Supplemental Information. Ca V 2.2 Gates Calcium-Independent. but Voltage-Dependent Secretion. in Mammalian Sensory Neurons

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1 Neuron, Volume 96 Supplemental Information Ca V 2.2 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, and Zhuan Zhou

2 Figure S1 (related to Figure 1). Typical CiVDS induced by standard depolarization pulses (200 ms) in soma of a neuron under whole-cell voltage clamp. The Cm baseline is indicated. The traces simultaneous recorded are membrane capacitance Cm (Cm), and membrane conductance (Gm), whole-cell access resistance (Gs), membrane current (Im), the stimulation membrane voltage (Vm). The cell was recorded under CiVDS conditions (Ca 2+ -free standard external solution (SE) and internal solution (SI) with 10 mm BAPTA, see Methods).

neurons before and after applying TTX to

or TEA to block voltage-gated K + channels

(B to E) Typical current traces (B and D)

3 Figure S2 (related to Figure 1). Voltage-gated Na + and K + channels are not responsible for CiVDS. (A) CiVDS from acutely isolated DRG neurons before and after applying TTX to block voltage-gated Na + channels (left) or TEA to block voltage-gated K + channels (right). (B to E) Typical current traces (B and D) and statistics (C and E) showing that TTX and TEA efficiently block Na + and K + currents in DRG neurons (n = 8 cells for TTX and 13 for TEA). Errorr bars, s.e.m.; ***P <0.001, Student s t-test (C and E).

4 Figure S3 (related to Figure 1). Ca 2+ -dependent secretion of DRG neurons increased after 3 days in culture. (A) Examples of Cm traces inducedd by a 200-ms depolarization in 2.5 mm Ca 2+ -containing solution from 0- and 3-day cultured DRG neurons. (B) Quantification of Cm jump and Ca 2+ current in (A) (n = 15 cells for 0 d and 19 for 3 d DRG neurons). Error bars, s.e.m.; *P <0.05, **P <0.01, Student s t-test (B).

5 Figure S4 (related to Figure 1). Effect of voltage-gated Ca 2+ channel overexpression on Ca 2+ -dependent secretion. (A) Cmm traces induced by a 200-ms depolarization in 2.5 mm Ca 2+ -containing solution from 3-day cultured DRG neurons expressing GFP-only (upper left), Ca V 2.2 (upper right), Ca V V1.2 (lower left), or Ca V V3.2 (lower right). (B and C) Quantification of Cm jump and Ca 2+ current as in (A) (n = 18 cells for GFP, 25 for Ca V 2.2, 9 for Ca V 1.2, and 8 for Ca V 3.2). Error bars, s.e.m.; ns, not significant, one-way ANOVA (B and C).

6 Figure S5 (related to Figure 2). Effect of Ca V 2.2 knockdown on Ca 2+ -dependent secretion. (A) mrna expression levels of different isoforms of voltage-gated Ca 2+ channels quantified with real-time PCR (n = 4 replicates). (B) Examples of Cm traces from DRG neurons infected with control (black) or two different shrna viruses (red and blue) (induced by a 200-ms depolarization in 2.5 mm Ca 2+ -containing solution) ). The DRG neurons were isolated from rats that had been infected with control or knockdown viruses for 3-4 weeks. (C) Quantification of Cm jump and Ca 2+ current as in (B) (n = 15 cells for control virus, 19 for sh-1, and 18 for sh-2). Error bars, s.e.m.; *P <0.05, **P <0.01, ***P <0.001, ns, not significant, one-way ANOVA (A and C).

7 Figure S6 (related to Figures 3 and 4). Effects of S4- and pore-mutated Ca V 2.2 overexpression on Ca 2+ -dependent secretion. (A) Representative Cm traces from 3-day cultured DRG neurons overexpressing WT (black) or S4-mutated (red) Ca V 2.2 (induced by a 200-ms depolarization in 2.5 mm Ca 2+ solution). (B) Quantificationn of Cm jump and Ca 2+ + current as in (A) (n = 18 cells for WT and 15 for S4 mutation). (C) Representative Cm traces from 3-day cultured DRG neurons expressing WT (black) or pore-mutated (grey) Ca V 2.2 (induced by a 200-ms depolarization in 2.5 mm Ca a 2+ solution). (D) Quantification of Cm jump and Ca 2+ current as in (C) (n = 18 cells for WT and 12 for pore mutation). Error bars, s.e.m.; *PP <0.05, **PP <0.01, ns, not significant, Student s t- test (B and D).

8 Figure S7 (related to Figure 5). BoNT/TeNT cleaves the SNARE complex and blocks Ca dependent secretion in DRG neurons. (A) Representative Cm traces from 24-h cultured DRG neurons overexpressing GFP-only control ( left) or that with botulinum toxin and tetanus toxin (BoNT/TeNT) (right) (induced by a 200-ms depolarization in 2.5 mm Ca 2+ solution). (B) Quantification of Cm jump and Ca 2+ current as in (A) (n = 20 cells for control and 19 for BoNT/TeNT). (C to F) Immuno-staining of SNAP-25 ( green) and VAMP2 (red) (C and E) and quantification ( D and F) showing thatt transfectionn with BoNT/TeNT decreased the expression levels of SNAP-25 (n = 36 cells from 3 replicates)

9 and VAMP2 (n = 28 cells from 3 replicates) in DRG neurons. Scale bars, 10 m. Error bars, s.e.m.; *P <0.05, ***P <0.001, ns, not significant, Student s t-test (B, D, and F).

11 Figure S8 (related to Figure 6). Effect of synprint on Ca 2+ -dependent secretion. (A) Representative Cm traces at 1 and 6 min after breaking into DRG neurons with control peptide (black, upper panel) or synprint peptide (grey, lower panel) (induced by a 200-ms depolarization in 2.5 mm Ca 2+ solution). (B and C) Quantification of Cm2/ Cm1 and Im2/Im1 in (A) (n = 16 cells for control and 15 for synprint). Cm2/ Cm1 are shown in (A). Im1 and Im2 represent the membrane current recorded from the same neuron in (A) at 1 and 6 min. (D and E) Representative current traces and I-V curves from HEK293A cells expressing WT (black, n = 10 cells) and synprint-truncated (Δsynprint) Ca V 2.2 (grey, n = 10 cells) (induced by a 200-ms depolarization in 2.5 mm Ca 2+ solution). (F) Representative Cm traces from 3-day cultured DRG neurons expressing WT (black) and Δsynprint Ca V 2.2 (grey) (induced by a 200-ms depolarization in 2.5 mm Ca 2+ solution). (G) Quantification of Cm jump and Ca 2+ current in (F) (n = 25 for WT Ca V 2.2 and 14 for Δsynprint). Error bars, s.e.m.; **P <0.01, ***P <0.001; ns, not significant, Student s t- test (B, C and G).

12 Figure S9 (related to Figure 7). TIRF images of the Ca 2+ -dependent release of Spyimage phluorin- and NPY-pHluorin-labeled vesicles from DRG neurons. (A) Typical of a DRG neuron (left) and fluorescence intensity of 2 events (right) showing the release of Spy-pHluorin-marked clear vesicles under electrical stimulation in 2.5 mm Ca 2+ - containing solution. Scale bar, 10 μm. (C) As in (A), but NPY-pHluorin replaced Spy- phluorin to image the release of dense-core vesicles. Scale bar, 10 μm. (B and D) Statistics of (A) and (C), showing the number of release events per celll in different time windows, without (Ctrl, 23 cells for Spy-pHluorin, 21 for NPY-pHluorin) or with the Cav2.2 antagonist (GVIA, 8 cells for Spy-pHluorin, 7 for NPY-pHluorin). Errorr bars, s.e.m.

by current injection in a DRG neuron before (Ctrl,

right). Current injection was 0.5 na for 1 s.

13 Figure S10 (related to Figure 7). ATP increased the number of action potentials in DRG neurons. (A) Representative action potentials (AP) generated by current injection in a DRG neuron before (Ctrl, left) and after treatment with 100 M ATP (ATP, right). Current injection was 0.5 na for 1 s. (B) Statistics showing that ATP increased the AP frequency in DRG neurons (n = 11 cells; **P <0.01, Student s t-test).

14 Movie S1 (Related to Figure S9). TIRF imagingg of depolarization-induced exocytosis of single Spy-pHluorin-labeled vesicles from DRG neurons in 2.5 mm Ca 2+ -containing bath solution.

15 Movie S2 (Related to Figure 7). TIRF imaging of depolarization-induced exocytosis of single Spy-pHluorin-labeled vesicles in Ca 2+ -free conditions.

16 Movie S3 (Related to Figure S9). TIRF imagingg of depolarization-induced exocytosis of single NPY-pHluorin-labeled vesicles from DRG neurons in 2.5 mm Ca 2+ -containing bath solution..

17 Movie S4 (Related to Figure 7). TIRF imaging of depolarization-induced exocytosis of single NPY-pHluorin-labeled vesicles in Ca 2+ -freee conditions.

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