Mutations of single residues in the complexin N-terminus exhibit distinct phenotypes in synaptic vesicle fusion.

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.

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

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. 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 show using a combination of biochemical, imaging and electrophysiological techniques that the binding of the proximal region of complexin (amino acids 1-10) to lipids is crucial for spontaneous synaptic vesicular release. Furthermore, we identify a single amino acid at position D15 which is structurally important since it not only is involved in spontaneous release but, when mutated, also decreases drastically the readily releasable pool, a function that was never attributed to complexin.

Complexin Suppresses Spontaneous Exocytosis by Capturing the Membrane-Proximal Regions of VAMP2 and SNAP25.

The neuronal protein complexin contains multiple domains that exert clamping and facilitatory functions to tune spontaneous and action potential-triggered synaptic release. We address the clamping mechanism and show that the accessory helix of complexin arrests assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that forms the core machinery of intracellular membrane fusion. In a reconstituted fusion assay, site- and stage-specific photo-cross-linking reveals that, prior to fusion, the complexin accessory helix laterally binds the membrane-proximal C-terminal ends of SNAP25 and VAMP2. Corresponding complexin interface mutants selectively increase spontaneous release of neurotransmitters in living neurons, implying that the accessory helix suppresses final zippering/assembly of the SNARE four-helix bundle by restraining VAMP2 and SNAP25.

Cis-retinoids and the chemistry of vision.

We discuss here principal biochemical transformations of retinoid molecules in the visual cycle. We focus our analysis on the accumulating evidence of alternate pathways and functional redundancies in the cycle. The efficiency of the visual cycle depends, on one hand, on fast regeneration of the photo-bleached chromophores. On the other hand, it is crucial that the cyclic process should be highly selective to avoid accumulation of byproducts. The state-of-the-art knowledge indicates that single enzymatically active components of the cycle are not strictly selective and may require chaperones to enhance their rates. It appears that protein-protein interactions significantly improve the biological stability of the visual cycle. In particular, synthesis of thermodynamically less stable 11-cis-retinoid conformers is favored by physical interactions of the isomerases present in the retina with cellular retinaldehyde binding protein.