Single residues in the complexin N-terminus exhibit distinct phenotypes in synaptic vesicle fusion

Abstract

The release of neurotransmitters at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Amongst those dedicated molecules the cytosolic complexins play an incompletely defined role as synaptic transmission regulators. Complexins are multidomain SNARE complex binding proteins which confer both inhibitory and stimulatory functions. Using systematic mutagenesis and combining reconstituted in vitro membrane fusion assays with electrophysiology in neurons, we deciphered the function of the N-terminus of complexin II (Cpx). The N-terminus (amino acid 1 - 27) starts with a region enriched in hydrophobic amino acids (1-12), which can lead to lipid binding. In contrast to mutants which maintain the hydrophobic character and the stimulatory function of Cpx, non-conservative exchanges largely perturbed spontaneous and evoked exocytosis. Mutants in the downstream region (amino acid 11-18) show differential effects. Cpx-A12W increased spontaneous release without affecting evoked release whereas replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release, but also impaired evoked release and surprisingly reduced the size of the readily releasable pool, a novel Cpx function, unanticipated from previous studies. Thus, the exact amino acid composition of the Cpx N-terminus fine tunes the degree of spontaneous and evoked neurotransmitter release.