SNARE zippering and synaptic strength.

Synapses vary widely in the probability of neurotransmitter release. We tested the hypothesis that the zippered state of the trans-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) complex determines initial release probability. We tested this hypothesis at phasic and tonic synapses which differ by 100-1000-fold in neurotransmitter release probability. We injected, presynaptically, three Clostridial neurotoxins which bind and cleave at different sites on VAMP to determine whether these sites were occluded by the zippering of the SNARE complex or open to proteolytic attack. Under low stimulation conditions, the catalytic light-chain fragment of botulinum B (BoNT/B-LC) inhibited evoked release at both phasic and tonic synapses and cleaved VAMP; however, neither BoNT/D-LC nor tetanus neurotoxin (TeNT-LC) were effective in these conditions. The susceptibility of VAMP to only BoNT/B-LC indicated that SNARE complexes at both phasic and tonic synapses were partially zippered only at the N-terminal end to approximately the zero-layer with the C-terminal end exposed under resting state. Therefore, the existence of the same partially zippered state of the trans-SNARE complex at both phasic and tonic synapses indicates that release probability is not determined solely by the zippered state of the trans-SNARE complex at least to the zero-layer

Susceptibility of VAMP to <i>Clostridial</i> neurotoxins.

<p><b><i>A</i></b>, Zippered states of the SNARE complex. <b><i>Ai</i></b>, SNARE complex tightly zippered beyond the zero-layer indicated by gray arrow head. VAMP (blue) is protected from cleavage because the binding (yellow and orange bars) and cleavage (scissors and white lines) sites of VAMP-specific <i>Clostridial</i> neurotoxins (TeNT, BoNT/B and BoNT/D) are occluded. <b><i>Aii</i></b>, Partially zippered SNARE complex. The binding sites of TeNT and BoNT/D are occluded but the binding and cleavage sites for BoNT/B are exposed such that VAMP is susceptible to cleavage. Green – syntaxin, red – SNAP25 (represents both SNARE binding motifs). <b><i>B</i></b>, <i>Clostridial</i> neurotoxins cleave crayfish VAMP <i>in-vitro</i>. Crayfish CNS protein sample was incubated with inactive or active neurotoxins (BoNT/B-LC (0.5 µg/μL), BoNT/D-LC (0.3 µg/μL) and TeNT-LC (0.5 µg/μL)) and stained for neuronal VAMP. The protein bands of 18 kDa represent VAMP. Cleaved VAMP does not appear on the blot when active neurotoxins were used because the VAMP antibody binds only to the uncleaved VAMP protein. Actin staining of 38 kDa below the VAMP blot shows that equal amounts (10 µg) of protein were loaded in each lane. <b><i>C</i></b>, Comparison of full-length crayfish VAMP amino acid sequence with VAMP sequences from other species. Crayfish VAMP is similar to VAMP from other species, especially in the conserved SNARE motif region (black bar). The cleavage sites of VAMP-specific neurotoxins are indicated in the alignment. The primary binding sites of the neurotoxins used in this study are indicated as boxed regions (V1 motif (aa 38–47) – TeNT and BoNT/D; V2 motif (aa 62–71) – BoNT/B) based on the human VAMP sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095130#pone.0095130-Arndt1" target="_blank">[29]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095130#pone.0095130-Foran1" target="_blank">[31]</a>. The multiple protein sequence alignment was performed using the online ClustalW2 Multiple Sequence Alignment tool (European Molecular Biology Laboratory - European Bioinformatics Institute, <a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>).</p

VAMP in phasic and tonic axonal terminals is susceptible only to BoNT/B-LC under low frequency stimulation.

<p>Immunostaining of VAMP and synapsin after the injection of inactive and active TeNT-LC (<i>A</i>), BoNT/D-LC (<i>B</i>) and BoNT/B-LC (<i>C</i>) into the phasic or tonic axon during the low frequency stimulation experiments. In <i>A-C</i>, arrows denote phasic terminals and arrow heads denote tonic terminals. The yellow areas in the merged image represent an overlap of VAMP and synapsin immunoreactivity. In <i>C</i>, the injected boutons contain only synapsin indicating that active BoNT/B-LC cleaved VAMP under low frequency stimulation. Note that only active BoNT/B-LC reduced VAMP immunoreactivity under low frequency stimulation conditions. In <i>B</i>,<i>C</i>, no tonic terminals were present in the phasic image with active neurotoxin. Scale bars – <i>A</i> and <i>B</i>: 19 µm; <i>C</i>: 10 µm.</p

Tonic EPSPs are inhibited using TeNT-LC (<i>B</i>), BoNT/D-LC (<i>C</i>) and BoNT/B-LC (<i>D</i>) under high frequency stimulation.

<p><b><i>A</i></b>, Example showing the tonic action potentials (APs) remain unchanged before (<i>i</i>) and after (<i>ii</i>) the injection of each neurotoxin (BoNT/B-LC used as the example). <b><i>E</i></b>, Percent difference between active and inactive neurotoxin, in which the inactive neurotoxin is the reference point at 0%. In <i>B-E,</i> a solid black line represents the time course of neurotoxin injection (90 min), the dotted lines represent the time course of each round of high frequency stimulation (40 min) and vertical arrows (↓) represent when EPSPs were recorded. Active neurotoxins: TeNT-LC (▪), BoNT/D-LC (▴) and BoNT/B-LC (•). Inactive neurotoxins: TeNT-LC (□), BoNT/D-LC (△) and BoNT/B-LC (○). Error bars represent S.E.M. The measured tonic (last) EPSP is encircled in black. An asterisk (*) denotes a significant difference (p<0.05) and ‘n’ represents sample size (active/inactive neurotoxin). Scale bars: horizontal – 10 ms; vertical – 10 mV (AP), 5 mV (EPSP).</p

The <i>trans</i>-SNARE complexes at phasic and tonic synapses are partially zippered under resting state.

<p>The N-terminal end of the SNARE complex is zippered whereas the C-terminal end is exposed. The binding and cleavage sites of BoNT/B are exposed but the shared binding site for TeNT and BoNT/D, and possibly the cleavage site for BoNT/D, are occluded. Scissors and white lines indicate neurotoxin cleavage site. Gray arrow head indicates the location of the zero-layer across all three SNARE proteins.</p

Tonic EPSPs are inhibited using BoNT/B-LC under low frequency stimulation.

<p><b><i>A</i></b>, Example showing the tonic action potentials (APs, 200 Hz) remain unchanged before (<i>i</i>) and after (<i>ii</i>) the injection of each neurotoxin (BoNT/B-LC used as the example). TeNT-LC (<i>B</i>) and BoNT/D-LC (<i>C</i>) did not have an effect on the tonic evoked response. <b><i>E</i></b>, Percent difference between active and inactive neurotoxin, in which the inactive neurotoxin is the reference point at 0%. In <i>B-E</i>, a solid black line represents the time course of neurotoxin injection (90 min) and vertical arrows (↓) represent when EPSPs were recorded. Active neurotoxins: TeNT-LC (▪), BoNT/D-LC (▴) and BoNT/B-LC (•). Inactive neurotoxins: TeNT-LC (□), BoNT/D-LC (△) and BoNT/B-LC (○). Error bars represent S.E.M. The measured tonic (last) EPSP is encircled in black (<i>Ai</i>). An asterisk (*) denotes a significant difference (p<0.05) and ‘n’ represents the sample size (active/inactive neurotoxin). Scale bars: horizontal – 10 ms; vertical – 10 mV (APs), 5 mV (EPSPs).</p

VAMP in phasic and tonic axonal terminals is susceptible to <i>Clostridial</i> neurotoxins under high frequency stimulation.

<p>Immunostaining of VAMP and synapsin following the high frequency stimulation experiments using inactive and active TeNT-LC (<i>A</i>), BoNT/D-LC (<i>B</i>) and BoNT/B-LC (<i>C</i>). In <i>A-C</i>, arrows denote phasic terminals and arrow heads denote tonic terminals. Areas of overlap between VAMP and synapsin immunoreactivity are denoted in yellow in the merged image. Note that all three active neurotoxins reduced VAMP immunoreactivity. In <i>A</i>-<i>C</i>, no tonic terminals were present in the phasic image with active neurotoxin. Scale bars: 10 µm.</p

Timeline of the physiological recordings for neurotoxin injection experiments.

<p><b><i>A</i></b>, Timeline of the low frequency stimulation experiment. <b><i>B</i></b>, Timeline of the high frequency stimulation experiment. In <i>A</i> and <i>B</i>, the black arrows () mark the time when test stimuli (Phasic – three single pulses at 0.1 Hz; tonic – three, 200 Hz, 15- pulse trains at 0.1 Hz) were applied following injection, and below each timeline is a diagram depicting the stimulation protocol for phasic and tonic recordings (in <i>B</i>, an example of the high frequency stimulation (HFS) protocol is given from time elapsed mark of 120 min to 180 min). Baseline recording (BR): Phasic – three single pulses at 0.1 Hz every 10 min; tonic – three, 200 Hz, 15-pulse trains at 0.1 Hz every 10 min. High frequency stimulation (HFS): Phasic – burst stimuli of 10 Hz for 2 min with an inter-burst interval of 2 min; tonic – burst stimuli of 150 Hz for 30 sec with an inter-burst interval of 10 sec. INJ – injection, RP – rest period.</p

Phasic EPSPs are inhibited using TeNT-LC (<i>B</i>), BoNT/D-LC (<i>C</i>) and BoNT/B-LC (<i>D</i>) under high frequency stimulation.

<p><b><i>A</i></b>, Example showing the phasic action potential (AP) remains unchanged before (<i>i</i>) and after (<i>ii</i>) the injection of each neurotoxin (BoNT/B-LC used as the example). <b><i>E</i></b>, Percent difference between active and inactive neurotoxin, in which the inactive neurotoxin is the reference point at 0%. In <i>B-E,</i> a solid black line represents the time course of neurotoxin injection (90 min), the dotted lines represent the time course of each round of high frequency stimulation (40 min) and vertical arrows (↓) represent when EPSPs were recorded. Active neurotoxins: TeNT-LC (▪), BoNT/D-LC (▴) and BoNT/B-LC (•). Inactive neurotoxins: TeNT-LC (□), BoNT/D-LC (△) and BoNT/B-LC (○). Error bars represent S.E.M. An asterisk (*) denotes a significant difference (p<0.05) and ‘n’ represents sample size (active/inactive neurotoxin). Scale bars: horizontal – 10 ms; vertical – 10 mV (AP), 5 mV (EPSP).</p

Phasic EPSPs are inhibited using BoNT/B-LC under low frequency stimulation.

<p><b><i>A</i></b>, Example showing the phasic action potential (AP) remains unchanged before (<i>i</i>) and after (<i>ii</i>) the injection of each neurotoxin (BoNT/B-LC used as the example). TeNT-LC (<i>B</i>) and BoNT/D-LC (<i>C</i>) did not have an effect on the phasic evoked response. <b><i>E</i></b>, Percent difference between active and inactive neurotoxin, in which the inactive neurotoxin is the reference point at 0%. In <i>B-E</i>, a solid black line represents the time course of neurotoxin injection (90 min) and vertical arrows (↓) represent when EPSPs were recorded. Active neurotoxins: TeNT-LC (▪), BoNT/D-LC (▴) and BoNT/B-LC (•). Inactive neurotoxins: TeNT-LC (□), BoNT/D-LC (△) and BoNT/B-LC (○). Error bars represent S.E.M. An asterisk (*) denotes a significant difference (p<0.05) and ‘n’ represents sample size (active/inactive neurotoxin). Scale bars: horizontal – 10 ms; vertical – 10 mV (AP), 5 mV (EPSP).</p