Complexin Has Opposite Effects on Two Modes of Synaptic Vesicle Fusion

Abstract

Summary Background Synaptic transmission can occur in a binary or graded fashion depending on whether transmitter release is triggered by action potentials or by gradual changes in membrane potential. Molecular differences of these two types of fusion events and their differential regulation in a physiological context have yet to be addressed. Complexin is a conserved SNARE-binding protein that has been proposed to regulate both spontaneous and stimulus-evoked synaptic vesicle (SV) fusion. Results Here, we examine complexin function at a graded synapse in C. elegans. Null complexin (cpx-1) mutants are viable although nervous system function is significantly impaired. Loss of CPX-1 results in a 3-fold increase in the rate of tonic synaptic transmission at the neuromuscular junction while stimulus-evoked SV fusion is decreased 10-fold. A truncated CPX-1 missing its C-terminal domain can rescue stimulus-evoked synaptic vesicle exocytosis but fails to suppress tonic activity, demonstrating that these two modes of exocytosis can be distinguished at the molecular level. A CPX-1 variant with impaired SNARE-binding also rescues evoked but not tonic neurotransmitter release. Finally, tonic but not evoked release can be rescued in a syntaxin point mutant by removing CPX-1. Rescue of either form of exocytosis partially restores locomotory behavior indicating that both types of synaptic transmission are relevant. Conclusion These observations suggest a dual role for CPX-1: suppressing SV exocytosis driven by low levels of endogenous neural activity while promoting synchronous fusion of SVs driven by a depolarizing stimulus. Thus, patterns of synaptic activity regulate complexin's inhibitory and permissive roles at a graded synapse.

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

“…In summary, our findings suggest that Cpx promotes Ca 2+ -dependent vesicle fusion by mechanisms that are independent of accessory domain interactions, and instead require residues within the central helix of Cpx. The independent effects on spontaneous vs. evoked release in both the SB and trans interaction-abolishing mutants indicate the Cpx clamping and promoting functions are genetically separable, consistent with prior studies (17,19,20,(31)(32)(33).…”

“…As such, the SB Cpx mutant may impede the transition of the Cpx central helix from an open to closed conformation with a single zippering SNARE complex. Within Cpx, therefore, the central α-helix is the primary domain that associates with SNARE complexes (28), and is absolutely required for all Cpx function (17,20,23). The N-terminal accessory domain is necessary for clamping functions, as demonstrated in this study and others (23,30,41).…”

“…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 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

“…Although the accessory domain is required for regulating spontaneous release, mutations of this domain do not affect the activating function of Cpx compared with wild-type neurons in rescue experiments with Cpx knockdown (28,29), and it can be entirely eliminated while still maintaining the activating function of Cpx in a reconstituted system (30). The α-helical central domain (amino acid residues 49-70) binds to the SNARE complex (31,32) and is essential for all functions of Cpx in all species studied to date, including priming (16,28,29,33), inhibiting spontaneous fusion (12,19,24,25,(34)(35)(36), and activation of Ca 2+ -triggered fusion (12-14, 19, 34, 36-38).…”