Volume 67, Issue 2, Pages 268-279 (July 2010) V-ATPase Membrane Sector Associates with Synaptobrevin to Modulate Neurotransmitter Release  Jérôme Di Giovanni, Sami Boudkkazi, Sumiko Mochida, Andrzej Bialowas, Nada Samari, Christian Lévêque, Fahamoe Youssouf, Aline Brechet, Cécile Iborra, Yves Maulet, Nicole Moutot, Dominique Debanne, Michael Seagar, Oussama El Far  Neuron  Volume 67, Issue 2, Pages 268-279 (July 2010) DOI: 10.1016/j.neuron.2010.06.024 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Determinants of c-Subunit Interaction with VAMP2 (A) Alignments of c-subunit recombinant constructions used in Y2H and their interaction with full-length VAMP2. (B) Summary of binary interactions between c-subunit and SNARE constructs. All experiments were reproduced at least four times. See also Figure S1. Neuron 2010 67, 268-279DOI: (10.1016/j.neuron.2010.06.024) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 The Membrane-Proximal Domain of VAMP2 Mediates Its Interaction with V0 c-Subunit Wild-type mutant and truncation constructs of GST-VAMP2 fusion proteins were immobilized on glutathione beads and used to pull down wild-type HSV-tagged c-subunit (c-2-155). After SDS-PAGE, samples were immunoblotted and bound c-2-155 was detected with anti-HSV antibodies. Arrowhead indicates position of c-2-155 monomer. (A) Pull-down of purified c-subunit (c-2-155) with either GST or GST-VAMP2. Left: Coomassie blue staining of 3 μg c-2-155. Right: specific binding of c-2-155 to GST-VAMP2-2-92 (GVΔ). Load lane contains 200 ng of purified c-2-155. Note the presence of c-2-155 oligomers that resisted SDS denaturation. (B) Mapping of c-2-155 binding site on VAMP2. Binding of c-2-155 to GST-VAMP2-28-92 (GV-28-92) but not to GV-2-31 or GV-28-76. Load lane contains 50 ng of purified c-2-155. (C) Tryptophan residues 89 and 90 on VAMP2 are crucial in mediating c-subunit interaction. c-2-155 binding to GV-W89,90A, GV-W89,90P, or GV-W90A is strongly reduced. (D) c-2-155 binding to immobilized GVΔ was monitored by ELISA with anti-HSV antibodies in the presence or absence of 1 or 5 μM of CaM or CAM1,2,3,4 and 0.5 mM Ca2+. Results are means ± SEM; n = 3. See also Figure S2. Neuron 2010 67, 268-279DOI: (10.1016/j.neuron.2010.06.024) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 c-Subunit Loop 3.4 Mediates Binding to VAMP2 (A) Binding of c-117-155 to VAMP2 immobilized on a sensor chip, monitored by SPR (Biacore). c-117-155 or c-117-155-L3.4s (100 nM) were injected (black bar) over captured His-VAMP2 (representative of four independent experiments). (B) Effect of L3.4, L3.4s, PH-L3.4, and PH-L3.4s on GVΔ binding to immobilized full-length c2-155 in ELISA. In contrast to L3.4s and PH-L3.4s, L3.4 as well as PH-L3.4 efficiently displaced binding. Results are mean ± SEM; n = 3. (C) Pull down of GVΔ and GV-W89,90P by immobilized PH-L3.4 or PH-L3.4s. 1 and 10 μM L3.4 displaced, respectively, 16% and 64% of GVΔ binding to PH-L3.4. See also Figure S3. Neuron 2010 67, 268-279DOI: (10.1016/j.neuron.2010.06.024) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 V0 c-Subunit Loop 3.4 Peptide Inhibits Neurotransmission at L5-L5 Excitatory Synapses (A) Effect of the wild-type L3.4 peptide. Top, representative examples of synaptic currents recorded 2 min (left) and 45 min (right in gray) after whole-cell access. Bottom, time course of the inhibition of the normalized synaptic current produced by the peptide. Dotted line represents the baseline level. (B) Effect of the scrambled L3.4 s peptide. Note the stability of synaptic transmission. (C) Mean of normalized EPSCs at 40 min recording ± SEM (∗p < 0.01, unpaired Student's t test). (D) Inhibition observed with bath application of bafilomycin. See also Figure S4. Neuron 2010 67, 268-279DOI: (10.1016/j.neuron.2010.06.024) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Differential Effects of L3.4 and Bafilomycin on Paired-Pulse Ratio and Synaptic Vesicle Proton Pump Activity (A) Changes in the paired-pulse ratio produced by L3.4. Top, representative examples of paired-pulse plasticity recorded 2 min (left) and 45 min (right in gray) after whole-cell access. Bottom, time course of the normalized paired-pulse ratio. Note the increase in the paired-pulse ratio. (B) Changes in the paired-pulse ratio produced by bafilomycin. Note the decrease in the paired-pulse ratio (red traces). (C) Mean of normalized PPR after 40 min of recording (∗∗p < 0.001, unpaired Student's t test). (D) Effects of L3.4 peptide or bafilomycin on synaptic vesicle acidification in vitro determined by monitoring acridine orange fluorescence. Arrows indicate addition of Mg2+/ATP. In control and L3.4, the reaction was stopped (arrowheads) by adding EDTA. See also Figure S5. Neuron 2010 67, 268-279DOI: (10.1016/j.neuron.2010.06.024) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Effect of L3.4 on Cholinergic Neurotransmission (A1) Excitatory postsynaptic potentials (EPSPs) from one representative experiment recorded 1 min before (black) and 5 (blue) and 20 (cyan) min after the start of 3 min presynaptic injection of L3.4 (1.5 mM in the injection pipette). (A2) The first order derivative of EPSPs before (black) and 20 min after (cyan) L3.4 injection shown in (A1). Pink dotted line indicates the peak of EPSPs. (A3) The peak amplitude of EPSPs shown in (A1) was normalized to that before the injection. (B) Smoothed values of normalized and averaged EPSP (n = 6–7). EPSP was recorded every 10 s in the presence of L3.4, L3.4s, VAMP77-94, or VAMP77-94m (1.5 mM of each peptide in the injection pipette). (C) Decrease in amplitudes of EPSP elicited by 0.1, 0.25, and 0.5 Hz (n = 5–8) action potentials at 20 min after peptide injection. Mean ± SEM. ∗∗p < 0.01; ∗p < 0.05, unpaired Student's t test. (D) Normalized EPSP amplitudes (mean ± SEM) during and after depletion of synaptic vesicles by 900 action potentials at 5 Hz. The line shows smoothed values. At 15 min after starting injection of 1.5 mM L3.4 (n = 7), EPSP amplitudes were measured every 1 s. After 1 min of EPSP recording, the train of stimuli (gray horizontal bar) was applied. (E) As in (D) but with L3.4s (n = 7). (F) Recovery of EPSP amplitude at 1 min and 5 min after the end of the train of stimulation in the presence of L3.4 (yellow) or L3.4s (green). At 1 min, p = 0.80 (L3.4 versus L3.4s). At 5 min, p = 0.67 (L3.4 versus L3.4s). Unpaired Student's t test. See also Figure S6. Neuron 2010 67, 268-279DOI: (10.1016/j.neuron.2010.06.024) Copyright © 2010 Elsevier Inc. Terms and Conditions