Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion

Abstract

Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the plasma membrane. Vesicles attach to membranes via a specialized protein machinery composed of membrane-attached (t-SNARE) and vesicle-attached (v-SNARE) proteins that zipper together to form a coiled-coil SNARE bundle that brings the two fusing membranes into close proximity. Neurotransmitter release may occur either in response to an action potential or through spontaneous fusion. A cytosolic protein, Complexin (Cpx), binds the SNARE complex and restricts spontaneous exocytosis by acting as a fusion clamp. We previously proposed a model in which the interaction between Cpx and the v-SNARE serves as a spring to prevent premature zippering of the SNARE complex, thereby reducing the likelihood of fusion. To test this model, we combined molecular-dynamics (MD) simulations and site-directed mutagenesis of Cpx and SNAREs in Drosophila. MD simulations of the Drosophila Cpx-SNARE complex demonstrated that Cpx's interaction with the v-SNARE promotes unraveling of the v-SNARE off the core SNARE bundle. We investigated clamping properties in the syx paralytic mutant, which has a single-point mutation in the t-SNARE and displays enhanced spontaneous release. MD simulations demonstrated an altered interaction of Cpx with the SNARE bundle that hindered v-SNARE unraveling by Cpx, thus compromising clamping. We used our model to predict mutations that should enhance the ability of Cpx to prevent full assembly of the SNARE complex. MD simulations predicted that a weakened interaction between the Cpx accessory helix and the v-SNARE would enhance Cpx flexibility and thus promote separation of SNAREs, reducing spontaneous fusion. We generated transgenic Drosophila with mutations in Cpx and the v-SNARE that disrupted a salt bridge between these two proteins. As predicted, both lines demonstrated a selective inhibition in spontaneous release, suggesting that Cpx acts as a fusion clamp that restricts full SNARE zippering.

Focal Macropatch Recordings of Synaptic Currents from the <em>Drosophila</em> Larval Neuromuscular Junction

“…This can be clearly illustrated by recordings of mEJCs from complexin null mutant ( Figure 6A). Spontaneous activity is drastically elevated in this mutant 32 , and therefore mEJPs recorded intracellularly overlap and cannot be clearly distinguished from each other (Figure 6B), while focal recordings 22 enable accurate detection of spontaneous release events ( Figure 4A).…”

“…In contrast, the activity of a limited number of the sites of release can be recorded focally by positioning a micropipette tip near neuronal terminals or synaptic varicosities. This technique was originally employed by Katz and Miledi 2 , and focal extracellular recordings have been successfully employed at several NMJ preparations, including frog 3,4,5 , mouse 6,7,8 , crustacean 9,10,11,12,13,14,15,16 , and Drosophila 17,18,19,20,21,22,23 . This approach was further developed by Dudel, who optimized macropatch recoding electrodes 24,25 .…”

“…The Drosophila larval NMJ has clearly defined synaptic boutons, and transgenic lines with genetically encoded neuronal fluorescent tags (see Table of Materials) are readily available. These advantages enabled us to record EJCs and mEJCs from a selected synaptic bouton 20,21,22 .…”

Electrophysiological analysis of synaptic transmission in Drosophila

“…This approach enabled reliable recordings of EJCs and mEJCs using fire polished electrodes of a tip diameter ranging from 1–3 to 10 µm, and it closely matches the loose‐patch‐clamp method . Focal macropatch recordings have been successfully employed at several NMJ preparations, including frog, mouse, crustacean, and Drosophila . Simultaneous recordings by two or three mactopatch electrodes from the same NMJ, as well as moving the electrode along the NMJ, demonstrated that a macropatch electrode predominantly records the activity of synaptic boutons under the electrode tip, although the boutons positioned under the electrode rim and those in the immediate proximity to the electrode tip can make a minor contribution to the recorded EJCs.…”

“…These advantages enable recordings of EJCs and mEJCs from a selected bouton (Figure (a) and (b)). Respectively, this technique is advantageous for quantitative studies of release kinetics in the mutants with enhanced spontaneous activity and altered release time‐course (Figure (c)), such the complexin null mutant . The rapid mEJC time‐course and limited number of release sites enable accurate detection of neurosecretory quanta, which can be performed automatically (Figure (d)) .…”