Position of Synaptotagmin I at the Membrane Interface:  Cooperative Interactions of Tandem C2 Domains†

Biochemistry ◽  
2006 ◽  
Vol 45 (32) ◽  
pp. 9668-9674 ◽  
Author(s):  
Dawn Z. Herrick ◽  
Stephenie Sterbling ◽  
Katie A. Rasch ◽  
Anne Hinderliter ◽  
David S. Cafiso
Keyword(s):  
Membrane Interface ◽  
C2 Domains ◽  
Cooperative Interactions ◽  
Synaptotagmin I

scholarly journals The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

2000 ◽  
Vol 150 (5) ◽  
pp. 1125-1136 ◽  
Author(s):  
Radhika C. Desai ◽  
Bimal Vyas ◽  
Cynthia A. Earles ◽  
J. Troy Littleton ◽  
Judith A. Kowalchyck ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Pc12 Cells ◽  
Conformational Changes ◽  
Cytoplasmic Domain ◽  
Dominant Negative ◽  
Fusion Pore ◽  
Vesicle Membrane ◽  
C2 Domains ◽  
Essential Step ◽  
Synaptotagmin I

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

scholarly journals Negative Coupling as a Mechanism for Signal Propagation between C2 Domains of Synaptotagmin I

PLoS ONE ◽  
2012 ◽  
Vol 7 (10) ◽  
pp. e46748 ◽  
Author(s):  
Michael E. Fealey ◽  
Jacob W. Gauer ◽  
Sarah C. Kempka ◽  
Katie Miller ◽  
Kamakshi Nayak ◽  
...  
Keyword(s):  
Signal Propagation ◽  
C2 Domains ◽  
Synaptotagmin I ◽  
Negative Coupling

scholarly journals The tandem C2 domains of synaptotagmin contain redundant Ca2+ binding sites that cooperate to engage t-SNAREs and trigger exocytosis

2001 ◽  
Vol 154 (6) ◽  
pp. 1117-1124 ◽  
Author(s):  
Cynthia A. Earles ◽  
Jihong Bai ◽  
Ping Wang ◽  
Edwin R. Chapman
Keyword(s):  
Membrane Fusion ◽  
Real Time ◽  
Pc12 Cells ◽  
Binding Sites ◽  
C2 Domain ◽  
C2 Domains ◽  
Synaptotagmin I ◽  
Target Membrane ◽  
Complete Disruption

Real-time voltammetry measurements from cracked PC12 cells were used to analyze the role of synaptotagmin–SNARE interactions during Ca2+-triggered exocytosis. The isolated C2A domain of synaptotagmin I neither binds SNAREs nor inhibits norepinephrine secretion. In contrast, two C2 domains in tandem (either C2A-C2B or C2A-C2A) bind strongly to SNAREs, displace native synaptotagmin from SNARE complexes, and rapidly inhibit exocytosis. The tandem C2 domains of synaptotagmin cooperate via a novel mechanism in which the disruptive effects of Ca2+ ligand mutations in one C2 domain can be partially alleviated by the presence of an adjacent C2 domain. Complete disruption of Ca2+-triggered membrane and target membrane SNARE interactions required simultaneous neutralization of Ca2+ ligands in both C2 domains of the protein. We conclude that synaptotagmin–SNARE interactions regulate membrane fusion and that cooperation between synaptotagmin's C2 domains is crucial to its function.

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 Lipid Targeting of Synaptotagmin I C2 Domains on Asymmetric Two-Phase Planar Bilayers

2010 ◽  
Vol 98 (3) ◽  
pp. 483a
Author(s):  
Chen Wan ◽  
Volker Kiessling ◽  
Lukas K. Tamm
Keyword(s):  
Two Phase ◽  
Planar Bilayers ◽  
C2 Domains ◽  
Synaptotagmin I

scholarly journals Partitioning of Synaptotagmin I C2 Domains between Liquid-Ordered and Liquid-Disordered Inner Leaflet Lipid Phases

Biochemistry ◽  
2011 ◽  
Vol 50 (13) ◽  
pp. 2478-2485 ◽  
Author(s):  
Chen Wan ◽  
Volker Kiessling ◽  
David S. Cafiso ◽  
Lukas K. Tamm
Keyword(s):  
C2 Domains ◽  
Synaptotagmin I ◽  
Liquid Ordered

scholarly journals Pull-down combined with proteomic strategy reveals functional diversity of synaptotagmin I

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2973
Author(s):  
Tianyao Guo ◽  
Zhigui Duan ◽  
Jia Chen ◽  
Chunliang Xie ◽  
Ying Wang ◽  
...  
Keyword(s):  
Synaptic Transmission ◽  
Functional Diversity ◽  
Membrane Trafficking ◽  
Metabolic Regulation ◽  
C2 Domains ◽  
Cellular Processes ◽  
Functional Regions ◽  
Synaptotagmin I ◽  
Vesicular Membrane ◽  
The Brain

Synaptotagmin I (Syt I) is most abundant in the brain and is involved in multiple cellular processes. Its two C2 domains, C2A and C2B, are the main functional regions. Our present study employed a pull-down combined with proteomic strategy to identify the C2 domain-interacting proteins to comprehensively understand the biological roles of the C2 domains and thus the functional diversity of Syt I. A total of 135 non-redundant proteins interacting with the C2 domains of Syt I were identified. Out of them, 32 and 64 proteins only bound to C2A or C2B domains, respectively, and 39 proteins bound to both of them. Compared with C2A, C2B could bind to many more proteins particularly those involved in synaptic transmission and metabolic regulation. Functional analysis indicated that Syt I may exert impacts by interacting with other proteins on multiple cellular processes, including vesicular membrane trafficking, synaptic transmission, metabolic regulation, catalysis, transmembrane transport and structure formation, etc. These results demonstrate that the functional diversity of Syt I is higher than previously expected, that its two domains may mediate the same and different cellular processes cooperatively or independently, and that C2B domain may play even more important roles than C2A in the functioning of Syt I. This work not only further deepened our understanding of the functional diversity of Syt I and the functional differences between its two C2 domains, but also provided important clues for the further related researches.

scholarly journals High affinity interactions of Pb2+ with Synaptotagmin I

2018 ◽  
Author(s):  
Sachin Katti ◽  
Bin Her ◽  
Atul K. Srivastava ◽  
Alexander B. Taylor ◽  
Steve W. Lockless ◽  
...  
Keyword(s):  
Rate Constants ◽  
C2 Domains ◽  
High Affinity ◽  
Synaptotagmin I ◽  
Diffusion Limited ◽  
Oxygen Coordination ◽  
Low Concentrations ◽  
Kinetic Trap ◽  
Synaptic Neurotransmission ◽  
Coordination Spheres

ABSTRACTLead (Pb) is a potent neurotoxin that disrupts synaptic neurotransmission. We report that Synaptotagmin I (SytI), a key regulator of Ca2+-evoked neurotransmitter release, has two high-affinity Pb2+ binding sites that belong to its cytosolic C2A and C2B domains. The crystal structures of Pb2+-complexed C2 domains revealed that protein-bound Pb2+ ions have holodirected coordination geometries and all-oxygen coordination spheres. The on-rate constants of Pb2+ binding to the C2 domains of SytI are comparable to those of Ca2+ and are diffusion-limited. In contrast, the off-rate constants are at least two orders of magnitude smaller, indicating that Pb2+ can serve as both thermodynamic and kinetic trap for the C2 domains. We demonstrate, using NMR spectroscopy, that population of these sites by Pb2+ ions inhibits further Ca2+ binding despite the existing coordination vacancies. Our work offers a unique insight into the bioinorganic chemistry of Pb(II) and suggests a mechanism by which low concentrations of Pb2+ ions can interfere with the Ca2+-dependent function of SytI in the cell.

scholarly journals High affinity interactions of Pb2+ with synaptotagmin I

Metallomics ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 1211-1222 ◽  
Author(s):  
Sachin Katti ◽  
Bin Her ◽  
Atul K. Srivastava ◽  
Alexander B. Taylor ◽  
Steve W. Lockless ◽  
...  
Keyword(s):  
Membrane Interactions ◽  
C2 Domains ◽  
High Affinity ◽  
Synaptotagmin I

Pb2+ binds C2 domains with high affinity, desensitizes them to Ca2+, and supports their membrane interactions.

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