Abstract
Complexins play activating and inhibitory functions in neurotransmitter release. The complexin accessory helix inhibits release and was proposed to insert into SNARE complexes to prevent their full assembly. This model was supported by 'superclamp' and 'poor-clamp' mutations that enhanced or decreased the complexin-I inhibitory activity in cell-cell fusion assays, and by the crystal structure of a superclamp mutant bound to a synaptobrevin-truncated SNARE complex. NMR studies now show that the complexin-I accessory helix does not insert into synaptobrevin-truncated SNARE complexes in solution, and electrophysiological data reveal that superclamp mutants have slightly stimulatory or no effects on neurotransmitter release, whereas a poor-clamp mutant inhibits release. Importantly, increasing or decreasing the negative charge of the complexin-I accessory helix inhibits or stimulates release, respectively. These results suggest a new model whereby the complexin accessory helix inhibits release through electrostatic (and perhaps steric) repulsion enabled by its location between the vesicle and plasma membranes.DOI: http://dx.doi.org/10.7554/eLife.02391.001.
Highlights
Neurotransmitter release is crucial for interneuronal communication and is exquisitely regulated by a sophisticated protein machinery (Sudhof, 2013)
To analyze interactions between Complexin I (CpxI) and soluble truncated sensitive factor attachment protein receptors (SNAREs) complexes that might mimic trans SNARE complexes partially assembled between two membranes (e.g., Figure 1C, insertion model), we used 1H-15N two-dimensional transverse relaxation optimized spectroscopy (TROSY) heteronuclear single quantum coherence (HSQC) spectra, which provide a powerful tool to study protein–protein interactions
As in the case of the synaptobrevin truncations (Figure 2C), the syntaxin-1 truncations in the SNARE complex caused changes in the center of the 1H-15N TROSY-HSQC spectrum of the bound 2H,15NCpxI(26-83), but the number of cross-peaks and their overall distribution remained similar (Figure 6B) and the well-resolved cross-peaks corresponding to A30 and Q38 exhibited small gradual shifts from their positions upon binding to the non-truncated SNARE complex to their free positions as syntaxin-1 was increasingly truncated (Figure 6C,D). These results strongly suggest that the shifts in the CpxI(26-83) accessory helix do not arise from interactions with the C-terminal half of the synaptobrevin SNARE motif but rather because the destabilization of the central helix caused by the truncations in syntaxin-1 is transferred into destabilization of the accessory helix
Summary
Neurotransmitter release is crucial for interneuronal communication and is exquisitely regulated by a sophisticated protein machinery (Sudhof, 2013). Great advances have been made in elucidating the mechanism of release (Brunger et al, 2009; Sorensen, 2009; Jahn and Fasshauer, 2012; Rizo and Sudhof, 2012) and basic aspects of this process have been reconstituted with eight central components of the release machinery (Ma et al, 2013), leading to a model with defined roles for each component.
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