Evolutionary Divergence of the C-terminal Domain of Complexin Accounts for Functional Disparities between Vertebrate and Invertebrate Complexins

Abstract

Complexin is a critical presynaptic protein that regulates both spontaneous and calcium-triggered neurotransmitter release in all synapses. Although the SNARE-binding central helix of complexin is highly conserved and required for all known complexin functions, the remainder of the protein has profoundly diverged across the animal kingdom. Striking disparities in complexin inhibitory activity are observed between vertebrate and invertebrate complexins but little is known about the source of these differences or their relevance to the underlying mechanism of complexin regulation. We found that mouse complexin 1 (mCpx1) failed to inhibit neurotransmitter secretion in Caenorhabditis elegans neuromuscular junctions lacking the worm complexin 1 (CPX-1). This lack of inhibition stemmed from differences in the C-terminal domain (CTD) of mCpx1. Previous studies revealed that the CTD selectively binds to highly curved membranes and directs complexin to synaptic vesicles. Although mouse and worm complexin have similar lipid binding affinity, their last few amino acids differ in both hydrophobicity and in lipid binding conformation, and these differences strongly impacted CPX-1 inhibitory function. Moreover, function was not maintained if a critical amphipathic helix in the worm CPX-1 CTD was replaced with the corresponding mCpx1 amphipathic helix. Invertebrate complexins generally shared more C-terminal similarity with vertebrate complexin 3 and 4 isoforms, and the amphipathic region of mouse complexin 3 significantly restored inhibitory function to worm CPX-1. We hypothesize that the CTD of complexin is essential in conferring an inhibitory function to complexin, and that this inhibitory activity has been attenuated in the vertebrate complexin 1 and 2 isoforms. Thus, evolutionary changes in the complexin CTD differentially shape its synaptic role across phylogeny.

Unique Structural Features of Membrane-Bound C-Terminal Domain Motifs Modulate Complexin Inhibitory Function

“…Although the significance of this observation is unclear, molecular dynamics simulations in an accompanying paper (Wragg et al, 2017) support a somewhat extended conformation for this region of the protein when bound to lipid membranes.…”

A C1-C2 Module in Munc13 Inhibits Calcium-Dependent Neurotransmitter Release

“…To investigate the contribution of calcium-triggered fusion to the observed hypersecretion phenotype in vivo, motor neurons were electrically silenced in intact behaving animals. The Drosophila HisCl histamine-gated chloride channel was expressed in cholinergic neurons to reversibly silence motor neurons in intact animals as described previously (Pokala et al, 2014; Wragg et al, 2017). Sensitivity to aldicarb was scored in the presence and absence of sub-maximal histamine in wild-type animals, and a significant delay in paralysis was driven by partial electrical silencing of cholinergic motor neurons (Figure S3).…”

Mechanism of neurotransmitter release coming into focus

“…132,136,146,148,149 In both mammalian Cpx1 and invertebrate complexin, the C-terminal region beyond the central helix contains a membranebinding motif that senses membrane curvature and helps to localize them to synaptic vesicles, which is important for inhibition of spontaneous release. [150][151][152][153] Indeed, sequence differences in the Cterminal region underlie the stronger suppression of spontaneous release by invertebrate complexins than mammalian Cpx1, 154 and the strong activity of Drosophila complexin in inhibiting spontaneous release depends on a C-terminal farnesylation motif that mediates membrane localization and is present in mammalian Cpx3 and Cpx4 but not Cpx1 and Cpx2. 155 Note however that the C-terminal region of Cpx1 contributes to both active and inhibitory roles in neurotransmitter release, 156 and may simply help to localize Cpx1 near the sites of fusion without having a direct active or inhibitory action.…”