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.

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 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 PKCα: a versatile key for decoding the cellular calcium toolkit

2006 ◽  
Vol 174 (4) ◽  
pp. 521-533 ◽  
Author(s):  
Gregor Reither ◽  
Michael Schaefer ◽  
Peter Lipp
Keyword(s):  
Signal Transduction ◽  
Protein Kinases ◽  
Essential Role ◽  
Flash Photolysis ◽  
Slow Phase ◽  
C2 Domain ◽  
Membrane Interactions ◽  
Binding Region ◽  
C2 Domains ◽  
Cellular Calcium

Conventional protein kinases C (cPKCs) play an essential role in signal transduction and are believed to integrate both global Ca2+ transients and diacylglycerol signals. We provide evidence that PKCα is a ubiquitous readout sensor for the cellular Ca2+ toolkit, including highly restricted elementary Ca2+ release. Threshold stimulations of cells with Ca2+-mobilizing agonists resulted in PKCα translocation events with limited spatial spreads (<4 μm) comprising two groups of lifetimes; brief events (400–1,500 ms) exclusively mediated by Ca2+–C2 domain membrane interactions and long-lasting events (>4 s) resulting from longer DAG-C1a domain–mediated membrane interactions. Although upon uncaging NP-EGTA, which is a caged Ca2+ compound, WT-PKCα displayed rapid membrane translocations within <250 ms, PKCα constructs with C2 domains mutated in their Ca2+-binding region lacked any Ca2+-dependent translocation. Flash photolysis of diazo-2, a photosensitive caged Ca2+ buffer, revealed a biphasic membrane dissociation (slow and fast period) of WT-PKCα. The slow phase was absent in cells expressing PKCα-constructs containing mutated C1a-domains with largely reduced DAG binding. Thus, two groups of PKCα membrane interactions coexist; C2- and C1a-mediated interactions with different lifetimes but rapid interconversion. We conclude that PKCα can readout very fast and, spatially and temporally, very complex cellular Ca2+ signals. Therefore, cPKCs are important transducers for the ubiquitous cellular Ca2+ signaling toolkit.

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 Single-molecule force spectroscopy of protein-membrane interactions

2017 ◽  
Author(s):  
Lu Ma ◽  
Yiying Cai ◽  
Yanghui Li ◽  
Junyi Jiao ◽  
Zhenyong Wu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Membrane Binding ◽  
Force Spectroscopy ◽  
Binding Energies ◽  
Membrane Interactions ◽  
Spatiotemporal Resolution ◽  
Single Molecule Force Spectroscopy ◽  
C2 Domains ◽  
Protein Membrane

AbstractMany biological processes rely on protein-membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2-7 pN and had binding energies of 4-14 kBT per C2 domain. Regulation by bilayer composition or Ca2+ recapitulated known properties of both proteins. The method can be widely applied to study protein-membrane interactions.

scholarly journals Single-molecule force spectroscopy of protein-membrane interactions

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Lu Ma ◽  
Yiying Cai ◽  
Yanghui Li ◽  
Junyi Jiao ◽  
Zhenyong Wu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Membrane Binding ◽  
Force Spectroscopy ◽  
Binding Energies ◽  
Membrane Interactions ◽  
Spatiotemporal Resolution ◽  
Single Molecule Force Spectroscopy ◽  
C2 Domains ◽  
Protein Membrane

Many biological processes rely on protein–membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach, single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2–7 pN and had binding energies of 4–14 kBT per C2 domain. Regulation by bilayer composition or Ca2+ recapitulated known properties of both proteins. The method can be widely applied to study protein–membrane interactions.

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 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
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