Complexin Controls Spontaneous and Evoked Neurotransmitter Release by Regulating the Timing and Properties of Synaptotagmin Activity

Abstract

Neurotransmitter release following synaptic vesicle (SV) fusion is the fundamental mechanism for neuronal communication. Synaptic exocytosis is a specialized form of intercellular communication that shares a common SNARE-mediated fusion mechanism with other membrane trafficking pathways. The regulation of synaptic vesicle fusion kinetics and short-term plasticity is critical for rapid encoding and transmission of signals across synapses. Several families of SNARE-binding proteins have evolved to regulate synaptic exocytosis, including Synaptotagmin (SYT) and Complexin (CPX). Here we demonstrate that Drosophila CPX controls evoked fusion occurring via the synchronous and asynchronous pathways. cpx−/− mutants show increased asynchronous release, while CPX overexpression largely eliminates the asynchronous component of fusion. We also find that SYT and CPX co-regulate the kinetics and Ca2+ cooperativity of neurotransmitter release. CPX functions as a positive regulator of release in part by coupling the Ca2+ sensor SYT to the fusion machinery and synchronizing its activity to speed fusion. In contrast, syt−/−; cpx−/− double mutants completely abolish the enhanced spontaneous release observe in cpx−/− mutants alone, indicating CPX acts as a fusion clamp to block premature exocytosis in part by preventing inappropriate activation of the SNARE machinery by SYT. CPX levels also control the size of synaptic vesicle pools, including the immediate releasable pool and the ready releasable pool – key elements of short-term plasticity that define the ability of synapses to sustain responses during burst firing. These observations indicate CPX regulates both spontaneous and evoked fusion by modulating the timing and properties of SYT activation during the synaptic vesicle cycle.

Genetic analysis of the Complexin trans-clamping model for cross-linking SNARE complexes in vivo

“…3). Although we cannot exclude the possibility that these mutants exert subtle effects on vesicle pool sizes, or vesicle release kinetics and synchronicity (19,21,31,35), our findings indicate a trans Cpx/SNARE array is not absolutely required for vesicle release following an action potential.…”

“…In addition to clamping release to prevent spontaneous fusion, Cpx also promotes Syt-dependent vesicle fusion (16,17,(19)(20)(21)(22). Ca 2+ -dependent triggering of release has been suggested to require a conformational switch in Cpx from an open (Cpx extends away from the SNARE surface at a ∼45°) to a closed (Cpx lies on the surface of a postfusion SNARE complex) state (Fig.…”

“…In addition to its role as a fusion clamp, Cpx also promotes Ca 2+ -triggered release in both Drosophila and mammals (16,18,21,29,30). cpx null mutants exhibited reduced evoked release, measured as evoked junctional potentials (EJPs) in both low and high Ca 2+ concentrations (16) (Fig.…”

“…In addition to enhanced minis, cpx mutants have reduced evoked release. Together with data from other genetic systems (17)(18)(19)(20)(21), an emerging model is that Cpx plays a dual role in release, acting as a vesicle fusion clamp that prevents spurious fusion of vesicles at some synapses, while simultaneously promoting synchronous release in response to an action potential at all synapses (16,17,(19)(20)(21)(22). Structurally, these dual activities are likely to require an α-helix motif found within Cpx that is divided into an accessory and central helix region (Fig.…”

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

“…The activating function of complexin is conserved across all species (mammals, Drosophila, and C. elegans) and different types of Ca 2+ -triggered synaptic vesicle fusion studied to date (7,12,16,28,(34)(35)(36)(37)(51)(52)(53). Regulation of spontaneous release by complexin is less conserved among species and varies depending on experimental conditions: for example, in Drosophila spontaneous release increases with knockout of complexin (14,54). Similarly, knockdown in cultured cortical neurons increases spontaneous release, although knockout of complexin in mice only affects spontaneous release depending on the particular neuronal cell type (12,16,27,28).…”