Re-examining how complexin inhibits neurotransmitter release

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.The instructions sent to, from and within the brain are rapidly transmitted along neurons in the form of electrical signals. These signals cannot pass across the small gaps—called synapses—that separate neighboring neurons. Instead, neurons release chemicals called neurotransmitters into the synapses, and these relay the signal to the next neuron.The neurotransmitters are stored inside neurons in small bubbles called vesicles. To release these neurotransmitters into the synapse, the membrane that encloses the vesicle fuses with the membrane that surrounds the neuron. To fuse the membranes, proteins embedded in the vesicle membrane interact with similar proteins in the neuron membrane to form a structure called a SNARE complex. Additional proteins control membrane fusion to ensure that the signal is passed to the other neuron at the right time and with the appropriate efficiency.Among these proteins are the complexins, which are often found attached to SNARE complexes. Although different parts of complexins can both help and hinder membrane fusion, a part known as an accessory helix is thought to have only one role—to stop the membranes from fusing together. Several models have been suggested for how the accessory helix interferes with fusion. However, after performing a range of analyses by diverse biophysical techniques, Trimbuch, Xu et al. suggest these models are unlikely to describe the process accurately.Instead, Trimbuch, Xu et al. propose a new model based on the electrostatic properties of two molecules that are both negatively charged. An accessory helix taken from a fruit fly complexin was more negatively charged than a mammalian version, and experiments showed it was also better at preventing the release of neurotransmitters. It is thought that the negative charges on the helix hold the membranes apart because the helix is located between the membranes, which are also negatively charged. Consistent with this model, Trimbuch, Xu et al. showed that the membranes fused more easily when some of the negative charges on the accessory helix were replaced with positive charges. The next challenges are to test the model further with additional studies, and to explain how other proteins work with complexins to control neurotransmitter release.