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

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.

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

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.

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

2019 ◽  
Vol 10 (1) ◽  
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.

scholarly journals Synaptotagmin-1 C2B domain interacts simultaneously with SNAREs and membranes to promote membrane fusion

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Shen Wang ◽  
Yun Li ◽  
Cong Ma
Keyword(s):  
Membrane Fusion ◽  
Neurotransmitter Release ◽  
Functional Significance ◽  
Underlying Mechanism ◽  
C2 Domains ◽  
Membrane Deformation ◽  
Synaptotagmin 1 ◽  
Bottom Face ◽  
Liposome Fusion

Synaptotagmin-1 (Syt1) acts as a Ca2+ sensor for neurotransmitter release through its C2 domains. It has been proposed that Syt1 promotes SNARE-dependent fusion mainly through its C2B domain, but the underlying mechanism is poorly understood. In this study, we show that the C2B domain interacts simultaneously with acidic membranes and SNARE complexes via the top Ca2+-binding loops, the side polybasic patch, and the bottom face in response to Ca2+. Disruption of the simultaneous interactions completely abrogates the triggering activity of the C2B domain in liposome fusion. We hypothesize that the simultaneous interactions endow the C2B domain with an ability to deform local membranes, and this membrane-deformation activity might underlie the functional significance of the Syt1 C2B domain in vivo.

scholarly journals Polybasic patches in both C2 domains of Synaptotagmin-1 are required for evoked neurotransmitter release

2021 ◽  
Author(s):  
Zhenyong Wu ◽  
Lu Ma ◽  
Nicholas A Courtney ◽  
Jie Zhu ◽  
Yongli Zhang ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Calcium Binding ◽  
Neurotransmitter Release ◽  
Transmembrane Domain ◽  
Snare Complex ◽  
Critical Feature ◽  
C2 Domains ◽  
Electrophysiological Recordings ◽  
Synaptotagmin 1

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem 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 not well understood. Calcium binding to C2B, but not to C2A, 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 (Syt1K189-192A and Syt1K326-327A) side-by-side to determine their relative contributions to Syt1 function in cultured cortical mouse neurons 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 readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.

scholarly journals Phosphatidylinositol 4,5 bisphosphate controls the cis and trans interactions of synaptotagmin 1

2019 ◽  
Author(s):  
S.B. Nyenhuis ◽  
A. Thapa ◽  
D. S. Cafiso
Keyword(s):  
Neurotransmitter Release ◽  
Plasma Membranes ◽  
Full Length ◽  
Membrane Insertion ◽  
Membrane Interface ◽  
Vesicle Membrane ◽  
C2 Domains ◽  
Synaptotagmin 1 ◽  
Site Directed Spin Labeling ◽  
Cis And Trans

AbstractSynaptotagmin 1 acts as the Ca2+-sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here we describe measurements on full-length membrane reconstituted synaptotagmin 1 using site-directed spin labeling where we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca2+; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate (PIP2), even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca2+, the Ca2+-binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS containing bilayers and will bind in a cis configuration to membranes containing PS even if a PIP2 membrane is presented in trans. The data are consistent with a bridging activity for Syt1 where the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca2+ and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca2+.

scholarly journals Functional and Biochemical Analysis of the C2 Domains of Synaptotagmin IV

1999 ◽  
Vol 10 (7) ◽  
pp. 2285-2295 ◽  
Author(s):  
David M. Thomas ◽  
Gregory D. Ferguson ◽  
Harvey R. Herschman ◽  
Lisa A. Elferink
Keyword(s):  
Pc12 Cells ◽  
Membrane Trafficking ◽  
Calcium Binding ◽  
Neurotransmitter Release ◽  
Biochemical Properties ◽  
Binding Properties ◽  
Independent Manner ◽  
C2 Domains ◽  
Calcium Dependent ◽  
C2a Domain

Synaptotagmins (Syts) are a family of vesicle proteins that have been implicated in both regulated neurosecretion and general membrane trafficking. Calcium-dependent interactions mediated through their C2 domains are proposed to contribute to the mechanism by which Syts trigger calcium-dependent neurotransmitter release. Syt IV is a novel member of the Syt family that is induced by cell depolarization and has a rapid rate of synthesis and a short half-life. Moreover, the C2A domain of Syt IV does not bind calcium. We have examined the biochemical and functional properties of the C2 domains of Syt IV. Consistent with its non–calcium binding properties, the C2A domain of Syt IV binds syntaxin isoforms in a calcium-independent manner. In neuroendocrine pheochromocytoma (PC12) cells, Syt IV colocalizes with Syt I in the tips of the neurites. Microinjection of the C2A domain reveals that calcium-independent interactions mediated through this domain of Syt IV inhibit calcium-mediated neurotransmitter release from PC12 cells. Conversely, the C2B domain of Syt IV contains calcium binding properties, which permit homo-oligomerization as well as hetero-oligomerization with Syt I. Our observation that different combinatorial interactions exist between Syt and syntaxin isoforms, coupled with the calcium stimulated hetero-oligomerization of Syt isoforms, suggests that the secretory machinery contains a vast repertoire of biochemical properties for sensing calcium and regulating neurotransmitter release accordingly.

scholarly journals A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release

2021 ◽  
Vol 220 (4) ◽  
Author(s):  
Lei Li ◽  
Haowen Liu ◽  
Mia Krout ◽  
Janet E. Richmond ◽  
Yu Wang ◽  
...  
Keyword(s):  
Neurotransmitter Release ◽  
Release Kinetics ◽  
Sensor System ◽  
C2 Domains ◽  
Loosely Coupled ◽  
C Elegans ◽  
Membrane Tethering ◽  
Synaptotagmin 1 ◽  
Tm Domain ◽  
Fast Release

Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.

scholarly journals Synaptotagmin-1 C2B domains cooperatively stabilize the fusion stalk via a master-servant mechanism

2021 ◽  
Author(s):  
Ary Lautaro Di Bartolo ◽  
Diego Masone
Keyword(s):  
Free Energy ◽  
Membrane Fusion ◽  
Membrane Binding ◽  
Vesicle Fusion ◽  
Fusion Process ◽  
Total Work ◽  
C2 Domains ◽  
Domain Interactions ◽  
Synaptotagmin 1 ◽  
Data Point

Synaptotagmin-1 is a low-affinity Ca2+ sensor that triggers synchronous vesicle fusion. It contains two similar C2 domains (C2A and C2B) that cooperate in membrane binding, being the C2B domain the main responsible for the membrane fusion process due to its polybasic patch KRLKKKKTTIKK (321-332). In this work, a master-servant mechanism between two identical C2B domains is shown to control the formation of the fusion stalk. Two regions in C2B are essential for the process, the well-known polybasic patch and a recently described pair of arginines (398,399). The master domain shows strong PIP2 interactions with its polybasic patch and its pair of arginines. At the same time, the servant analogously cooperates with the master to reduce the total work to form the fusion stalk. The strategic mutation (T328E,T329E) in both master and servant domains disrupts the cooperative mechanism, drastically increasing the free energy needed to induce the fusion stalk, however with negligible effects on the master domain interactions with PIP2. These data point to a difference in the behavior of the servant domain, which is unable to sustain its PIP2 interactions neither through its polybasic patch nor through its pair of arginines, in the end losing its ability to assist the master in the formation of the fusion stalk.

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