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 proteins that bind SNARE complexes, conferring both inhibitory and stimulatory functions. Using systematic mutagenesis and comparing reconstituted in vitro membrane fusion assays with electrophysiology in cultured neurons from mice of either sex, 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 binds lipids. Mutants maintaining this hydrophobic character retained the stimulatory function of Cpx, whereas exchanges introducing charged residues perturbed both spontaneous and evoked exocytosis. Mutants in the more distal region of the N-terminal domain (amino acid 11-18) showed a spectrum of effects. On one hand, mutation of residue A12 increased spontaneous release without affecting evoked release. On the other hand, replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release, but also impaired evoked release. Most surprising, this substitution reduced the size of the readily releasable pool, a novel function for Cpx at mammalian synapses. Thus, the exact amino acid composition of the Cpx N-terminus fine tunes the degree of spontaneous and evoked neurotransmitter release.
Significance Statement We describe in this work the importance of the N-terminal domain of the small regulatory cytosolic protein complexin in spontaneous and evoked glutamatergic neurotransmitter release at hippocampal mouse neurons. We use biochemical assays to screen for amino acids of interest in the complexin N-terminus and test these residues for functional relevance in spontaneous and Ca2+-triggered synaptic vesicle exocytosis using electrophysiology assays and site-directed mutagenesis. In addition to identifying crucial residues for clamping spontaneous release and promoting Ca2+-evoked transmission, we identify a single amino acid at position D15 which determines synaptic vesicle priming, a function that was never before attributed to complexin at vertebrate synapses.
Footnotes
The authors declare no competing interests.
This work was funded by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) project 278001972-TRR186 (to E.T., Ja.M., S.B., T.T., T.H.S., and C.R.). We are grateful to Berit Söhl-Kielczynski, Bettina Brokowski, Katja Pötschke, Ursula Göbel, Lara Braun, Susanne Kreye and Heike Lerch for excellent technical assistance. We also thank Denisa Jamecna and Doris Höglinger for sharing their expertise in lipid crosslinking using click chemistry and Daniela Schweinfurth and Britta Brügger for sharing their knowledge in protein-lipid extraction. We are grateful to Andrew Plested for comments on the manuscript. We thank the services of the Charité viral core facility for virus production and the electron microscopy core facility for technical support. Molecular graphics were performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from the National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.