Distinct Domains of Complexins Bind SNARE Complexes and Clamp Fusion in Vitro

Abstract

In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca 2؉ ) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.

Complexin Controls the Force Transfer from SNARE Complexes to Membranes in Fusion

“…In vertebrate autapses, deletion of complexin selectively impairs fast synchronous neurotransmitter release without changing asynchronous or spontaneous release [7,10]. In in vitro fusion assays, conversely, addition of complexin causes a general block of SNARE-dependent fusion, indicating that complexin is a SNARE clamp [11][12][13][14]. In Drosophila neuromuscular synapses, deletion of complexin produces a >20-fold increase in spontaneous release but only a small decrease in evoked release [15].…”

“…Complexin binds to SNARE complexes via its central α-helix, which inserts in an anti-parallel orientation into a groove formed by synaptobrevin/VAMP and syntaxin-1 [8,9]. Although multiple approaches have revealed an essential role of complexin in synaptic fusion [7,[10][11][12][13][14][15], the nature of this role remains unclear. In vertebrate autapses, deletion of complexin selectively impairs fast synchronous neurotransmitter release without changing asynchronous or spontaneous release [7,10].…”

“…S4). Thus, the complexin knock-down greatly impaired fast synchronous but not asynchronous synaptic vesicle fusion, and increased spontaneous fusion, thereby reconciling the divergent phenotypes observed in vertebrate autapses, Drosophila neuromuscular junctions, and in vitro fusion assays [7,[10][11][12][13][14][15].We next examined which complexin sequences mediate spontaneous and evoked fusion (Fig. 1C).…”

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

“…Most of this uncertainty is based on differences in approach. For example, in vitro fusion assays initially only described a clamping function of complexin, thus emphasizing this role (19)(20)(21)(22). Analysis of mouse complexin double-knockout (DKO) neurons lacking Cpx1 and Cpx2, conversely, initially demonstrated only a Ca 2+ -triggering function of complexin, thus suggesting that mammalian complexins primarily function in this role (4,13).…”

Complexin 2

“…Since then several mutations and truncations of complexin 1 have been produced and studied in brain (9,18,19,30).…”