Publications by authors named "Alexander J Groffen"

29 Publications

  • Page 1 of 1

Ca sensor proteins in spontaneous release and synaptic plasticity: Limited contribution of Doc2c, rabphilin-3a and synaptotagmin 7 in hippocampal glutamatergic neurons.

Mol Cell Neurosci 2021 04 19;112:103613. Epub 2021 Mar 19.

Department of Functional Genomics, Faculty of Science, Center for Neurogenomics and Cognitive Research, VU University, De Boelelaan 1085, 1081HV Amsterdam, the Netherlands; Department of Clinical Genetics, VU Medical Center, De Boelelaan 1085, 1081HV Amsterdam, the Netherlands. Electronic address:

Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca and a number of Ca sensors including synaptotagmins (Syts) and Double C domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C domain architecture, but are functionally diverse in their subcellular location, Ca-binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca-sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca sensors that may be implicated in the spontaneous release of glutamate.
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http://dx.doi.org/10.1016/j.mcn.2021.103613DOI Listing
April 2021

Doc2 Proteins Are Not Required for the Increased Spontaneous Release Rate in Synaptotagmin-1-Deficient Neurons.

J Neurosci 2020 03 25;40(13):2606-2617. Epub 2020 Feb 25.

Department of Clinical Genetics, Center for Neurogenomics and Cognitive Research, Amsterdam UMC, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands

Regulated secretion is controlled by Ca sensors with different affinities and subcellular distributions. Inactivation of (synaptotagmin-1), the main Ca sensor for synchronous neurotransmission in many neurons, enhances asynchronous and spontaneous release rates, suggesting that Syt1 inhibits other sensors with higher Ca 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.) Neurotransmission in the brain is regulated by presynaptic Ca concentrations. Multiple Ca 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 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.
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http://dx.doi.org/10.1523/JNEUROSCI.0309-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7096138PMC
March 2020

Synaptotagmin-1 and Doc2b Exhibit Distinct Membrane-Remodeling Mechanisms.

Biophys J 2020 02 25;118(3):643-656. Epub 2019 Dec 25.

Department of Physics and Astronomy and LaserLab, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands. Electronic address:

Synaptotagmin-1 (Syt1) is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared with that of Doc2b, which contains a conserved CAB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble CAB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both CAB fragments cause a reduction in the membrane-bending modulus, as measured by a method based on atomic force microscopy. This lowering of the energy required for membrane deformation may contribute to Ca-induced fusion.
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http://dx.doi.org/10.1016/j.bpj.2019.12.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002981PMC
February 2020

Doc2b Ca binding site mutants enhance synaptic release at rest at the expense of sustained synaptic strength.

Sci Rep 2019 10 8;9(1):14408. Epub 2019 Oct 8.

Department of Functional Genomics, Faculty of Science, Center for Neurogenomics and Cognitive Research, Vrije Universiteit, De Boelelaan 1085, 1081HV, Amsterdam, The Netherlands.

Communication between neurons involves presynaptic neurotransmitter release which can be evoked by action potentials or occur spontaneously as a result of stochastic vesicle fusion. The Ca-binding double C proteins Doc2a and -b were implicated in spontaneous and asynchronous evoked release, but the mechanism remains unclear. Here, we compared wildtype Doc2b with two Ca binding site mutants named DN and 6A, previously classified as gain- and loss-of-function mutants. They carry the substitutions D218,220N or D163,218,220,303,357,359A respectively. We found that both mutants bound phospholipids at low Ca concentrations and were membrane-associated in resting neurons, thus mimicking a Ca-activated state. Their overexpression in hippocampal primary cultured neurons had similar effects on spontaneous and evoked release, inducing high mEPSC frequencies and increased short-term depression. Together, these data suggest that the DN and 6A mutants both act as gain-of-function mutants at resting conditions.
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http://dx.doi.org/10.1038/s41598-019-50684-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783474PMC
October 2019

SICT: automated detection and supervised inspection of fast Ca transients.

Sci Rep 2018 10 19;8(1):15523. Epub 2018 Oct 19.

Department of Functional Genomics, Faculty of Science, Center for Neurogenomics and Cognitive Research, Vrije Universiteit, De Boelelaan 1085, 1081HV, Amsterdam, The Netherlands.

Recent advances in live Ca imaging with increasing spatial and temporal resolution offer unprecedented opportunities, but also generate an unmet need for data processing. Here we developed SICT, a MATLAB program that automatically identifies rapid Ca rises in time-lapse movies with low signal-to-noise ratios, using fluorescent indicators. A graphical user interface allows visual inspection of automatically detected events, reducing manual labour to less than 10% while maintaining quality control. The detection performance was tested using synthetic data with various signal-to-noise ratios. The event inspection phase was evaluated by four human observers. Reliability of the method was demonstrated in a direct comparison between manual and SICT-aided analysis. As a test case in cultured neurons, SICT detected an increase in frequency and duration of spontaneous Ca transients in the presence of caffeine. This new method speeds up the analysis of elementary Ca transients.
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http://dx.doi.org/10.1038/s41598-018-33847-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195629PMC
October 2018

Transcription Factor 2I Regulates Neuronal Development via TRPC3 in 7q11.23 Disorder Models.

Mol Neurobiol 2019 May 17;56(5):3313-3325. Epub 2018 Aug 17.

Department of Physiology, University of Toronto, 1 King's College Circle, 3306 Medical Sciences Building, Toronto, ON, M5S 1A8, Canada.

Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7q11.23) are neurodevelopmental disorders caused by the deletion and duplication, respectively, of ~ 25 protein-coding genes on chromosome 7q11.23. The general transcription factor 2I (GTF2I, protein TFII-I) is one of these proteins and has been implicated in the neurodevelopmental phenotypes of WS and Dup7q11.23. Here, we investigated the effect of copy number alterations in Gtf2i on neuronal maturation and intracellular calcium entry mechanisms known to be associated with this process. Mice with a single copy of Gtf2i (Gtf2i) had increased axonal outgrowth and increased TRPC3-mediated calcium entry upon carbachol stimulation. In contrast, mice with 3 copies of Gtf2i (Gtf2i) had decreases in axon outgrowth and in TRPC3-mediated calcium entry. The underlying mechanism was that TFII-I did not affect TRPC3 protein expression, while it regulated TRPC3 membrane translocation. Together, our results provide novel functional insight into the cellular mechanisms that underlie neuronal maturation in the context of the 7q11.23 disorders.
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http://dx.doi.org/10.1007/s12035-018-1290-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6477017PMC
May 2019

Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs.

Elife 2017 12 23;6. Epub 2017 Dec 23.

Neuronal Secretion Group, Department of Neuroscience, University of Copenhagen, København, Denmark.

Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B - inhibition of release during sustained calcium elevations - depends on an overlapping protein domain (the MID-domain), but is separate from its Ca-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.
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http://dx.doi.org/10.7554/eLife.27000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5758110PMC
December 2017

δ-Catenin () missense mutation in familial cortical myoclonic tremor and epilepsy.

Neurology 2017 Dec 10;89(23):2341-2350. Epub 2017 Nov 10.

From the Departments of Neurology and Clinical Neurophysiology (A.-F.v.R., A.B., M.A.J.T.), Academic Medical Centre, Amsterdam Neuroscience, University of Amsterdam; Departments of Functional Genomics and Clinical Genetics (A.J.G.), CNCR, Neuroscience Campus Amsterdam, VU University and VU Medical Centre; Departments of Human Genetics (B.d.V., S.K., L.S.V., A.M.J.M.v.d.M.), Clinical Genetics (G.W.E.S.), and Neurology (A.M.J.M.v.d.M.), Leiden University Medical Centre; and Department of Neurology (P.M.C.C., M.A.J.T.), University Medical Centre Groningen, University of Groningen, the Netherlands.

Objective: To identify the causative gene in a large Dutch family with familial cortical myoclonic tremor and epilepsy (FCMTE).

Methods: We performed exome sequencing for 3 patients of our FCMTE family. Next, we performed knock-down (shRNA) and rescue experiments by overexpressing wild-type and mutant human δ-catenin (CTNND2) proteins in cortical mouse neurons and compared the results with morphologic abnormalities in the postmortem FCMTE brain.

Results: We identified a missense mutation, p.Glu1044Lys, in the gene that cosegregated with the FCMTE phenotype. The knock-down of in cultured cortical mouse neurons revealed increased neurite outgrowth that was rescued by overexpression of wild-type, but not mutant, CTNND2 and was reminiscent of the morphologic abnormalities observed in cerebellar Purkinje cells from patients with FCMTE.

Conclusions: We propose as the causal gene in FCMTE3. Functional testing of the mutant protein revealed abnormal neuronal sprouting, consistent with the abnormal cerebellar Purkinje cell morphology in patients with FCMTE.
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http://dx.doi.org/10.1212/WNL.0000000000004709DOI Listing
December 2017

Tomosyn associates with secretory vesicles in neurons through its N- and C-terminal domains.

PLoS One 2017 26;12(7):e0180912. Epub 2017 Jul 26.

Department of Functional Genomics, Centre for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands.

The secretory pathway in neurons requires efficient targeting of cargos and regulatory proteins to their release sites. Tomosyn contributes to synapse function by regulating synaptic vesicle (SV) and dense-core vesicle (DCV) secretion. While there is large support for the presynaptic accumulation of tomosyn in fixed preparations, alternative subcellular locations have been suggested. Here we studied the dynamic distribution of tomosyn-1 (Stxbp5) and tomosyn-2 (Stxbp5l) in mouse hippocampal neurons and observed a mixed diffuse and punctate localization pattern of both isoforms. Tomosyn-1 accumulations were present in axons and dendrites. As expected, tomosyn-1 was expressed in about 75% of the presynaptic terminals. Interestingly, also bidirectional moving tomosyn-1 and -2 puncta were observed. Despite the lack of a membrane anchor these puncta co-migrated with synapsin and neuropeptide Y, markers for respectively SVs and DCVs. Genetic blockade of two known tomosyn interactions with synaptotagmin-1 and its cognate SNAREs did not abolish its vesicular co-migration, suggesting an interplay of protein interactions mediated by the WD40 and SNARE domains. We hypothesize that the vesicle-binding properties of tomosyns may control the delivery, pan-synaptic sharing and secretion of neuronal signaling molecules, exceeding its canonical role at the plasma membrane.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0180912PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5529015PMC
October 2017

Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes.

Nat Commun 2015 Sep 23;6:8387. Epub 2015 Sep 23.

Department of Functional Genomics and Clinical Genetics, CNCR, NCA, VU University and VU Medical Center, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands.

Ca(2+)-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca(2+)-dependent association of their tandem C2 domains with phosphatidylserine may induce membrane curvature and thereby enhance fusion. Second, their association with SNARE complexes may inhibit membrane fusion in the absence of a Ca(2+) trigger. Here we present a method using two optically trapped beads coated with SNARE-free synthetic membranes to elucidate the direct role of the C2AB domain of the soluble Ca(2+)-sensor Doc2b. Contacting membranes are often coupled by a Doc2b-coated membrane stalk that resists forces up to 600 pN upon bead separation. Stalk formation depends strictly on Ca(2+) and phosphatidylserine. Real-time fluorescence imaging shows phospholipid but not content mixing, indicating membrane hemifusion. Thus, Doc2b acts directly on membranes and stabilizes the hemifusion intermediate in this cell-free system. In living cells, this mechanism may co-occur with progressive SNARE complex assembly, together defining Ca(2+)-secretion coupling.
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http://dx.doi.org/10.1038/ncomms9387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598560PMC
September 2015

Identification of a Dutch founder mutation in MUSK causing fetal akinesia deformation sequence.

Eur J Hum Genet 2015 Sep 24;23(9):1151-7. Epub 2014 Dec 24.

1] Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands [2] Department of Functional Genomics, Center for Neurogenomics and Cognition Research, VU University, Amsterdam, The Netherlands.

Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. FADS can result from mutations in CHRNG, CHRNA1, CHRND, DOK7 and RAPSN; however, these genes only account for a minority of cases. Here we identify MUSK as a novel cause of lethal FADS. Fourteen affected fetuses from a Dutch genetic isolate were traced back to common ancestors 11 generations ago. Homozygosity mapping in two fetuses revealed MUSK as a candidate gene. All tested cases carried an identical homozygous variant c.1724T>C; p.(Ile575Thr) in the intracellular domain of MUSK. The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers. Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings. A functional assay in myocytes derived from human fetuses confirmed that the variant blocks MUSK-dependent motor endplate formation. Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.
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http://dx.doi.org/10.1038/ejhg.2014.273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538208PMC
September 2015

Two male adults with pathogenic AUTS2 variants, including a two-base pair deletion, further delineate the AUTS2 syndrome.

Eur J Hum Genet 2015 Jun 10;23(6):803-7. Epub 2014 Sep 10.

Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands.

AUTS2 syndrome is characterized by low birth weight, feeding difficulties, intellectual disability, microcephaly and mild dysmorphic features. All affected individuals thus far were caused by chromosomal rearrangements, variants at the base pair level disrupting AUTS2 have not yet been described. Here we present the full clinical description of two affected men with intragenic AUTS2 variants (one two-base pair deletion in exon 7 and one deletion of exon 6). Both variants are de novo and are predicted to cause a frameshift of the full-length transcript but are unlikely to affect the shorter 3' transcript starting in exon 9. The similarities between the phenotypes of both men are striking and further support that AUTS2 syndrome is a single gene disorder.
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http://dx.doi.org/10.1038/ejhg.2014.173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4795056PMC
June 2015

DOC2 isoforms play dual roles in insulin secretion and insulin-stimulated glucose uptake.

Diabetologia 2014 Oct 9;57(10):2173-82. Epub 2014 Jul 9.

Diabetes and Obesity Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia.

Aims/hypothesis: Glucose-stimulated insulin secretion (GSIS) and insulin-stimulated glucose uptake are processes that rely on regulated intracellular vesicle transport and vesicle fusion with the plasma membrane. DOC2A and DOC2B are calcium-sensitive proteins that were identified as key components of vesicle exocytosis in neurons. Our aim was to investigate the role of DOC2 isoforms in glucose homeostasis, insulin secretion and insulin action.

Methods: DOC2 expression was measured by RT-PCR and western blotting. Body weight, glucose tolerance, insulin action and GSIS were assessed in wild-type (WT), Doc2a (-/-) (Doc2aKO), Doc2b (-/-) (Doc2bKO) and Doc2a (-/-)/Doc2b (-/-) (Doc2a/Doc2bKO) mice in vivo. In vitro GSIS and glucose uptake were assessed in isolated tissues, and exocytotic proteins measured by western blotting. GLUT4 translocation was assessed by epifluorescence microscopy.

Results: Doc2b mRNA was detected in all tissues tested, whereas Doc2a was only detected in islets and the brain. Doc2aKO and Doc2bKO mice had minor glucose intolerance, while Doc2a/Doc2bKO mice showed pronounced glucose intolerance. GSIS was markedly impaired in Doc2a/Doc2bKO mice in vivo, and in isolated Doc2a/Doc2bKO islets in vitro. In contrast, Doc2bKO mice had only subtle defects in insulin secretion in vivo. Insulin action was impaired to a similar degree in both Doc2bKO and Doc2a/Doc2bKO mice. In vitro insulin-stimulated glucose transport and GLUT4 vesicle fusion were defective in adipocytes derived from Doc2bKO mice. Surprisingly, insulin action was not altered in muscle isolated from DOC2-null mice.

Conclusions/interpretation: Our study identifies a critical role for DOC2B in insulin-stimulated glucose uptake in adipocytes, and for the synergistic regulation of GSIS by DOC2A and DOC2B in beta cells.
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http://dx.doi.org/10.1007/s00125-014-3312-yDOI Listing
October 2014

Tomosyn-2 is required for normal motor performance in mice and sustains neurotransmission at motor endplates.

Brain Struct Funct 2015 Jul 18;220(4):1971-82. Epub 2014 Apr 18.

Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, 1081 HV, Amsterdam, The Netherlands.

Tomosyn-1 (STXBP5) is a soluble NSF attachment protein receptor complex-binding protein that inhibits vesicle fusion, but the role of tomosyn-2 (STXBP5L) in the mammalian nervous system is still unclear. Here we generated tomosyn-2 null (Tom2(KO/KO)) mice, which showed impaired motor performance. This was accompanied by synaptic changes at the neuromuscular junction, including enhanced spontaneous acetylcholine release frequency and faster depression of muscle motor endplate potentials during repetitive stimulation. The postsynaptic geometric arrangement and function of acetylcholine receptors were normal. We conclude that tomosyn-2 supports motor performance by regulation of transmitter release willingness to sustain synaptic strength during high-frequency transmission, which makes this gene a candidate for involvement in neuromuscular disorders.
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http://dx.doi.org/10.1007/s00429-014-0766-0DOI Listing
July 2015

Tomosyn interacts with the SUMO E3 ligase PIASγ.

PLoS One 2014 10;9(3):e91697. Epub 2014 Mar 10.

Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, Amsterdam, Netherlands; Department of Clinical Genetics, VU Medical Center, Amsterdam, Netherlands.

Protein modification by Small Ubiquitin-like MOdifier (SUMO) entities is involved in a number of neuronal functions, including synaptogenesis and synaptic plasticity. Tomosyn-1 (syntaxin-binding protein 5; STXPB5) binds to t-SNARE (Soluble NSF Attachment Protein Receptor) proteins to regulate neurotransmission and is one of the few neuronal SUMO substrate proteins identified. Here we used yeast two-hybrid screening to show that tomosyn-1 interacts with the SUMO E3 ligase PIASγ (Protein Inhibitor of Activated STAT; PIAS4 or ZMIZ6). This novel interaction involved the C-terminus of tomosyn-1 and the N-terminus of PIASγ. It was confirmed by two-way immunoprecipitation experiments using the full-length proteins expressed in HEK293T cells. Tomosyn-1 was preferentially modified by the SUMO-2/3 isoform. PIASγ-dependent modification of tomosyn-1 with SUMO-2/3 presents a novel mechanism to adapt secretory strength to the dynamic synaptic environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091697PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948876PMC
March 2016

Doc2b synchronizes secretion from chromaffin cells by stimulating fast and inhibiting sustained release.

J Neurosci 2013 Oct;33(42):16459-70

Department of Neuroscience and Pharmacology, Faculty of Health Sciences, and Lundbeck Foundation Center for Biomembranes in Nanomedicine, University of Copenhagen, 2200 Copenhagen N, Denmark, Center for Neurogenomics and Cognitive Research, Department of Functional Genomics, Neuroscience Campus Amsterdam, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands, and VU Medical Center, 1081 HV Amsterdam, The Netherlands.

Synaptotagmin-1 and -7 constitute the main calcium sensors mediating SNARE-dependent exocytosis in mouse chromaffin cells, but the role of a closely related calcium-binding protein, Doc2b, remains enigmatic. We investigated its role in chromaffin cells using Doc2b knock-out mice and high temporal resolution measurements of exocytosis. We found that the calcium dependence of vesicle priming and release triggering remained unchanged, ruling out an obligatory role for Doc2b in those processes. However, in the absence of Doc2b, release was shifted from the readily releasable pool to the subsequent sustained component. Conversely, upon overexpression of Doc2b, the sustained component was largely inhibited whereas the readily releasable pool was augmented. Electron microscopy revealed an increase in the total number of vesicles upon Doc2b overexpression, ruling out vesicle depletion as the cause for the reduced sustained component. Further experiments showed that, in the absence of Doc2b, the refilling of the readily releasable vesicle pools is faster, but incomplete. Faster refilling leads to an increase in the sustained component as newly primed vesicles fuse while the [Ca(2+)]i following stimulation is still high. We conclude that Doc2b acts to inhibit vesicle priming during prolonged calcium elevations, thus protecting unprimed vesicles from fusing prematurely, and redirecting them to refill the readily releasable pool after relaxation of the calcium signal. In sum, Doc2b favors fast, synchronized release, and limits out-of-phase secretion.
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http://dx.doi.org/10.1523/JNEUROSCI.2656-13.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618527PMC
October 2013

TRIM3 regulates the motility of the kinesin motor protein KIF21B.

PLoS One 2013 24;8(9):e75603. Epub 2013 Sep 24.

Department of Molecular Neurogenetics, Center for Molecular Neurobiology, ZMNH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Kinesin superfamily proteins (KIFs) are molecular motors that transport cellular cargo along the microtubule cytoskeleton. KIF21B is a neuronal kinesin that is highly enriched in dendrites. The regulation and specificity of microtubule transport involves the binding of motors to individual cargo adapters and accessory proteins. Moreover, posttranslational modifications of either the motor protein, their cargos or tubulin regulate motility, cargo recognition and the binding or unloading of cargos. Here we show that the ubiquitin E3 ligase TRIM3, also known as BERP, interacts with KIF21B via its RBCC domain. TRIM3 is found at intracellular and Golgi-derived vesicles and co-localizes with the KIF21B motor in neurons. Trim3 gene deletion in mice and TRIM3 overexpression in cultured neurons both suggested that the E3-ligase function of TRIM3 is not involved in KIF21B degradation, however TRIM3 depletion reduces the motility of the motor. Together, our data suggest that TRIM3 is a regulator in the modulation of KIF21B motor function.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075603PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3782429PMC
July 2014

Exonic deletions in AUTS2 cause a syndromic form of intellectual disability and suggest a critical role for the C terminus.

Am J Hum Genet 2013 Feb 17;92(2):210-20. Epub 2013 Jan 17.

Department of Clinical Genetics, VU University Medical Center, Amsterdam 1007 MB, The Netherlands.

Genomic rearrangements involving AUTS2 (7q11.22) are associated with autism and intellectual disability (ID), although evidence for causality is limited. By combining the results of diagnostic testing of 49,684 individuals, we identified 24 microdeletions that affect at least one exon of AUTS2, as well as one translocation and one inversion each with a breakpoint within the AUTS2 locus. Comparison of 17 well-characterized individuals enabled identification of a variable syndromic phenotype including ID, autism, short stature, microcephaly, cerebral palsy, and facial dysmorphisms. The dysmorphic features were more pronounced in persons with 3'AUTS2 deletions. This part of the gene is shown to encode a C-terminal isoform (with an alternative transcription start site) expressed in the human brain. Consistent with our genetic data, suppression of auts2 in zebrafish embryos caused microcephaly that could be rescued by either the full-length or the C-terminal isoform of AUTS2. Our observations demonstrate a causal role of AUTS2 in neurocognitive disorders, establish a hitherto unappreciated syndromic phenotype at this locus, and show how transcriptional complexity can underpin human pathology. The zebrafish model provides a valuable tool for investigating the etiology of AUTS2 syndrome and facilitating gene-function analysis in the future.
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http://dx.doi.org/10.1016/j.ajhg.2012.12.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567268PMC
February 2013

Genetic and phenotypic heterogeneity in sporadic and familial forms of paroxysmal dyskinesia.

J Neurol 2013 Jan 30;260(1):93-9. Epub 2012 Jun 30.

Departments of Clinical Genetics and Functional Genomics, Center of Neurogenomics and Cognitive Research (CNCR), VU University and VU Medical Center, Amsterdam, The Netherlands.

Paroxysmal dyskinesia (PxD) is a group of movement disorders characterized by recurrent episodes of involuntary movements. Familial paroxysmal kinesigenic dyskinesia (PKD) is caused by PRRT2 mutations, but a distinct etiology has been suggested for sporadic PKD. Here we describe a cohort of patients collected from our movement disorders outpatient clinic in the period 1996-2011. Fifteen patients with sporadic PxD and 23 subjects from three pedigrees with familial PKD were screened for mutations in candidate genes. PRRT2 mutations co-segregated with PKD in two families and occurred in two sporadic cases of PKD. No mutations were detected in patients with non-kinesigenic or exertion-induced dyskinesia, and none in other candidate genes including PNKD1 (MR-1) and SLC2A1 (GLUT1). Thus, PRRT2 mutations also cause sporadic PKD as might be expected given the variable expressivity and reduced penetrance observed in familial PKD. Further genetic heterogeneity is suggested by the absence of candidate gene mutations in both sporadic and familial PKD suggesting a contribution of other genes or non-coding regions.
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http://dx.doi.org/10.1007/s00415-012-6592-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535363PMC
January 2013

Doc2b is a key effector of insulin secretion and skeletal muscle insulin sensitivity.

Diabetes 2012 Oct 14;61(10):2424-32. Epub 2012 Jun 14.

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.

Exocytosis of intracellular vesicles, such as insulin granules, is carried out by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. An additional regulatory protein, Doc2b (double C2 domain), has recently been implicated in exocytosis from clonal β-cells and 3T3-L1 adipocytes. Here, we investigated the role of Doc2b in insulin secretion, insulin sensitivity, and the maintenance of whole-body glucose homeostasis. Doc2b heterozygous (Doc2b(+/-)) and homozygous (Doc2b(-/-)) knockout mice exhibited significant whole-body glucose intolerance and peripheral insulin resistance, compared with wild-type littermates. Correspondingly, Doc2b(+/-) and Doc2b(-/-) mice exhibited decreased responsiveness of pancreatic islets to glucose in vivo, with significant attenuation of both phases of insulin secretion ex vivo. Peripheral insulin resistance correlated with ablated insulin-stimulated glucose uptake and GLUT4 vesicle translocation in skeletal muscle from Doc2b-deficient mice, which was coupled to impairments in Munc18c-syntaxin 4 dissociation and in SNARE complex assembly. Hence, Doc2b is a key positive regulator of Munc18c-syntaxin 4-mediated insulin secretion as well as of insulin responsiveness in skeletal muscle, and thus a key effector for glucose homeostasis in vivo. Doc2b's actions in glucose homeostasis may be related to its ability to bind Munc18c and/or directly promote fusion of insulin granules and GLUT4 vesicles in a stimulus-dependent manner.
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http://dx.doi.org/10.2337/db11-1525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3447898PMC
October 2012

Paroxysmal kinesigenic dyskinesia: cortical or non-cortical origin.

Parkinsonism Relat Disord 2012 Jun 30;18(5):645-8. Epub 2012 Mar 30.

Department of Neurology, Sint Lucas Andreas Hospital, Amsterdam, The Netherlands.

Paroxysmal kinesigenic dyskinesia (PKD) is characterized by involuntary dystonia and/or chorea triggered by a sudden movement. Cases are usually familial with an autosomal dominant inheritance. Hypotheses regarding the pathogenesis of PKD focus on the controversy whether PKD has a cortical or non-cortical origin. A combined familial trait of PKD and benign familial infantile seizures has been reported as the infantile convulsions and paroxysmal choreoathetosis (ICCA) syndrome. Here, we report a family diagnosed with ICCA syndrome with an Arg217STOP mutation. The index patient showed interictal EEG focal changes compatible with paroxysmal dystonic movements of his contralateral leg. This might support cortical involvement in PKD.
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http://dx.doi.org/10.1016/j.parkreldis.2012.03.006DOI Listing
June 2012

Deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and early postnatal lethality.

PLoS One 2011 29;6(11):e28137. Epub 2011 Nov 29.

Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Neuroscience Campus Amsterdam, VU University, Amsterdam, The Netherlands.

The serotonin (5-HT) system densely innervates many brain areas and is important for proper brain development. To specifically ablate the 5-HT system we generated mutant mice carrying a floxed Munc18-1 gene and Cre recombinase driven by the 5-HT-specific serotonin reuptake transporter (SERT) promoter. The majority of mutant mice died within a few days after birth. Immunohistochemical analysis of brains of these mice showed that initially 5-HT neurons are formed and the cortex is innervated with 5-HT projections. From embryonic day 16 onwards, however, 5-HT neurons started to degenerate and at postnatal day 2 hardly any 5-HT projections were present in the cortex. The 5-HT system of mice heterozygous for the floxed Munc18-1 allele was indistinguishable from control mice. These data show that deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and suggests that the 5-HT system is important for postnatal survival.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028137PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3226659PMC
April 2012

Multiple Ca2+ sensors in secretion: teammates, competitors or autocrats?

Trends Neurosci 2011 Sep 9;34(9):487-97. Epub 2011 Aug 9.

Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, Vrije Universiteit Amsterdam and VU Medical Center, 1081 HV Amsterdam, The Netherlands.

Regulated neurotransmitter secretion depends on Ca(2+) sensors, C2 domain proteins that associate with phospholipids and soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) complexes to trigger release upon Ca(2+) binding. Ca(2+) sensors are thought to prevent spontaneous fusion at rest (clamping) and to promote fusion upon Ca(2+) activation. At least eight, often coexpressed, Ca(2+) sensors have been identified in mammals. Accumulating evidence suggests that multiple Ca(2+) sensors interact, rather than work autonomously, to produce the complex secretory response observed in neurons and secretory cells. In this review, we present several working models to describe how different sensors might be arranged to mediate synchronous, asynchronous and spontaneous neurotransmitter release. We discuss the scenario that different Ca(2+) sensors typically act on one shared vesicle pool and compete for binding the multiple SNARE complexes that are likely to assemble at single vesicles, to exert both clamping and fusion-promoting functions.
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http://dx.doi.org/10.1016/j.tins.2011.07.003DOI Listing
September 2011

Doc2b is a high-affinity Ca2+ sensor for spontaneous neurotransmitter release.

Science 2010 Mar 11;327(5973):1614-8. Epub 2010 Feb 11.

Department of Functional Genomics, CNCR, Neuroscience Campus Amsterdam, VU University and VU Medical Center, Amsterdam, 1081 HV, Netherlands.

Synaptic vesicle fusion in brain synapses occurs in phases that are either tightly coupled to action potentials (synchronous), immediately following action potentials (asynchronous), or as stochastic events in the absence of action potentials (spontaneous). Synaptotagmin-1, -2, and -9 are vesicle-associated Ca2+ sensors for synchronous release. Here we found that double C2 domain (Doc2) proteins act as Ca2+ sensors to trigger spontaneous release. Although Doc2 proteins are cytosolic, they function analogously to synaptotagmin-1 but with a higher Ca2+ sensitivity. Doc2 proteins bound to N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complexes in competition with synaptotagmin-1. Thus, different classes of multiple C2 domain-containing molecules trigger synchronous versus spontaneous fusion, which suggests a general mechanism for synaptic vesicle fusion triggered by the combined actions of SNAREs and multiple C2 domain-containing proteins.
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http://dx.doi.org/10.1126/science.1183765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846320PMC
March 2010

Chronic activation of the 5-HT(2) receptor reduces 5-HT neurite density as studied in organotypic slice cultures.

Brain Res 2009 Dec 1;1302:1-9. Epub 2009 Sep 1.

Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, Amsterdam, The Netherlands.

The serotonin system densely innervates the brain and is implicated in psychopathological processes. Here we studied the effect of serotonin and serotonin pharmacological compounds on the outgrowth of serotonergic projections using organotypic slice co-cultures of hippocampus and dorsal raphe nuclei. Immunocytochemical analysis showed that several serotonergic neurites had grown into the target slice within 7 days in culture, after which the neurite density stabilized. These projections expressed the serotonin-synthesizing enzyme Tryptophan hydroxylase and the serotonin transporter and contained several serotonin-positive varicosities that also accumulated presynaptic markers. Chronic application of a 5-HT(2) agonist reduced the serotonergic neurite density, without effects on survival of serotonergic neurons. In contrast, application of a 5-HT(1A) agonist or the serotonin transporter inhibitor fluoxetine did not affect serotonergic neurite density. We conclude that serotonergic connectivity was reproduced in vitro and that the serotonin neurite density is inhibited by chronic activation of the 5-HT(2) receptor.
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http://dx.doi.org/10.1016/j.brainres.2009.08.071DOI Listing
December 2009

DOC2B acts as a calcium switch and enhances vesicle fusion.

J Neurosci 2008 Jul;28(27):6794-806

Department of Neurobiology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusion-competent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release.
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http://dx.doi.org/10.1523/JNEUROSCI.0538-08.2008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2673511PMC
July 2008

DOC2A and DOC2B are sensors for neuronal activity with unique calcium-dependent and kinetic properties.

J Neurochem 2006 May 3;97(3):818-33. Epub 2006 Mar 3.

Department of Functional Genomics, Center for Neurogenomics and Cognition Research, Vrije Universiteit (VU) and VU Medical Centre, Amsterdam, the Netherlands.

Elevation of the intracellular calcium concentration ([Ca2+]i) to levels below 1 microm alters synaptic transmission and induces short-term plasticity. To identify calcium sensors involved in this signalling, we investigated soluble C2 domain-containing proteins and found that both DOC2A and DOC2B are modulated by submicromolar calcium levels. Fluorescent-tagged DOC2A and DOC2B translocated to plasma membranes after [Ca2+]i elevation. DOC2B translocation preceded DOC2A translocation in cells co-expressing both isoforms. Half-maximal translocation occurred at 450 and 175 nm[Ca2+]i for DOC2A and DOC2B, respectively. This large difference in calcium sensitivity was accompanied by a modest kinetic difference (halftimes, respectively, 2.6 and 2.0 s). The calcium sensitivity of DOC2 isoforms can be explained by predicted topologies of their C2A domains. Consistently, neutralization of aspartates D218 and D220 in DOC2B changed its calcium affinity. In neurones, both DOC2 isoforms were reversibly recruited to the plasma membrane during trains of action potentials. Consistent with its higher calcium sensitivity, DOC2B translocated at lower depolarization frequencies. Styryl dye uptake experiments in hippocampal neurones suggest that the overexpression of mutated DOC2B alters the synaptic activity. We conclude that both DOC2A and DOC2B are regulated by neuronal activity, and hypothesize that their calcium-dependent translocation may regulate synaptic activity.
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http://dx.doi.org/10.1111/j.1471-4159.2006.03755.xDOI Listing
May 2006

Two distinct genes drive expression of seven tomosyn isoforms in the mammalian brain, sharing a conserved structure with a unique variable domain.

J Neurochem 2005 Feb;92(3):554-68

Department of Functional Genomics, Center for Neurogenomics and Cognition Research, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands.

Tomosyn was previously identified as a syntaxin-binding protein that inhibits soluble NSF (n-ethylmaleimide-sensitive fusion protein) attachment protein receptor (SNARE)-mediated secretion. We set out to investigate the distribution of tomosyn mRNA in the mammalian brain and found evidence for the presence of two paralogous genes designated tomosyn-1 and -2. In a collection of tomosyn-2 cDNA clones, we observed four splice variants (named xb-, b-, m- and s-tomosyn-2) derived from the skipping of exons 19 and 21. This feature is conserved with tomosyn-1 that encodes three splice variants. To compare the expression pattern of tomosyn-1 and -2, we performed in situ hybridization experiments with gene-specific probes. Both genes were expressed in the nervous system, clearly following distinct spatial and developmental expression patterns. Real-time quantitative PCR experiments indicated that tomosyn-1 expression was up-regulated less than threefold between developmental stages E10 and P12, whereas tomosyn-2 expression increased 31-fold. Not only the transcription level, but also the splice composition of tomosyn-2 mRNA shifted during development. We conclude that two distinct genes drive expression of seven tomosyn isoforms. Their expression patterns support a role in regulating neuronal secretion. All isoforms share conserved WD40 and SNARE domains separated by a hypervariable module, the function of which remains to be clarified.
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http://dx.doi.org/10.1111/j.1471-4159.2004.02890.xDOI Listing
February 2005

Ca(2+)-induced recruitment of the secretory vesicle protein DOC2B to the target membrane.

J Biol Chem 2004 May 21;279(22):23740-7. Epub 2004 Mar 21.

Department of Functional Genomics, Center for Neurogenomics and Cognition Research, Vrije Universiteit, De Boelelaan 1085, Amsterdam 1081 HV, The Netherlands.

Ca(2+)-dependent fusion of transport vesicles at their target can be enhanced by intracellular Ca2+ and diacylglycerol. Diacylglycerol induces translocation of the vesicle priming factor Munc13 and association of the secretory vesicle protein DOC2B to the membrane. Here we demonstrate that a rise in intracellular Ca2+ is sufficient for a Munc13-independent recruitment of DOC2B to the target membrane. This novel mechanism occurred readily in the absence of Munc13 and was not influenced by DOC2B mutations that abolish Munc13 binding. Purified DOC2B (expressed as a bacterial fusion protein) bound phospholipids in a Ca(2+)-dependent way, suggesting that the translocation is the result of a C2 domain activation mechanism. Ca(2+)-induced translocation was also observed in cultured neurons expressing DOC2B-enhanced green fluorescent protein. In this case, however, various degrees of membrane association occurred under resting conditions, suggesting that physiological Ca2+ concentrations modulate DOC2B localization. Depolarization of the neurons induced a complete translocation of DOC2B-enhanced green fluorescent protein to the target membrane within 5 s. We hypothesize that this novel Ca(2+)-induced activity of DOC2B functions synergistically with diacylglycerol-induced Munc13 binding to enhance exocytosis during episodes of high secretory activity.
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http://dx.doi.org/10.1074/jbc.M400731200DOI Listing
May 2004