Mutations that disrupt Ca2+-binding activity endow Doc2β with novel functional properties during synaptic transmission

Double C2-domain protein (Doc2) is a Ca2+-binding protein implicated in asynchronous and spontaneous neurotransmitter release. Here we demonstrate that each of its C2 domains senses Ca2+; moreover, the tethered tandem C2 domains display properties distinct from the isolated domains. We confirm that overexpression of a mutant form of Doc2β, in which two acidic Ca2+ ligands in the C2A domain and two in the C2B domain have been neutralized, results in markedly enhanced asynchronous release in synaptotagmin 1–knockout neurons. Unlike wild-type (wt) Doc2β, which translocates to the plasma membrane in response to increases in [Ca2+]i, the quadruple Ca2+-ligand mutant does not bind Ca2+ but is constitutively associated with the plasma membrane; this effect is due to substitution of Ca2+ ligands in the C2A domain. When overexpressed in wt neurons, Doc2β affects only asynchronous release; in contrast, Doc2β Ca2+-ligand mutants that constitutively localize to the plasma membrane enhance both the fast and slow components of synaptic transmission by increasing the readily releasable vesicle pool size; these mutants also increase the frequency of spontaneous release events. Thus, mutations in the C2A domain of Doc2β that were intended to disrupt Ca2+ binding result in an anomalous enhancement of constitutive membrane-binding activity and endow Doc2β with novel functional properties.

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Structural elements that underlie Doc2β function during asynchronous synaptic transmission

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1502288112 ◽

2015 ◽

Vol 112 (31) ◽

pp. E4316-E4325 ◽

Cited By ~ 11

Author(s):

Renhao Xue ◽

Jon D. Gaffaney ◽

Edwin R. Chapman

Keyword(s):

Plasma Membrane ◽

Synaptic Transmission ◽

Neurotransmitter Release ◽

Lipid Binding ◽

Slow Phase ◽

Structural Elements ◽

Excitatory Postsynaptic Current ◽

Synaptotagmin 1 ◽

Asynchronous Release

Double C2-like domain-containing proteins alpha and beta (Doc2α and Doc2β) are tandem C2-domain proteins proposed to function as Ca2+ sensors for asynchronous neurotransmitter release. Here, we systematically analyze each of the negatively charged residues that mediate binding of Ca2+ to the β isoform. The Ca2+ ligands in the C2A domain were dispensable for Ca2+-dependent translocation to the plasma membrane, with one exception: neutralization of D220 resulted in constitutive translocation. In contrast, three of the five Ca2+ ligands in the C2B domain are required for translocation. Importantly, translocation was correlated with the ability of the mutants to enhance asynchronous release when overexpressed in neurons. Finally, replacement of specific Ca2+/lipid-binding loops of synaptotagmin 1, a Ca2+ sensor for synchronous release, with corresponding loops from Doc2β, resulted in chimeras that yielded slower kinetics in vitro and slower excitatory postsynaptic current decays in neurons. Together, these data reveal the key determinants of Doc2β that underlie its function during the slow phase of synaptic transmission.

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Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin

Nature Communications ◽

10.1038/s41467-019-12015-w ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 13

Author(s):

Nicholas A. Courtney ◽

Huan Bao ◽

Joseph S. Briguglio ◽

Edwin R. Chapman

Keyword(s):

Synaptic Vesicle ◽

Vesicle Fusion ◽

Snare Proteins ◽

Major Focus ◽

C2 Domains ◽

Releasable Pool ◽

Readily Releasable Pool ◽

Synaptotagmin 1 ◽

Synaptic Vesicle Fusion ◽

C2a Domain

Abstract Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.

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Drosophila Synaptotagmin 7 negatively regulates synaptic vesicle fusion and replenishment in a dosage-dependent manner

10.1101/2020.01.30.926246 ◽

2020 ◽

Author(s):

Zhuo Guan ◽

Mónica C. Quiñones-Frías ◽

Yulia Akbergenova ◽

J. Troy Littleton

Keyword(s):

Plasma Membrane ◽

Synaptic Vesicle ◽

Active Zone ◽

Neurotransmitter Release ◽

Dependent Manner ◽

Short Term ◽

Sensor Model ◽

Synaptotagmin 1 ◽

Asynchronous Release ◽

Synaptic Vesicle Fusion

AbstractSynchronous neurotransmitter release is triggered by Ca2+ binding to the synaptic vesicle protein Synaptotagmin 1, while asynchronous fusion and short-term facilitation is hypothesized to be mediated by plasma membrane-localized Synaptotagmin 7 (SYT7). We generated mutations in Drosophila Syt7 to determine if it plays a conserved role as the Ca2+ sensor for these processes. Electrophysiology and quantal imaging revealed evoked release was elevated 2-fold. Syt7 mutants also had a larger pool of readily-releasable vesicles, faster recovery following stimulation, and robust facilitation. Syt1/Syt7 double mutants displayed more release than Syt1 mutants alone, indicating SYT7 does not mediate the residual asynchronous release remaining in the absence of SYT1. SYT7 localizes to an internal membrane tubular network within the peri-active zone, but does not enrich at release sites. These findings indicate the two Ca2+ sensor model of SYT1 and SYT7 mediating all phases of neurotransmitter release and facilitation is not applicable at Drosophila synapses.

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Exophilin4/Slp2-a Targets Glucagon Granules to the Plasma Membrane through Unique Ca2+-inhibitory Phospholipid-binding Activity of the C2A Domain

Molecular Biology of the Cell ◽

10.1091/mbc.e06-10-0914 ◽

2007 ◽

Vol 18 (2) ◽

pp. 688-696 ◽

Cited By ~ 30

Author(s):

Miao Yu ◽

Kazuo Kasai ◽

Kazuaki Nagashima ◽

Seiji Torii ◽

Hiromi Yokota-Hashimoto ◽

...

Keyword(s):

Plasma Membrane ◽

Secretory Granules ◽

Cell Types ◽

Binding Activity ◽

Effector Proteins ◽

Membrane Phospholipids ◽

Β Cells ◽

Phospholipid Binding ◽

Lysosome Related Organelles ◽

C2a Domain

Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic β cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic α cells, in contrast to another effector, granuphilin, in β cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca2+ concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca2+-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca2+-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.

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Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells

The Journal of Cell Biology ◽

10.1083/jcb.200302060 ◽

2003 ◽

Vol 162 (2) ◽

pp. 199-209 ◽

Cited By ~ 84

Author(s):

Ward C. Tucker ◽

J. Michael Edwardson ◽

Jihong Bai ◽

Hyun-Jung Kim ◽

Thomas F.J. Martin ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Pc12 Cells ◽

Functional Significance ◽

Synaptic Vesicles ◽

Membrane Traffic ◽

Binding Activity ◽

C2 Domains ◽

Expression Levels ◽

Target Molecules

T he synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+–syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I–XI to interfere with endogenous syt–effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin–SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)–binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.

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Synaptotagmin-1 membrane binding is driven by the C2B domain and assisted cooperatively by the C2A domain

Scientific Reports ◽

10.1038/s41598-020-74923-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Clémence Gruget ◽

Oscar Bello ◽

Jeff Coleman ◽

Shyam S. Krishnakumar ◽

Eric Perez ◽

...

Keyword(s):

Neurotransmitter Release ◽

Mutational Analysis ◽

Membrane Binding ◽

Surface Force ◽

Membrane Interaction ◽

C2 Domains ◽

Binding Energetics ◽

Synaptotagmin 1 ◽

Site Binding ◽

C2a Domain

Abstract Synaptotagmin interaction with anionic lipid (phosphatidylserine/phosphatidylinositol) containing membranes, both in the absence and presence of calcium ions (Ca2+), is critical to its central role in orchestrating neurotransmitter release. The molecular surfaces involved, namely the conserved polylysine motif in the C2B domain and Ca2+-binding aliphatic loops on both C2A and C2B domains, are known. Here we use surface force apparatus combined with systematic mutational analysis of the functional surfaces to directly measure Syt1-membrane interaction and fully map the site-binding energetics of Syt1 both in the absence and presence of Ca2+. By correlating energetics data with the molecular rearrangements measured during confinement, we find that both C2 domains cooperate in membrane binding, with the C2B domain functioning as the main energetic driver, and the C2A domain acting as a facilitator.

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Cooperativity and Avidity in Membrane Binding by C2AB Tandem Domains of Synaptotagmins 1 and 7

10.1101/393702 ◽

2018 ◽

Author(s):

H. Tran ◽

L. Anderson ◽

J. Knight

Keyword(s):

Hormone Secretion ◽

Membrane Binding ◽

Dissociation Rate ◽

Dissociation Kinetics ◽

Kinetic Measurements ◽

C2 Domains ◽

Synaptotagmin 1 ◽

Fusion Pores ◽

Fluorescence Kinetic ◽

C2a Domain

AbstractSynaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together serve as a Ca2+ sensor to bind membranes and promote the stabilization of exocytotic fusion pores. Functionally, Syt-1 triggers fast release of neurotransmitters, while Syt-7 is involved in lower-Ca2+ processes such as hormone secretion. Evidence suggests that Syt-1 C2 domains bind membranes cooperatively, penetrating farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously reported that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we have investigated the effects of C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. We report Ca2+ sensitivities, dissociation kinetics, and membrane insertion using liposomes approximating physiological lipid compositions. Equilibrium Ca2+ titrations confirm that the Syt-7 C2AB tandem has a greater Ca2+ sensitivity of membrane binding than either of its individual domains. Stopped-flow fluorescence kinetic measurements show that Syt-1 C2AB dissociates from liposome membranes much more slowly than either of its isolated C2 domains, suggesting that the two C2 domains of Syt-1 bind membranes cooperatively. In contrast, the dominant population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, indicating a lack of cooperativity, while only a small subpopulation dissociates at a slower rate. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts more deeply into membranes as part of the C2AB tandem, similarly to Syt-1. Overall, these measurements are consistent with a model in which the structural linkage of C2A and C2B impacts the membrane-binding geometry of synaptotagmin C2B domains, but imparts little or no cooperativity to Syt-7 membrane binding and dissociation events that are dominated by its C2A domain.

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