Abstract
The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C2A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch.
Highlights
Ca2+ binding by synaptotagmin triggers the fast, synchronous fusion of maximally-primed synaptic vesicles thereby releasing neurotransmitter onto the postsynaptic cell [1, 2]
We show two hydrophobic residues of the C2A domain are absolutely required for synaptotagmin-triggered neurotransmitter release
After over twenty years of research, we demonstrate that the C2A domain of synaptotagmin is an essential component of the Ca2+ sensor for triggering synaptic transmission in vivo
Summary
Ca2+ binding by synaptotagmin triggers the fast, synchronous fusion of maximally-primed synaptic vesicles thereby releasing neurotransmitter onto the postsynaptic cell [1, 2]. Synaptotagmin is an integral membrane protein found on synaptic vesicles whose cytosolic domain is composed of two Ca2+-binding C2 domains, C2A and C2B [Fig 1A and 1B, [7]]. One end of each C2 domain contains three loops of amino acids, two of which form a Ca2+-binding pocket: loops 1 and 3 contain five negatively-charged aspartate residues that coordinate Ca2+[8, 9]. Prior to Ca2+ influx, the net negative charge of each Ca2+-binding pocket results in electrostatic repulsion of the negatively-charged presynaptic membrane, preventing fusion.
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