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The Journal of Neuroscience, July 23, 2008, 28(30):7458-7466; doi:10.1523/JNEUROSCI.0197-08.2008

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Cellular/Molecular
Ca²⁺-Dependent, Phospholipid-Binding Residues of Synaptotagmin Are Critical for Excitation–Secretion Coupling In Vivo

Brie E. Paddock,¹ Amelia R. Striegel,¹ Enfu Hui,² Edwin R. Chapman,² and Noreen E. Reist¹

¹Molecular, Cellular, and Integrative Neuroscience Program, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523, and ²Howard Hughes Medical Institute and Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706

Correspondence should be addressed to Dr. Noreen E. Reist, Molecular, Cellular, and Integrative Neuroscience Program, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1617. Email: reist{at}lamar.colostate.edu

Synaptotagmin I is the Ca²⁺ sensor for fast, synchronous release of neurotransmitter; however, the molecular interactions that couple Ca²⁺ binding to membrane fusion remain unclear. The structure of synaptotagmin is dominated by two C₂ domains that interact with negatively charged membranes after binding Ca²⁺. In vitro work has implicated a conserved basic residue at the tip of loop 3 of the Ca²⁺-binding pocket in both C₂ domains in coordinating this electrostatic interaction with anionic membranes. Although results from cultured cells suggest that the basic residue of the C₂A domain is functionally significant, such studies provide contradictory results regarding the importance of the C₂B basic residue during vesicle fusion. To directly test the functional significance of each of these residues at an intact synapse in vivo, we neutralized either the C₂A or the C₂B basic residue and assessed synaptic transmission at the Drosophila neuromuscular junction. The conserved basic residues at the tip of the Ca²⁺-binding pocket of both the C₂A and C₂B domains mediate Ca²⁺-dependent interactions with anionic membranes and are required for efficient evoked transmitter release. Our results directly support the hypothesis that the interactions between synaptotagmin and the presynaptic membrane, which are mediated by the basic residues at the tip of both the C₂A and C₂B Ca²⁺-binding pockets, are critical for coupling Ca²⁺ influx with vesicle fusion during synaptic transmission in vivo. Our model for synaptotagmin's direct role in coupling Ca²⁺ binding to vesicle fusion incorporates this finding with results from multiple in vitro and in vivo studies.

Key words: synaptotagmin; synaptic vesicle fusion; anionic phospholipid interactions; site-directed mutagenesis; electrophysiology; calcium dependence; Western analysis; immunohistochemistry; Drosophila

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Received May 15, 2007; revised June 3, 2008; accepted June 5, 2008.

Correspondence should be addressed to Dr. Noreen E. Reist, Molecular, Cellular, and Integrative Neuroscience Program, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1617. Email: reist{at}lamar.colostate.edu

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