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Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release, composed of a single-pass transmembrane domain linked to two C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is poorly understood. Calcium binding to C2B is critical for synchronous release, and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here, we asked what critical feature of C2A may be responsible for its functional role and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1 K189-192A and Syt1 K326-327A ) side by side to determine their relative contributions to Syt1 function in cultured cortical neurons from mice of either sex and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The size of the readily releasable vesicle pool and the rate of spontaneous release were unaffected, so both patches are likely required specifically for synchronization of release. We suggest these patches contribute to cooperative membrane binding, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release. SIGNIFICANCE STATEMENT Synaptotagmin-1 is a vesicular calcium sensor required for synchronous neurotransmitter release. Its tandem cytosolic C2 domains (C2A and C2B) bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. How calcium binding to Synaptotagmin-1 leads to release and the relative contributions of the C2 domains are unclear. Combining electrophysiological recordings from cultured neurons and optical tweezers measurements of single C2 domain-membrane interactions, we show that conserved polybasic regions in both domains contribute to membrane binding cooperatively, and both are required for evoked release, likely by increasing the overall affinity of Synaptotagmin-1 for acidic membranes., (Copyright © 2022 the authors.)

The molecular mechanisms underlying somatodendritic dopamine (DA) release remain unresolved, despite the passing of decades since its discovery. Our previous work showed robust release of somatodendritic DA in submillimolar extracellular Ca 2+ concentration ([Ca 2+ ] o ). Here we tested the hypothesis that the high-affinity Ca 2+ sensor synaptotagmin 7 (Syt7), is a key determinant of somatodendritic DA release and its Ca 2+ dependence. Somatodendritic DA release from SNc DA neurons was assessed using whole-cell recording in midbrain slices from male and female mice to monitor evoked DA-dependent D2 receptor-mediated inhibitory currents (D2ICs). Single-cell application of an antibody to Syt7 (Syt7 Ab) decreased pulse train-evoked D2ICs, revealing a functional role for Syt7. The assessment of the Ca 2+ dependence of pulse train-evoked D2ICs confirmed robust DA release in submillimolar [Ca 2+ ] o in wild-type (WT) neurons, but loss of this sensitivity with intracellular Syt7 Ab or in Syt7 knock-out (KO) mice. In millimolar [Ca 2+ ] o , pulse train-evoked D2ICs in Syt7 KOs showed a greater reduction in decreased [Ca 2+ ] o than seen in WT mice; the effect on single pulse-evoked DA release, however, did not differ between genotypes. Single-cell application of a Syt1 Ab had no effect on train-evoked D2ICs in WT SNc DA neurons, but did cause a decrease in D2IC amplitude in Syt7 KOs, indicating a functional substitution of Syt1 for Syt7. In addition, Syt1 Ab decreased single pulse-evoked D2ICs in WT cells, indicating the involvement of Syt1 in tonic DA release. Thus, Syt7 and Syt1 play complementary roles in somatodendritic DA release from SNc DA neurons. SIGNIFICANCE STATEMENT The respective Ca 2+ dependence of somatodendritic and axonal dopamine (DA) release differs, resulting in the persistence of somatodendritic DA release in submillimolar Ca 2+ concentrations too low to support axonal release. We demonstrate that synaptotagmin7 (Syt7), a high-affinity Ca 2+ sensor, underlies phasic somatodendritic DA release and its Ca 2+ sensitivity in the substantia nigra pars compacta. In contrast, we found that synaptotagmin 1 (Syt1), the Ca 2+ sensor underlying axonal DA release, plays a role in tonic, but not phasic, somatodendritic DA release in wild-type mice. However, Syt1 can facilitate phasic DA release after Syt7 deletion. Thus, we show that both Syt1 and Syt7 act as Ca 2+ sensors subserving different aspects of somatodendritic DA release processes., (Copyright © 2022 the authors.)

The epilepsy-linked gene SV2A , has a number of potential roles in the synaptic vesicle (SV) life cycle. However, how loss of SV2A function translates into presynaptic dysfunction and ultimately seizure activity is still undetermined. In this study, we examined whether the first SV2A mutation identified in human disease (R383Q) could provide information regarding which SV2A-dependent events are critical in the translation to epilepsy. We utilized a molecular replacement strategy in which exogenous SV2A was expressed in mouse neuronal cultures of either sex, which had been depleted of endogenous SV2A to mimic the homozygous human condition. We found that the R383Q mutation resulted in a mislocalization of SV2A from SVs to the plasma membrane, but had no effect on its activity-dependent trafficking. This SV2A mutant displayed reduced mobility when stranded on the plasma membrane and reduced binding to its interaction partner synaptotagmin-1 (Syt1). Furthermore, the R383Q mutant failed to rescue reduced expression and dysfunctional activity-dependent trafficking of Syt1 in the absence of endogenous SV2A. This suggests that the inability to control Syt1 expression and trafficking at the presynapse may be key in the transition from loss of SV2A function to seizure activity. SIGNIFICANCE STATEMENT SV2A is a synaptic vesicle (SV) protein, the absence or dysfunction of which is linked to epilepsy. However, the series of molecular events that result in this neurological disorder is still undetermined. We demonstrate here that the first human mutation in SV2A identified in an individual with epilepsy displays reduced binding to synaptotagmin-1 (Syt1), an SV protein essential for synchronous neurotransmitter release. Furthermore, this mutant cannot correct alterations in both Syt1 expression and trafficking when expressed in the absence of endogenous SV2A (to mimic the homozygous human condition). This suggests that the inability to control Syt1 expression and trafficking may be key in the transition from loss of SV2A function to seizure activity., (Copyright © 2020 the authors.)

Regulated secretion is controlled by Ca 2+ sensors with different affinities and subcellular distributions. Inactivation of Syt1 (synaptotagmin-1), the main Ca 2+ sensor for synchronous neurotransmission in many neurons, enhances asynchronous and spontaneous release rates, suggesting that Syt1 inhibits other sensors with higher Ca 2+ affinities and/or lower cooperativities. Such sensors could include Doc2a and Doc2b, which have been implicated in spontaneous and asynchronous neurotransmitter release and compete with Syt1 for binding SNARE complexes. Here, we tested this hypothesis using triple-knock-out mice. Inactivation of Doc2a and Doc2b in Syt1-deficient neurons did not reduce the high spontaneous release rate. Overexpression of Doc2b variants in triple-knock-out neurons reduced spontaneous release but did not rescue synchronous release. A chimeric construct in which the C2AB domain of Syt1 was substituted by that of Doc2b did not support synchronous release either. Conversely, the soluble C2AB domain of Syt1 did not affect spontaneous release. We conclude that the high spontaneous release rate in synaptotagmin-deficient neurons does not involve the binding of Doc2 proteins to Syt1 binding sites in the SNARE complex. Instead, our results suggest that the C2AB domains of Syt1 and Doc2b specifically support synchronous and spontaneous release by separate mechanisms. (Both male and female neurons were studied without sex determination.) SIGNIFICANCE STATEMENT Neurotransmission in the brain is regulated by presynaptic Ca 2+ concentrations. Multiple Ca 2+ sensor proteins contribute to synchronous (Syt1, Syt2), asynchronous (Syt7), and spontaneous (Doc2a/Doc2b) phases of neurotransmitter release. Genetic ablation of synchronous release was previously shown to affect other release phases, suggesting that multiple sensors may compete for similar release sites, together encoding stimulus-secretion coupling over a large range of synaptic Ca 2+ concentrations. Here, we investigated the extent of functional overlap between Syt1, Doc2a, and Doc2b by reintroducing wild-type and mutant proteins in triple-knock-out neurons, and conclude that the sensors are highly specialized for different phases of release., (Copyright © 2020 the authors.)

Major signaling molecules initially characterized as key early developmental regulators are also essential for the plasticity of the nervous system. Previously, the Wingless (Wg)/Wnt pathway was shown to underlie the structural and electrophysiological changes during activity-dependent synaptic plasticity at the Drosophila neuromuscular junction. A challenge remains to understand how this signal mediates the cellular changes underlying this plasticity. Here, we focus on the actin regulator Cortactin, a major organizer of protrusion, membrane mobility, and invasiveness, and define its new role in synaptic plasticity. We show that Cortactin is present presynaptically and postsynaptically at the Drosophila NMJ and that it is a presynaptic regulator of rapid activity-dependent modifications in synaptic structure. Furthermore, animals lacking presynaptic Cortactin show a decrease in spontaneous release frequency, and presynaptic Cortactin is necessary for the rapid potentiation of spontaneous release frequency that takes place during activity-dependent plasticity. Most interestingly, Cortactin levels increase at stimulated synaptic terminals and this increase requires neuronal activity, de novo transcription and depends on Wg/Wnt expression. Because it is not simply the presence of Cortactin in the presynaptic terminal but its increase that is necessary for the full range of activity-dependent plasticity, we conclude that it probably plays a direct and important role in the regulation of this process. SIGNIFICANCE STATEMENT In the nervous system, changes in activity that lead to modifications in synaptic structure and function are referred to as synaptic plasticity and are thought to be the basis of learning and memory. The secreted Wingless/Wnt molecule is a potent regulator of synaptic plasticity in both vertebrates and invertebrates. Understanding the molecular mechanisms that underlie these plastic changes is a major gap in our knowledge. Here, we identify a presynaptic effector molecule of the Wingless/Wnt signal, Cortactin. We show that this molecule is a potent regulator of modifications in synaptic structure and is necessary for the electrophysiological changes taking place during synaptic plasticity., (Copyright © 2017 the authors 0270-6474/17/372203-13$15.00/0.)

Whether interactions between synaptotagmin-1 (syt-1) and the soluble NSF attachment protein receptors (SNAREs) are required during neurotransmission is debated. We examined five SNAP-25 mutations designed to interfere with syt-1 interactions. One mutation, D51/E52/E55A, targeted negative charges within region II of the primary interface (Zhou et al., 2015); two mutations targeted region I (D166A and D166/E170A) and one mutation targeted both (D51/E52/E55/D166A). The final mutation (D186/D193A) targeted C-terminal residues not expected to interact with syt-1. An in vitro assay showed that the region I, region II, and region I+II (D51/E52/E55/D166A) mutants markedly reduced the attachment between syt-1 and t-SNARE-carrying vesicles in the absence of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. In the presence of PI(4,5)P 2 , vesicle attachment was unaffected by mutation. When expressed in Snap-25-null mouse autaptic neurons, region I mutations reduced the size of the readily releasable pool of vesicles, whereas the region II mutation reduced vesicular release probability. Combining both in the D51/E52/E55/D166A mutation abrogated evoked release. These data point to a division of labor between region I (vesicle priming) and region II (evoked release). Spontaneous release was disinhibited by region I mutations and found to correlate with defective complexin (Cpx) clamping in an in vitro fusion assay, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Mutation in region II (D51/E52/E55A) also unclamped release, but this effect could be overcome by synaptotagmin overexpression, arguing against an obligatory role in clamping. We conclude that three synaptic release functions of syt-1, vesicle priming, spontaneous release clamping, and evoked release triggering, depend on direct SNARE complex interaction., Significance Statement: The function of synaptotagmin-1 (syt-1):soluble NSF attachment protein receptor (SNARE) interactions during neurotransmission remains unclear. We mutated SNAP-25 within the recently identified region I and region II of the primary synaptotagmin:SNARE interface. Using in vitro assays and rescue experiments in autaptic neurons, we show that interactions within region II of the primary interface are necessary for synchronized calcium-triggered release, whereas region I is involved in vesicle priming. Spontaneous release was disinhibited by region I mutation and found to correlate with defective complexin (Cpx) clamping in vitro, pointing to an interdependent role of synaptotagmin and Cpx in release clamping. Therefore, vesicle priming, clamping spontaneous release, and eliciting evoked release are three different functions of syt-1 that involve different interaction modes with the SNARE complex., (Copyright © 2016 the authors 0270-6474/16/3611865-16$15.00/0.)

Synaptotagmin-1 (Syt1) is the principal Ca(2+) sensor for vesicle fusion and is also essential for vesicle docking in chromaffin cells. Docking depends on interactions of the Syt1-C2B domain with the t-SNARE SNAP25/Syntaxin1 complex and/or plasma membrane phospholipids. Here, we investigated the role of the positively charged "bottom" region of the C2B domain, proposed to help crosslink membranes, in vesicle docking and secretion in mouse chromaffin cells and in cell-free assays. We expressed a double mutation shown previously to interfere with lipid mixing between proteoliposomes and with synaptic transmission, Syt1-R398/399Q (RQ), in syt1 null mutant cells. Ultrastructural morphometry revealed that Syt1-RQ fully restored the docking defect observed previously in syt1 null mutant cells, similar to wild type Syt1 (Syt1-wt). Small unilamellar lipid vesicles (SUVs) that contained the v-SNARE Synaptobrevin2 and Syt1-R398/399Q also docked to t-SNARE-containing giant vesicles (GUVs), similar to Syt1-wt. However, unlike Syt1-wt, Syt1-RQ-induced docking was strictly PI(4,5)P2-dependent. Unlike docking, neither synchronized secretion in chromaffin cells nor Ca(2+)-triggered SUV-GUV fusion was restored by the Syt1 mutants. Finally, overexpressing the RQ-mutant in wild type cells produced no effect on either docking or secretion. We conclude that the positively charged bottom region in the C2B domain--and, by inference, Syt1-mediated membrane crosslinking--is required for triggering fusion, but not for docking. Secretory vesicles dock by multiple, PI(4,5)P2-dependent and PI(4,5)P2-independent mechanisms. The R398/399 mutations selectively disrupt the latter and hereby help to discriminate protein regions involved in different aspects of Syt1 function in docking and fusion., Significance Statement: This study provides new insights in how the two opposite sides of the C2B domain of Synaptotagmin-1 participate in secretory vesicle fusion, and in more upstream steps, especially vesicle docking. We show that the "bottom" surface of the C2B domain is required for triggering fusion, but not for docking. Synaptotagmin-1 promotes docking by multiple, PI(4,5)P2-dependent and PI(4,5)P2-independent mechanisms. Mutations in the C2B bottom surface (R398/399) selectively disrupt the latter. These mutations help to discriminate protein regions involved in different aspects of Synaptotagmin-1 function in docking and fusion., (Copyright © 2015 the authors 0270-6474/15/3514173-11$15.00/0.)

SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.

Synaptic vesicle fusion mediates communication between neurons and is triggered by rapid influx of Ca(2+). The Ca(2+)-triggering step for fusion is regulated by the synaptic vesicle transmembrane protein Synaptotagmin 1 (Syt1). Syt1 contains two cytoplasmic C2 domains, termed C2A and C2B, which coordinate Ca(2+) binding. Although C2A and C2B share similar topology, binding of Ca(2+) ions to the C2B domain has been suggested as the only critical trigger for evoked vesicle release. If and how C2A domain function is coordinated with C2B remain unclear. In this study, we generated a panel of Syt1 chimeric constructs in Drosophila to delineate the unique and shared functions of each C2 domain in regulation of synaptic vesicle fusion. Expression of Syt 1 transgenes containing only individual C2 domains, or dual C2A-C2A or C2B-C2B chimeras, failed to restore Syt1 function in a syt1(-/-) null mutant background, indicating both C2A and C2B are specifically required to support fast synchronous release. Mutations that disrupted Ca(2+) binding to both C2 domains failed to rescue evoked release, but supported synaptic vesicle docking and endocytosis, indicating that these functions of Syt1 are Ca(2+)-independent. The dual C2 domain Ca(2+)-binding mutant also enhanced spontaneous fusion while dramatically increasing evoked release when coexpressed with native Syt1. Together, these data indicate that synaptic transmission can be regulated by Syt1 multimerization and that both C2 domains of Syt1 are uniquely required for modulating Ca(2+)-independent spontaneous fusion and Ca(2+)-dependent synchronous release.

Cochlear inner hair cells (IHCs) use Ca(2+)-dependent exocytosis of glutamate to signal sound information. Otoferlin (Otof), a C(2) domain protein essential for IHC exocytosis and hearing, may serve as a Ca(2+) sensor in vesicle fusion in IHCs that seem to lack the classical neuronal Ca(2+) sensors synaptotagmin 1 (Syt1) and Syt2. Support for the Ca(2+) sensor of fusion hypothesis for otoferlin function comes from biochemical experiments, but additional roles in late exocytosis upstream of fusion have been indicated by physiological studies. Here, we tested the functional equivalence of otoferlin and Syt1 in three neurosecretory model systems: auditory IHCs, adrenal chromaffin cells, and hippocampal neurons. Long-term and short-term ectopic expression of Syt1 in IHCs of Otof (-/-) mice by viral gene transfer in the embryonic inner ear and organotypic culture failed to rescue their Ca(2+) influx-triggered exocytosis. Conversely, virally mediated overexpression of otoferlin did not restore phasic exocytosis in Syt1-deficient chromaffin cells or neurons but enhanced asynchronous release in the latter. We further tested exocytosis in Otof (-/-) hippocampal neurons and in Syt1(-/-) IHCs but found no deficits in vesicle fusion. Expression analysis of different synaptotagmin isoforms indicated that Syt1 and Syt2 are absent from mature IHCs. Our data argue against a simple functional equivalence of the two C(2) domain proteins in exocytosis of IHC ribbon synapses, chromaffin cells, and hippocampal synapses.

In pre-hearing mice, vesicle exocytosis at cochlear inner hair cell (IHC) ribbon synapses is triggered by spontaneous Ca(2+) spikes. At the onset of hearing, IHC exocytosis is then exclusively driven by graded potentials, and is characterized by higher Ca(2+) efficiency and improved synchronization of vesicular release. The molecular players involved in this transition are still unknown. Here we addressed the involvement of synaptotagmins and otoferlin as putative Ca(2+) sensors in IHC exocytosis during postnatal maturation of the cochlea. Using cell capacitance measurements, we showed that Ca(2+)-evoked exocytosis in mouse IHCs switches from an otoferlin-independent to an otoferlin-dependent mechanism at postnatal day 4. During this early exocytotic period, several synaptotagmins (Syts), including Syt1, Syt2 and Syt7, were detected in IHCs. The exocytotic response as well as the release of the readily releasable vesicle pool (RRP) was, however, unchanged in newborn mutant mice lacking Syt1, Syt2 or Syt7 (Syt1(-/-), Syt2(-/-) and Syt7(-/-) mice). We only found a defect in RRP recovery in Syt1(-/-) mice which was apparent as a strongly reduced response to repetitive stimulations. In post-hearing Syt2(-/-) and Syt7(-/-) mutant mice, IHC synaptic exocytosis was unaffected. The transient expression of Syt1 and Syt2, which were no longer detected in IHCs after the onset of hearing, indicates that these two most common Ca(2+)-sensors in CNS synapses are not involved in mature IHCs. We suggest that otoferlin underlies highly efficient Ca(2+)-dependent membrane-membrane fusion, a process likely essential to increase the probability and synchrony of vesicle fusion events at the mature IHC ribbon synapse.

Several presynaptic proteins involved in neurotransmitter release in the CNS have been implicated in schizophrenia in human clinical genetic studies, in postmortem studies, and in studies of putative animal models of schizophrenia. The presynaptic protein RIM1alpha mediates presynaptic plasticity and cognitive function. We now demonstrate that mice deficient in RIM1alpha exhibit abnormalities in multiple schizophrenia-relevant behavioral tasks including prepulse inhibition, response to psychotomimetic drugs, and social interaction. These schizophrenia-relevant behavioral findings are relatively selective to RIM1alpha-deficient mice, as mice bearing mutations in the RIM1alpha binding partners Rab3A or synaptotagmin 1 only show decreased prepulse inhibition. In addition to RIM1alpha's involvement in multiple behavioral abnormalities, these data suggest that alterations in presynaptic forms of short-term plasticity are linked to alterations in prepulse inhibition, a measure of sensorimotor gating.

Synaptotagmin-I (syt-I) is required for rapid neurotransmitter release in mouse hippocampal neurons. However, contradictory results have been reported regarding evoked and spontaneous secretion from syt-I knock-out (KO) neurons. Here, we compared synaptic transmission in two different hippocampal neuron preparations: autaptic cultures in which a single isolated cell innervates itself, and dissociated mass cultures in which individual cells are innervated by neighboring cells. In autaptic cultures, the total extent of evoked release, size of readily releasable pool of synaptic vesicles, and release probability were unchanged in syt-I KO neurons. In contrast, in cultures containing multiple interconnected neurons, total evoked release, the number of docked vesicles, and release probability, were significantly reduced in syt-I KO neurons. Using a micronetwork system in which we varied the number of cells on an island, we found that the frequency of spontaneous synaptic vesicle fusion events (minis) was unchanged in syt-I KO neurons when two or fewer cells were present on an island. However, in micronetworks composed of three or more neurons, mini frequency was increased threefold to fivefold in syt-I KO neurons compared with wild type. Moreover, interneuronal synapses exhibited higher rates of spontaneous release than autaptic synapses. This higher rate was attributable to an increase in release probability because excitatory hippocampal neurons in micronetworks formed a set number of synapses per cell regardless of the number of connected neurons. Thus, aspects of synaptic transmission differ between autaptic and dissociated cultures, and the synaptic transmission phenotype, resulting from loss of syt-I, is dictated by the connectivity of neurons.

Synaptotagmin I is the Ca(2+) sensor for fast, synchronous release of neurotransmitter; however, the molecular interactions that couple Ca(2+) binding to membrane fusion remain unclear. The structure of synaptotagmin is dominated by two C(2) domains that interact with negatively charged membranes after binding Ca(2+). In vitro work has implicated a conserved basic residue at the tip of loop 3 of the Ca(2+)-binding pocket in both C(2) domains in coordinating this electrostatic interaction with anionic membranes. Although results from cultured cells suggest that the basic residue of the C(2)A domain is functionally significant, such studies provide contradictory results regarding the importance of the C(2)B basic residue during vesicle fusion. To directly test the functional significance of each of these residues at an intact synapse in vivo, we neutralized either the C(2)A or the C(2)B basic residue and assessed synaptic transmission at the Drosophila neuromuscular junction. The conserved basic residues at the tip of the Ca(2+)-binding pocket of both the C(2)A and C(2)B domains mediate Ca(2+)-dependent interactions with anionic membranes and are required for efficient evoked transmitter release. Our results directly support the hypothesis that the interactions between synaptotagmin and the presynaptic membrane, which are mediated by the basic residues at the tip of both the C(2)A and C(2)B Ca(2+)-binding pockets, are critical for coupling Ca(2+) influx with vesicle fusion during synaptic transmission in vivo. Our model for synaptotagmin's direct role in coupling Ca(2+) binding to vesicle fusion incorporates this finding with results from multiple in vitro and in vivo studies.

Synaptotagmin-1, the Ca2+ sensor for fast neurotransmitter release, was proposed to function by Ca2+-dependent phospholipid binding and/or by Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex binding. Extensive in vivo data support the first hypothesis, but testing the second hypothesis has been difficult because no synaptotagmin-1 mutation is known that selectively interferes with SNARE complex binding. Using knock-in mice that carry aspartate-to-asparagine substitutions in a Ca2+-binding site of synaptotagmin-1 (the D232N or D238N substitutions), we now show that the D232N mutation dramatically increases Ca2+-dependent SNARE complex binding by native synaptotagmin-1, but leaves phospholipid binding unchanged. In contrast, the adjacent D238N mutation does not significantly affect SNARE complex binding, but decreases phospholipid binding. Electrophysiological recordings revealed that the D232N mutation increased Ca2+-triggered release, whereas the D238N mutation decreased release. These data establish that fast vesicle exocytosis is driven by a dual Ca2+-dependent activity of synaptotagmin-1, namely Ca2+-dependent binding both to SNARE complexes and to phospholipids.

Synaptotagmin IV (Syt IV) is a brain-specific isoform of the synaptotagmin family, the levels of which are strongly elevated after seizure activity. The dominant hypothesis of Syt IV function states that Syt IV upregulation is a neuroprotective mechanism for reducing neurotransmitter release. To test this hypothesis in mammalian CNS synapses, Syt IV was overexpressed in cultured mouse hippocampal neurons, and acute effects on fast excitatory neurotransmission were assessed. We found neurotransmission unaltered with respect to basal release probability, Ca2+ dependence of release, short-term plasticity, and fusion pore kinetics. In contrast, expression of a mutant Syt I with diminished Ca2+ affinity (R233Q) reduced release probability and altered the Ca2+ dependence of release, thus demonstrating the sensitivity of the system to changes in neurotransmission resulting from changes to the Ca2+ sensor. Together, these data refute the dominant model that Syt IV functions as an inhibitor of neurotransmitter release in mammalian neurons.

Synaptotagmins comprise a large protein family, of which synaptotagmin 1 (Syt1) is a Ca2+ sensor for fast exocytosis, and its close relative, synaptotagmin 2 (Syt2), is assumed to serve similar functions. Chromaffin cells express Syt1 but not Syt2. We compared secretion from chromaffin cells from Syt1 null mice overexpressing either Syt isoform. High time-resolution capacitance measurement showed that Syt1 null cells lack the exocytotic phase corresponding to the readily-releasable pool (RRP) of vesicles. Comparison with the amperometric signal confirmed that the missing phase of exocytosis consists of catecholamine-containing vesicles. Overexpression of Syt1 rescued the RRP and increased its size above wild-type values, whereas the size of the slowly releasable pool decreased, indicating that the availability of Syt1 regulates the relative size of the two releasable pools. The RRP was also rescued by Syt2 overexpression, but the kinetics of fusion was slightly slower than in cells expressing Syt1. Biochemical experiments showed that Syt2 has a slightly lower Ca2+ affinity for phospholipid binding than Syt1 because of a difference in the C2A domain. These data constitute evidence for the function of Syt1 and Syt2 as alternative, but not identical, calcium-sensors for RRP fusion. By overexpression of Syt1 mutated in the shared PKC/calcium/calmodulin-dependent kinase phosphorylation site, we show that phorbol esters act independently and upstream of Syt1 to regulate the size of the releasable pools. We conclude that exocytosis from mouse chromaffin cells can be modified by the differential expression of Syt isoforms and by Syt abundance but not by phosphorylation of Syt1.