SNAP-25 with mutations in the zero layer supports normal membrane fusion kinetics
Margaret E. Graham1,
Philip Washbourne2,*,
Michael C. Wilson2 and
Robert D. Burgoyne1,
1 The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK
2 Department of Neurosciences, University of New Mexico, Albuquerque, New Mexico, USA
* Present address: Center for Neuroscience, University of California at Davis, Davis, California, USA
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Fig. 1. The organisation of SNARE helices and mutated residues in SNAP-25B. (A) Domain structure of SNAP-25B. (B) Alignment of the SNAP-25B helices that participate in the core complex. Residues are indicated that are mutated in the BoNT/E-resistant construct, Em, and in the Q/E mutants. (C) The structure of the core SNARE complex showing the helices from VAMP (red), syntaxin 1 (blue) and SNAP-25 (green).
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Fig. 2. Effect of expression of SNAP-25 constructs on exocytosis in BoNT/E treated PC12 cells. The cells were co-transfected with a plasmid encoding growth hormone (GH) along with control vector (pcDNA3), plasmid encoding toxin-resistant mutant (Em2) or Em2 containing additional mutations as indicated. (A) Expression of SNAP-25 constructs demonstrated by blotting with anti-HA three days after transfection. (B) Transfected cells were permeabilised with 20 µM digitonin for 6 minutes and treated with recombinant BoNT/E light chain for 40 minutes. The cells were then challenged with 0 or 10 µM Ca2+ as indicated and GH release over a 20 minute period assayed. Total GH content for each well was determined and GH release is shown as a percentage of total GH expressed.
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Fig. 3. Effect of Q/E mutations in SNAP-25 on the time course and Ca2+-dependency of exocytosis in PC12 cells. The cells were transfected with the GH plasmid along with the Em2 plasmid or Em2 harbouring the double Q/E mutations. After 3 days, the cells were permeabilised with digitonin and treated with BoNT/E light chain followed by incubation with Ca2+ to stimulate exocytosis. (A) Time course of GH release in response to 10 µM Ca2+ from cells co-transfected with either Em2 or the double mutant. (B) Ca2+-dependency of GH release from cells transfected with Em2 or the double Q/E mutant.
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Fig. 4. Effect of Q/E mutations in SNAP-25 on the extent of exocytosis in adrenal chromaffin cells measured using amperometry. Adrenal chromaffin cells were transfected with a plasmid-encoding GFP-BoNT/E light chain along with the Em2 plasmid or Em2 containing the double Q/E mutations. After 3-5 days the cells were stimulated by local application of 20 µM digitonin and 10 µM Ca2+ and responses measured using carbon-fibre amperometry. Non-transfected cells were assayed as controls in the same dishes as GFP-expressing transfected cells and using the same carbon-fibres. (A) Mean numbers of amperometric spikes elicited in BoNT/E light-chain expressing cells (n=9 cells) compared to controls (n=10 cells) in the same dishes. (B) Mean numbers of spikes in control cells (n=39) or in cells co-transfected with Em2 (n=31 cells) or Em2 containing the double Q/E mutations (n=40 cells). Typical traces are shown from a non-transfected cell (C) or from cells co-transfected with GFP-BoNT/E light chain and Em2 (D) or the double Q/E mutant (E).
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Fig. 5. Effect of Q/E mutations of the kinetics of single granule release events. Data was taken from chromaffin cells following transfection. Non-transfected cells in the same dishes were used as controls for comparison with cells expressing Em2 or the double Q/E mutations. Identified spikes were analysed using Origin and the rise time to peak determined for each spike. (A) Values of rise time shown as mean±s.e.m. (B-D) Frequency distribution of rise-times for control (n=727 spikes), Em2 expressing (n=440 spikes) and double-mutant expressing (n=343 spikes) cells.
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Fig. 6. Effect of Q/R mutations in SNAP-25 on the extent of exocytosis in PC12 cells. PC12 cells were transfected with a plasmid-encoding growth hormone along with control plasmid, plasmid encoding Em2 or plasmid encoding Em2 containing the double Q/R mutations. After 3 days the cells were permeabilised, treated with BoNT/E for 40 minutes and responses to 0 or 10 µM Ca2+ determined. (A) GH release shown as a percentage of total GH expressed. (B) Immunoblot with anti-HA showing the expression of Em2 and the double Q/R mutant in the same cells used for the GH assay.
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Fig. 7. Schematic model of SNARE complex states before and during exocytosis. This model is based on previous data from adrenal chromaffin cells (Xu et al., 1998; Xu et al., 1999b), synapses (Hua and Charlton, 1999), liposome fusion (Weber et al., 2000) and the data presented in this paper. In this model, secretory vesicles initially associate with the plasma membrane via a loose SNARE complex in which the SNAREs are sensitive to clostridial neurotoxins. Disassembly of cis SNARE complexes may be important prior to exocytosis to free SNAREs for assembly into the initial trans complex (Graham and Burgoyne, 2000; Xu et al., 1999a). Conversion to a ‘tight’ complex resistant to tetanus toxin precedes fusion, and fusion itself is driven by a complex that is fully toxin-resistant but not zipped up into the stable complex. The formation of the stable SNARE complex occurs only after full fusion has been completed and then it be can disassembled by the action of  -SNAP and NSF.
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© The Company of Biologists Ltd 2001
