Dissection of SNARE-driven membrane fusion and neuroexocytosis by wedging small hydrophobic molecules into the SNARE zipper

Abstract

Neuronal SNARE proteins mediate neurotransmitter release at the synapse by facilitating the fusion of vesicles to the presynaptic plasma membrane. Cognate v-SNAREs and t-SNAREs from the vesicle and the plasma membrane, respectively, zip up and bring about the apposition of two membranes attached at the Cterminal ends. Here, we demonstrate that SNARE zippering can be modulated in the midways by wedging with small hydrophobic molecules. Myricetin, which intercalated into the hydrophobic inner core near the middle of the SNARE complex, stopped SNARE zippering in motion and accumulated the trans-complex, where the N-terminal region of v-SNARE VAMP2 is in the coiled coil with the frayed C-terminal region. Delphinidin and cyanidin inhibited N-terminal nucleation of SNARE zippering. Neuronal SNARE complex in PC12 cells showed the same pattern of vulnerability to small hydrophobic molecules. We propose that the half-zipped trans-SNARE complex is a crucial intermediate waiting for a calcium trigger that leads to fusion pore opening.polyphenol | hemifusion | neurotransmission | neuron N eurotransmitter release at the synapse, which serves as the brain's major form of cell-cell communication, requires the fusion of synaptic vesicles with the presynaptic plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate this synaptic fusion event (1-5), and the formation of a four-helical bundle (6-8) is believed to generate the force required for fusion. A zipper model has been proposed for SNARE complex formation, initiating assembly at the N-terminal region and zipping toward the C-terminal membrane-proximal region (6-9). To account for fast neuroexocytosis, the SNAREs in primed readily releasable vesicles have been proposed as being partially zipped in the trans-configuration bridging the two membranes.Although the structure of the fully assembled cis-SNARE complex, which is believed to represent the postfusion state, has been determined (10), the structure of the trans-complex is poorly understood and is purely imaginary, most likely because of its inherently transient nature. Precisely linking the degrees of SNARE zippering to specific stages of membrane fusion seems to be prerequisite for determining the structure of the trans-complex and for providing answers to the questions of how fast fusion is controlled in neurons and how the trans-complexes set up the readily releasable vesicles with other regulatory proteins.Here, we show that certain small hydrophobic molecules (SHM) enable layer-by-layer control of SNARE zippering by wedging into various points of the SNARE zipper. SNAREmediated membrane fusion is dissected via this wedge-like action of SHMs. Analysis of the captured replication fork-like structure allowed us to understand the basic architecture of the putative trans-complex. Results SNARE-Driven Membrane Fusion Can Be Controlled by SHMs withDifferent Modes of Action. As an initial step to examine the feasibility of whether SHM works as a wedge for the SNARE zipp...

A botulinum neurotoxin-like function ofPotentilla chinensisextract that inhibits neuronal SNARE complex formation, membrane fusion, neuroexocytosis, and muscle contraction

“…Generally, the effect of extracts on SNARE-driven membrane fusion was abolished when the colors were removed. It is likely that polyphenols are associated with the activity as we have demonstrated that some polyphenolic compounds wedge into SNARE complex and stop neuroexocytosis (Yang et al, 2010). Intriguingly, however, Potentilla chinensis extract did not exhibit such a strong color or order, and could be dissolved well in water as high as 300 mg/L which is 30 times higher concentration than those that inhibited approximately 90% neuroexocytosis ( Figure 3A).…”

“…We added 1% n-octyl-d-glucopyranoside (OG) to syntaxin 1a and VAMP2. The purified proteins were examined by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the purity was at least 90% for all proteins ( Figure 1B) (Jung et al, 2008;Yang et al, 2010).…”

“…Protein expression and purification procedures for neuronal SNARE proteins have been described elsewhere (Yang et al, 2010). In brief, a pGEX-2T vector encoding a thrombin-cleavable amino-terminal glutathione S-transferase (GST)-tag was used to express the following constructs: full-length syntaxin 1a (amino acids 1-288), the core domain of syntaxin 1a (SynH3, amino acid 168-266), full-length VAMP2 (amino acids 1-116), the core domain of VAMP2 (VpS, 1-96), and SNAP-25 (amino acids 1-206) ( Figure 1A).…”

“…Delphinidin and cyanidin inhibited N-terminal nucleation of SNARE zippering while myricetin allowed N-terminal half zippering but stopped further zippering. Stopped zippering resulted in arrest of fusion reaction at hemifusion stage (Yang et al, 2010).…”

“…Accordingly, the initial motivation of this study was to find small inhibitors from natural resources that might be alternatives for use in cosmetics or therapeutics. Since we recently demonstrated that several polyphenols inhibited SNARE complex formation by wedging into the hydrophobic inner core (Yang et al, 2010), it is highly probable that natural resources contain more potent SNARE inhibitors. It is notable that myricetin, delphinidin, and cyanidin were most potent neuroexocytosis blockers among polyphenols tested.…”