Deconstructing complexin function in activating and clamping Ca ²⁺ -triggered exocytosis by comparing knockout and knockdown phenotypes

Abstract

Complexin, a presynaptic protein that avidly binds to assembled SNARE complexes, is widely acknowledged to activate Ca 2+ -triggered exocytosis. In addition, studies of invertebrate complexin mutants and of mouse neurons with a double knockdown (DKD) of complexin-1 and -2 suggested that complexin maintains the readily releasable pool (RRP) of vesicles and clamps spontaneous exocytosis. In contrast, studies of mouse neurons with a double knockout (DKO) of complexin-1 and -2, largely carried out in hippocampal autapses, did not detect changes in the RRP size or in spontaneous exocytosis. To clarify complexin function, we here directly compared in two different preparations, cultured cortical and olfactory bulb neurons, the phenotypes of complexin DKD and DKO neurons. We find that complexin-deficient DKD and DKO neurons invariably exhibit a ∼50% decrease in vesicle priming. Moreover, the DKD consistently increased spontaneous exocytosis, but the DKO did so in cortical but not olfactory bulb neurons. Furthermore, the complexin DKD but not the complexin DKO caused a compensatory increase in complexin-3 and -4 mRNA levels; overexpression of complexin-3 but not complexin-1 increased spontaneous exocytosis. Complexin-3 but not complexin-1 contains a C-terminal lipid anchor attaching it to the plasma membrane; addition of a similar lipid anchor to complexin-1 converted complexin-1 from a clamp into an activator of spontaneous exocytosis. Viewed together, our data suggest that complexin generally functions in priming and Ca 2+ triggering of exocytosis, and additionally contributes to the control of spontaneous exocytosis dependent on the developmental history of a neuron and on the subcellular localization of the complexin. synaptic transmission | neurotransmitter release | synaptotagmin | SNARE protein | membrane fusion

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

“…Although Cpx was present throughout neurons, there was a preference for synaptic localization ( Fig. 2 A and C fused to the C-terminal palmitoylated sequence of cysteine-string protein-α (CSPα), a sequence that targets synaptic vesicles (46); and Cpx 1-86-CAIM is a chimera of Cpx fused to the C-terminal isoprenylation sequence (CAIM) that targets the plasma membrane (28). Immunocytochemistry experiments, similar to above, were again performed in lentivirus-infected neurons (Fig.…”

“…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).…”

“…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). A split pair of N-terminal and central domain fragments of Cpx is sufficient to activate Ca 2+ -triggered release using our reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery (30).…”

“…The accessory domain (amino acid residues 28-48) regulates spontaneous fusion in neurons and it suppresses Ca 2+ -independent fusion in reconstituted systems (12,14,19,(22)(23)(24)(25)(26)(27). 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).…”

Targeting Synaptic Pathology with a Novel Affinity Mass Spectrometry Approach

“…Apart from the SNARE complex proteins, several other proteins that are known or suspected to interact with the SNARE complex (e.g. complexin-1, complexin-2, syntaxin-binding protein-1, and synaptotagmin-1) were co-immunoprecipitated with SNAP-25 (supplemental Table S2) (31)(32)(33).…”