1. Molecular determinants of complexin clamping and activation function
- Author
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Manindra Bera, Sathish Ramakrishnan, Jeff Coleman, Shyam S Krishnakumar, and James E Rothman
- Subjects
synaptic vesicle ,membrane fusion ,calcium regulation ,complexin ,synaptotagmin ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al., 2020). Here, using the same in vitro single-vesicle fusion assay, we determine the molecular details of the Complexin-mediated fusion clamp and its role in Ca2+-activation. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins enhances this functionality. The C-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Independent of their clamping functions, the accessory-central helical domains of Complexin also contribute to rapid Ca2+-synchronized vesicle release by increasing the probability of fusion from the clamped state.
- Published
- 2022
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