE complex interaction after docking but before fusion would allow the complex to be stabilized and primed for Syt1-membrane interactions triggered by Ca2+. Syt1 could also interact with the plasma membrane in such a scenario via its polybasic C2B stretch, which lies on the opposite side of the SNARE binding surface. This complex could reduce spontaneous and asynchronous release, with Syt1 sandwiched between the plasma membrane and the SNARE complex. Ca2+-triggered lipid binding would be predicted to rotate the Syt1 C2B domain downward and towards the membrane, facilitating full SNARE zippering and rapid fusion (Guan, 2017).

It is interesting to note that the core residues in Syt1 essential for SNARE binding are not conserved in Syt7, a homolog that has been implicated as the asynchronous Ca2+ sensor for synaptic vesicle exocytosis. Of the five key residues identified on the Syt1-SNARE binding surface (S332, R334, E348, Y391 and A455), only S332 is conserved in Syt7. More surprisingly, Syt7 contains homologous mutations identified in the screen (R334C and E348K) in its native C2B sequence that indicates it is unlikely to engage the SNARE complex in the same manner as Syt1. Analysis of differences in mammalian Syt1 and Syt7 also argue for distinct C2B-dependent properties for the two proteins. It will be interesting in future studies to determine if differential SNARE binding between Syt1 and Syt7 contributes to their distinct roles in regulating synchronous and asynchronous phases of synaptic vesicle fusion (Guan, 2017).

Beyond the data defining an evolutionarily conserved role for Syt1-SNARE interactions in synaptic vesicle fusion, the work also provides insights into how dominant-negative C2B Ca2+ binding mutations disrupt neurotransmitter release. Earlier studies in Drosophila indicated that mutating the Ca2+ binding aspartate residues that line the C2B lipid penetration loops resulted in a dominant-negative disruption of release. Expression of this DN-Syt1 in Drosophila results in pupal lethality, and formed the basis for the initial screen for intragenic suppressors. It is important to note that the intragenic suppressor screen is unlikely to have hit all the key residues required for normal Syt1 function, as this study specifically screened for mutations that block the DN effects of the mutant C2B protein as opposed to native Syt1 activity. The importance of defining how Syt1 with defective C2B Ca2+ binding disrupts release is highlighted by recent findings that indicate this effect is conserved in humans. Two multigenerational families were identified with autosomal dominant mutations in the Ca2+ binding pocket of the C2B domain of human Syt2, a Syt1 homolog enriched in the PNS. These patients display peripheral neuropathy and dysfunctional synaptic transmission at neuromuscular junctions (NMJs). From the genetic suppressor screen, it is clear that the mutant DN Syt1 protein is likely to engage and disrupt core mechanisms of Syt1 that lead to the reduction in neurotransmitter release. The screen revealed that altering SNARE binding (discussed above), C2A domain Ca2+ binding (D229N), C2B domain lipid interactions (P363S), multimerization and/or C2A-C2B domain interactions (R250H), and the polybasic C2B surface regulated Ca2+-independent lipid binding (K379E) all lead to a suppression of the dominant-negative effects on release. Each of these interactions also function during the normal synaptic vesicle cycle based on in vivo rescue experiments with the point mutants expressed in the wildtype Syt1 protein lacking the Ca2+ binding C2B mutations (Guan, 2017).

Given the positioning of the R250H mutation near the dimer surface observed in the Syt1 crystal structure, this residue was explored in more detail. Molecular modeling indicated the Syt1 dimer is likely to be stable in vivo, with dimer stability disrupted by the R250H amino acid substitution. If R250H disrupts the ability of mutant Syt1 to bind to and inactivate endogenous Syt1, that could prevent the ability of the mutant protein to interfere with release. Prior studies of Drosophila Syt1 have suggested the protein is likely to function as a multimer, consistent with a dimerization defect. An alternative model is that R250 plays a key role in stabilizing the interaction between the C2A and C2B domains, such that loss of an intramolecular C2A-C2B domain interaction blocks the DN effects on release. Single molecule experiments indicate that Syt1 in solution represents an ensemble of C2 conformations, and a subsequent modeling study suggested these conformations have tightly interacting C2 domains. Recent observations indicate that a stable C2A-C2B interface is also important for Syt1 activity both in vitro and in vivo. Molecular modeling indicates the normal C2A-C2B monomer stability of Syt1 is disrupted by R250H, consistent with a potential non-dimerization role for this residue as well. Consequentially, this mutation is likely to destabilize Syt1 conformations that are functional in vivo. However other possibilities cannot be ruled out of other possible effects of R250 on Syt1 function. The R250 residue is situated in proximity to the C2A Ca2+ binding pocket and could potentially compromise C2A lipid binding as well. Future studies will be required to differentiate which of these functions of R250 are key for Syt1 activity. In vivo, Syt1 R250 fails to rescue the amount of synchronous fusion, although the timing of release and the suppression of asynchronous events are normal. There is also a striking defect in the ability of the R250 mutation to suppress the enhanced spontaneous release and to properly modulate synaptic vesicle density. As such, R250-mediated interactions regulate both the exocytotic and endocytotic functions of Syt1 in vivo. In summary, this genetic analysis of suppressors of Syt1 C2B Ca2+ binding mutations have highlighted a key role for multiple Syt1 functions during neurotransmitter release, including an essential role for the Syt1-SNARE binding surface during the final stages of fusion (Guan, 2017).


cDNA clone length - 2180

Bases in 5' UTR - 359

Exons - 11

Bases in 3' UTR - 398


Amino Acids - 474

Structural Domains

Synaptotagmin (p65) is an abundant synaptic vesicle protein that contains two copies of a sequence that is homologous to the regulatory region of protein kinase C. Full length cDNAs encoding human and Drosophila synaptotagmins were characterized to study its structural and functional conservation in evolution. The deduced amino acid sequences for human and rat synaptotagmins show 97% identity, whereas Drosophila and rat synaptotagmins are only 57% identical but exhibit a selective conservation of the two internal repeats that are homologous to the regulatory region of protein kinase C (78% invariant residues in all three species). The two internal repeats of synaptotagmin are only slightly more homologous to each other than to protein kinase C, and the differences between the repeats are conserved in evolution, suggesting that they might not be functionally equivalent. The cytoplasmic domains of human and Drosophila synaptotagmins produced as recombinant proteins in Escherichia coli specifically bind phosphatidylserine similar to rat synaptotagmin. They also hemagglutinate trypsinized erythrocytes at nanomolar concentrations. Hemagglutination is inhibited both by negatively charged phospholipids and by a recombinant fragment from rat synaptotagmin that contains only a single copy of the two internal repeats. Together these results demonstrate that synaptotagmin is highly conserved in evolution compatible with a function in the trafficking of synaptic vesicles at the active zone. The similarity of the phospholipid binding properties of the cytoplasmic domains of rat, human, and Drosophila synaptotagmins and the selective conservation of the sequences that are homologous to protein kinase C suggest that these are instrumental in phospholipid binding. The human gene for synaptotagmin was mapped by Southern blot analysis of DNA from somatic cell hybrids to chromosome 12 region cen-q21, and the Drosophila gene by in situ hybridization to 23B (Perin, 1991).

Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 18 February 2003

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