1. Volume 96, Issue 6, Pages 1317-1326.e4 (December 2017)
CaV2.2 Gates Calcium-Independent but Voltage-Dependent Secretion in Mammalian Sensory Neurons 
Zuying Chai, Changhe Wang, Rong Huang, Yuan Wang, Xiaoyu Zhang, Qihui Wu, Yeshi Wang, Xi Wu, Lianghong Zheng, Chen Zhang, Wei Guo, Wei Xiong, Jiuping Ding, Feipeng Zhu, Zhuan Zhou 
Neuron 
Volume 96, Issue 6, Pages e4 (December 2017)
DOI: /j.neuron
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2. Figure 1 CaV2.2 Is Necessary for CiVDS
(A) Typical Cm traces induced by a standard depolarization of 200 ms from –70 to 0 mV (arrows) in Ca2+-free solution (CiVDS) from acutely isolated DRG neurons before and after applying control solution (Ctrl, left) or the CaV2.2 blocker ω-conotoxin GVIA (GVIA, right).
(B) Quantification of the Cm jump after puffing drugs (ΔCm2). Except for the cases of control solution (n = 29 cells) and GVIA (n = 29 cells), we used tetrodotoxin (TTX, n = 15 cells) and TEA (n = 17 cells) to block voltage-gated Na+ and K+ channels.
(C) Left: CiVDS from DRG neurons acutely isolated (0 days) or cultured for 3 days. Right: quantification of the Cm jump at 0 (n = 27 cells) and 3 days (n = 30 cells).
(D) Representative western blots and statistical data showing the expression levels of CaV2.2 from 0- and 3-day DRG neurons (n = 4 replicates).
(E) Left: typical current traces induced by a 200-ms depolarization from 0 day and 3 day DRG neurons in 2.5 mM Ca2+-containing solution before and after puffing GVIA (the reduced current by GVIA was CaV2.2 current). Right: quantitative plot of current versus voltage (I-V) curve of the CaV2.2 current at the left (n = 7 cells each).
(F) CiVDS from 3-day cultured DRG neurons overexpressing GFP only (left, GFP-OE) or together with CaV2.2 (right, Cav2.2 OE). The CaV2.2 plasmid was co-expressed with the α2δ1 and β3 auxiliary subunits.
(G) Statistics of Cm jumps in (F) with separate OE of GFP, voltage-gated N-type (CaV2.2), L-type (CaV1.2), or T-type (CaV3.2) Ca2+ channels (n = 30 cells for GFP, 27 for CaV2.2, 20 for CaV1.2, and 12 for CaV3.2).
Error bars, SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant; one-way ANOVA (B and G) and Student’s t test (C–E).
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3. Figure 2 In Vivo KD of CaV2.2 Blocked CiVDS
(A) Typical western blots showing the expression levels of CaV2.2 from DRG neurons infected with control (scrambled, Sc) adeno-associated virus (AAV), or two KD viruses (sh-1 and sh-2, n = 4 replicates).
(B) Schematic of the injection of AAV into DRG neurons in vivo. AAV was directly injected into the L5 DRG after removing the dorsal vertebra.
(C) Immunofluorescence of CaV2.2 in DRG neurons acutely isolated from rats infected with two KD viruses for 3–4 weeks. The fluorescence intensity of CaV2.2 in KD cells was normalized to control cells (n = 19 cells for control and sh-1 and 20 cells for control and sh-2). Scale bars, 20 μm.
(D and E) Typical traces (D) and statistics (E) CiVDS from acutely isolated DRG neurons infected with Sc and two KD viruses (n = 15 for control virus, 17 for sh-1, and 21 for sh-2).
Error bars, SEM; ∗∗p < 0.01; one-way ANOVA (A and E) and Student’s t test (C).
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4. Figure 3 The Voltage-Sensitive Region of CaV2.2 Is Critical for CiVDS
(A) Schematic of mutated sites in the voltage-sensitive segment (S4) of CaV2.2.
(B) Typical current traces and I-V curves from HEK293A cells expressing WT (black, n = 12 cells) and S4-mutated (red, n = 14 cells) CaV2.2 (currents induced by 200-ms depolarization in 2.5 mM Ca2+-containing solution).
(C) Immunoblots of membrane surface and total expression of WT and S4-mutated CaV2.2 in HEK293A cells (top) and statistics (bottom, n = 5 replicates).
(D and E) CiVDS from 3-day cultured DRG neurons overexpressing WT or S4-mutated CaV2.2 (D) and statistics (E) (n = 30 cells for WT and 17 cells for S4 mutation).
Error bars, SEM; ∗∗p < 0.01; Student’s t test (C and E).
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5. Figure 4 CiVDS Is Independent of the Pore Region of CaV2.2
(A) Schematic of mutated sites in the pore region of CaV2.2.
(B) Typical [Ca2+]i (top) and current membrane current (Im) traces (bottom) recorded from HEK293A cells expressing WT or pore region-mutated CaV2.2 (induced by a 500-ms depolarization in 2.5 mM Ca2+-containing solution).
(C) Quantification of (B) (ampl, amplitude) (n = 8 cells for each).
(D) Immunoblots of membrane surface and total expression of WT and pore region-mutated CaV2.2 in HEK293A cells (top) and statistics (bottom, n = 4 replicates).
(E and F) CiVDS from 3-day cultured DRG neurons overexpressing WT or pore region-mutated CaV2.2 (E) and statistics (F) (n = 30 cells for WT and 12 cells for pore mutation).
Error bars, SEM; ∗p < 0.05; Student’s t test (C, D, and F).
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6. Figure 5 CiVDS Is SNARE Complex-Dependent
(A) Co-immunoprecipitation (coIP) of CaV2.2 with SNAP-25 in DRG tissue. Protein lysates collected from DRG tissue were immunoprecipitated with anti-CaV2.2 (top) or anti-SNAP-25 (bottom), followed by immunoblotting (IB) with antibodies as indicated (n = 3 replicates).
(B) CoIP of CaV2.2 with SNAP-25 in HEK293A cells. FLAG-CaV2.2 was co-expressed with SNAP-25, and then the cell lysates were immunoprecipitated with anti-FLAG (left) or anti-SNAP-25 (right), followed by IB with the indicated antibodies (n = 3 replicates). HEK293A cells expressing FLAG-CaV2.2 or SNAP-25 only were used as controls.
(C) Immunostaining of endogenous SNAP-25 (green) and CaV2.2 (red) in an acutely isolated DRG neuron. Bottom: enlargements of the boxes at the top. Arrowheads indicate co-localization of SNAP25 and Cav2.2. Scale bar, 20 μm.
(D and E) CiVDS from 3-day cultured DRG neurons overexpressing GFP (left) or SNAP-25 (right) (D) and quantification (E) (n = 30 cells for GFP and 17 cells for SNAP-25).
(F and G) CiVDS from 24-hr cultured DRG neurons overexpressing control (left) or botulinum toxin/tetanus toxin (right) (F) and statistics (G) (n = 22 for control and 24 for toxins).
Error bars, SEM; ∗p < 0.05, ∗∗∗p < 0.001, Student’s t test (E and G).
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7. Figure 6
Synprint, the Linker between CaV2.2 and SNARE Protein in DRG Neurons, Is Essential for CiVDS
(A) Cartoon showing the binding site between CaV2.2 and SNAP-25. Red indicates the synprint site (amino acids 772–856) located between domains II and III of CaV2.2.
(B and C) CoIP of WT CaV2.2 or synprint-truncated CaV2.2 (Δsynprint) with SNAP-25. FLAG-CaV2.2 or FLAG-Δsynprint was co-expressed with SNAP-25 in HEK293A cells, and the lysates were immunoprecipitated with anti-FLAG (B) or anti-SNAP-25 (C), followed by IB with the indicated antibodies (n = 3 replicates).
(D) CiVDS recordings from acutely isolated DRG neurons 1 and 6 min after they had been whole cell-dialyzed with control peptide (n = 9 cells) or synprint peptide (n = 13 cells).
(E) Statistics of ΔCm2/ΔCm1 in (D).
(F and G) CiVDS from 3-day cultured DRG neurons overexpressing WT or Δsynprint CaV2.2 (F) and statistics (G) (n = 27 for WT and 15 for Δsynprint).
Error bars, SEM; ∗∗p < 0.01, ∗∗∗p < 0.001; Student’s t test (E and G).
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8. Figure 7 ATP Is Released from DRG Neurons via Both CDS and CiVDS
(A) Cartoon showing the luciferase assay used to detect ATP release from acutely isolated DRG neurons via CiVDS or CDS. For CiVDS, 100 mM KCl in Ca2+-free solution (0Ca 100K) was used to stimulate DRG neurons. For CDS, 0Ca caffeine (20 mM) was used to increase [Ca2+]i by releasing intracellular Ca2+ from the endoplasmic reticulum.
(B) Left: summary of ATP concentrations in different solutions. An antagonist of CaV2.2, ω-conotoxin-GVIA, was used to test the effect of CaV2.2 on CiVDS-mediated ATP release (n = 5 replicates). Right: leak of cytosol LDH detected only after adding digitonin (n = 4 replicates).
(C) Representative EM images showing a docked clear vesicle (arrows, left), a large dense-core vesicle (black arrows, right), and a clathrin-coated pit (white arrowheads, right), all of similar size (∼100 nm), in a DRG neuron. Scale bar, 500 nm.
(D) Statistics for docked clear vesicles/section of DRG neurons incubated in 0Ca solution (n = 12 sections from 3 cells) or 0Ca 100K solution (n = 10 sections from 3 cells) for 2 min.
(E) Left: TIRF image showing the exocytosis of Syp-pHluorin-marked vesicles following whole-cell depolarization in a 0Ca bath. Top right: cartoon of a fusing vesicle labeled by Spy-pHluorin. Bottom: two typical events (intensity versus time) marked in the left image.
(F) Statistics showing release events in 0Ca solution before, during, and after depolarization with or without the CaV2.2 antagonist (∗∗∗p < 0.001; two-way ANOVA; n = 17 cells for control, 11 cells for GVIA).
(G and H) As in (E) and (F), but Spy-pHluorin was replaced with NPY-pHluorin to indicate dense-core vesicle release (∗∗∗p < 0.001; two-way ANOVA; n = 13 cells for control, 8 cells for GVIA).
Error bars, SEM; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; Student’s t test (B and D).
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9. Figure 8 Model of Mechanism of CiVDS
The voltage-gated Ca2+ channel CaV2.2 binds with SNARE proteins through the synprint region (red) on the plasma membrane of DRG neurons (top). When an AP arrives, it drives a conformational change of the CaV2.2-SNARE complex and then triggers “CiVDS-vesicle” fusion and ATP release even without Ca2+ (bottom).
Neuron  , e4DOI: ( /j.neuron )
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