N-terminal domain of complexin independently activates calcium-triggered fusion

Complexin activates Ca²⁺-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca²⁺-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca²⁺-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca²⁺-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca²⁺-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca²⁺-triggered release.Neurotransmitter release occurs upon fusion of synaptic vesicles with the plasma membrane (1, 2). Synaptic vesicle fusion is orchestrated by the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) fusion proteins (3, 4), in conjunction with synaptotagmin, complexin, and other synaptic proteins. The Ca²⁺-sensor synaptotagmin is essential for Ca²⁺-triggered release (5–8). Neuronal SNARE proteins form a ternary complex consisting of synaptobrevin/vesicle-associated membrane protein (VAMP2), syntaxin, and synaptosomal-associated protein 25 (SNAP-25). The main isoform synaptotagmin-1 is involved in synchronous release, and forms a conserved Ca²⁺-independent interface with the ternary SNARE complex (9), along with Ca²⁺-dependent interactions with the plasma membrane, and potentially other interfaces with the SNARE complex (10). Complexin is a small cytosolic α-helical protein abundant in the presynaptic terminal (11) that interacts with the SNARE complex (12) and the membrane (13).Complexin has at least two functions: It “activates” (i.e., greatly enhances) Ca²⁺-triggered synchronous neurotransmitter release by cooperating with synaptotagmin, and it regulates spontaneous release in the presynaptic terminal (recently reviewed in refs. 14–16). The activating function of complexin is conserved across all species (mammals, Drosophila, and Caenorhabditis elegans) and different types of Ca²⁺-triggered synaptic vesicle fusion studied to date (11, 17–26). Complexin also regulates spontaneous neurotransmitter release, although this effect is less conserved among species and varies depending on experimental conditions: for example, in Drosophila, spontaneous release increases with knockout of complexin (27, 28). Likewise, knockdown of complexin in cultured cortical neurons increases spontaneous release, although knockout of complexin in mice only affects spontaneous release depending on the particular neuronal cell type (20, 23, 24, 29). Exactly how complexin can exhibit these dual effects on Ca²⁺-triggered and spontaneous synaptic vesicle fusion remains enigmatic; however, it is known that different domains of complexin play different roles in Ca²⁺-triggered and spontaneous vesicle fusion, as summarized in the following paragraphs.Here, we focus on the complexin-1 isoform (referred to as Cpx in the following). Cpx can be divided into four domains (Fig. 1A, Bottom) that are involved in different functions. The N-terminal domain (residues 1–27) of Cpx is important for activation of synchronous Ca²⁺-triggered release in murine neurons (20, 30, 31) and in isolated chromaffin cells (32). However, N-terminal truncation of Cpx in C. elegans neuromuscular junctions does not decrease Ca²⁺-triggered release, but rather increases spontaneous release (21, 22), perhaps suggesting that reduction of activation may have been masked by a simultaneous increase of spontaneous fusion in these previous experiments.Open in a separate windowFig. 1.Cpx (26–83) fragment reduces spontaneous fusion similar to wild-type Cpx. (A) Schematic diagram of the single-vesicle content mixing assay (35) (Methods) and domain diagrams of Cpx and Cpx fragments used in this figure. PM, vesicles with reconstituted syntaxin-1A and SNAP-25A that mimic the plasma membrane; SV, vesicles with reconstituted synaptobrevin-2 and synaptotagmin-1 that mimic synaptic vesicles. The bar graphs show the effects of 2 μM Cpx or Cpx fragments on the SV/PM vesicle association count during the first acquisition periods (Methods) (B), the average probability of spontaneous fusion events per second (C), the amplitude of the first 1-s time bin (probability of a fusion event in that bin) upon Ca²⁺ injection (D), and the decay rate (1/τ) of the histogram upon Ca²⁺ injection (E). The fusion probabilities and amplitudes were normalized with respect to the corresponding number of analyzed SV/PM vesicle pairs (Methods). Individual histograms are in Figs. S1 and ​andS2.S2. The error bars in B–D are SDs for multiple independent repeat experiments (20, 29, 30, 33–38). Although the accessory domain is required for regulating spontaneous release, mutations of this domain do not affect the activating function of Cpx for Ca²⁺-triggered release compared with wild-type neurons in rescue experiments of Cpx knockdown (23, 39).The central domain of Cpx (residues 49–70) is essential for all functions of complexins in all species studied to date, including priming (23, 24, 39, 40), inhibiting spontaneous release (18, 20–22, 35, 37, 38), and activation of Ca²⁺-triggered release (17, 18, 20, 22, 30, 31, 35, 41).The C-terminal domain binds to phospholipids (24, 42), and it is important for vesicle priming in neurons (24, 32, 43). Moreover, Cpx without the C-terminal domain does not reduce spontaneous release in neuronal cultures, but it still activates Ca²⁺-triggered release in neuronal cultures (24) and in a reconstituted system (35). The C-terminal domain is sensitive to membrane curvature, and it may thus localize Cpx to the synaptic membrane (13, 44).Structurally, in isolation, both the N- and C-terminal domains of Cpx are largely flexible, although the accessory and central domains have α-helical propensity (45). The α-helical central domain of Cpx binds to the groove between the synaptobrevin-2 and syntaxin-1A α-helices in the center of the neuronal SNARE complex (12, 46). Cpx has two conformations when bound to the ternary SNARE complex, one of which induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of Cpx (47).Cpx has been studied extensively with reconstituted systems (35, 38, 48–52). The single-vesicle fusion assay described by Lai et al. (35) qualitatively reproduced the effects of synaptotagmin-1 and Cpx in both spontaneous and Ca²⁺-triggered release that have been observed in cortical neuronal cultures (9, 35).Here, we conducted single-vesicle fusion and single-molecule membrane-binding experiments to obtain new insights into the function of the Cpx N-terminal domain. We found that the N-terminal domain can be physically separated from the accessory and central domains of Cpx and still preserve its role in activating Ca²⁺-triggered release. The N-terminal domain interacts with membranes, an interaction that is enhanced by the presence of SNARE complex. Moreover, the N-terminal domain of full-length Cpx can be functionally substituted by the fusion peptide of influenza virus hemagglutinin (HA), suggesting that similar fusion elements and principles are used in different contexts of biological membrane fusion.