Volume 22, Issue 8, Pages 1956-1964 (February 2018) BK Potassium Channels Suppress Cavα2δ Subunit Function to Reduce Inflammatory and Neuropathic Pain  Fang-Xiong Zhang, Vinicius M. Gadotti, Ivana A. Souza, Lina Chen, Gerald W. Zamponi  Cell Reports  Volume 22, Issue 8, Pages 1956-1964 (February 2018) DOI: 10.1016/j.celrep.2018.01.073 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 22, 1956-1964DOI: (10.1016/j.celrep.2018.01.073) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 BK Channel Coexpression Reduces the Plasma Membrane Expression and Currents of HVA Cav Channels (A) Representative confocal images of tsA-201 cells expressing Cav2.2 (α1B-GFP + Cavβ2a + Cavα2δ-1), with co-expression of either empty vector (left) or the BK channel (right). The arrow indicates the cell surface and the arrowhead indicates intracellular α1B-GFP. Scale bar, 10 μm. (B) Statistical analysis of plasma membrane expression of α1B-GFP (plasma membrane fluorescence of total fluorescence) in tsA-201 cells in the presence of empty vector (0.564 ± 0.027; n = 32) or full-length BK channels (0.220 ± 0.022; n = 24; ∗∗∗p < 0.001). Error bars in all figures indicate SEM. (C) Co-localization of Cav2.2 (α1B-GFP) and mCherry-Rab7 in tsA-201 cells, in the presence of either empty vector (top) or full-length BK channel (bottom). The arrow denotes the cell surface and the arrowhead indicates intracellular α1B-GFP. Scale bar, 10 μm. (D) Current-voltage relationships for Cav2.2 (α1B + Cavβ2a + Cavα2δ-1), after coexpression with either empty vector (peak current density −217.17 ± 26.03 pA/pF at 0 mV; n = 25) or the BK channel (−130.86 ± 17.93 pA/pF at +5 mV; n = 25; p = 0.009). Currents in all figures were normalized to cell capacitance. (E) Current-voltage relationships for Cav2.1 (α1A + Cavβ1b + Cavα2δ-1) after coexpression with either empty vector (−244.27 ± 36.79 pA/pF at −20 mV; n = 20) or BK (−136.67 ± 15.28 pA/pF at −5 mV; n = 20; p = 0.010). (F) Current-voltage relationships for Cav1.2 (α1C + Cavβ1b + Cavα2δ-1) after coexpression with either empty vector (−110.86 ± 11.58 pA/pF at −5 mV; n = 10) or BK (−26.42 ± 3.61 pA/pF at +10 mV; n = 10; p < 0.001). (G) Current-voltage relationships for Cav3.2 (without Cavβ and Cavα2δ), in the presence of either empty vector (−57.66 ± 3.27 pA/pF at −15 mV; n = 9) or BK (−64.99 ± 7.05 pA/pF at −15 mV; n = 9; p = 0.359). Cell Reports 2018 22, 1956-1964DOI: (10.1016/j.celrep.2018.01.073) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 The BK Channel N Terminus Reduces the Plasma Membrane Expression and Currents of HVA Cav Channels (A) The N-terminal peptide sequence of the human BK channel. Letters in bold show predicted initiation methionines (M1, M25, and M66). (B) Statistical analysis of the plasma membrane expression of α1B-GFP (plasma membrane fluorescence of total fluorescence) in tsA-201 cells expressing Cav2.2(α1B-GFP), with either empty vector (0.564 ± 0.027; n = 32), or BK(Δ1-24) (0.604 ± 0.025; n = 20; p = 0.302 versus Cav2.2), or BK(Δ1-65) (0.510 ± 0.021; n = 28; p = 0.120 versus Cav2.2). (C) Current-voltage relationships for Cav2.2, with either empty vector (−188.65 ± 23.55 pA/pF at +5 mV; n = 15) or BK(Δ1-24) (−172.52 ± 16.10 pA/pF at +5 mV; n = 15; p = 0.576 versus Cav2.2), or BK(Δ1-65) (−173.09 ± 17.31 pA/pF at +5 mV; n = 15; p = 0.597 versus Cav2.2). (D) Statistical analysis of the plasma membrane expression of α1B-GFP (of total) in tsA-201 cells overexpressing Cav2.2(α1B-GFP), with either empty vector (0.531 ± 0.028; n = 21) or BK(1-86) (0.154 ± 0.019; n = 21; ∗∗∗p < 0.001 versus Cav2.2.), or BK(1-65) (0.534 ± 0.042; n = 22; p = 0.960 versus Cav2.2), or BK(66-86) (0.452 ± 0.035; n = 20; p = 0.086 versus Cav2.2). The fragments of the BK channel (BK fragment) were anchored to the plasma membrane by covalent linkage to glycosylphosphatidylinositol (GPI), with signal peptide (SP) from cellular prion protein linked at N-terminal. (E) Current-voltage relationships for Cav2.2 coexpressed with either empty vector (−161.27 ± 11.78 pA/pF at +5 mV; n = 15) or BK(1-86) (−69.04 ± 11.68 pA/pF at +15 mV; n = 15; p < 0.001 versus Cav2.2), or BK(1-65) (−153.27 ± 9.93 pA/pF at +5 mV; n = 15; p = 0.608 versus Cav2.2), or BK(66-86) (−147.32 ± 13.10 pA/pF at +5 mV; n = 15; p = 0.435 versus Cav2.2). (F) Current-voltage relationships for Cav2.2 coexpressed with either empty vector (−191.81 ± 10.71 pA/pF at +5 mV; n = 15) or BK(N3A) (−76.28 ± 7.96 pA/pF at +15 mV; n = 15; p < 0.001 versus Cav2.2), or BK(N3D) (−186.71 ± 9.46 pA/pF at +5 mV; n = 15; p = 0.734 versus Cav2.2). (G) Current-voltage relationships for the BK channel and BK(N3D) expressed in tsA-201 cells (p = 0.311, two-way ANOVA). In all panels, Cav2.2 refers to combination of α1B (tagged with GFP only when indicated), Cavβ2a, and Cavα2δ-1. Cell Reports 2018 22, 1956-1964DOI: (10.1016/j.celrep.2018.01.073) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 BK Channel Reduces HVA Currents via the Cavα2δ-1 Subunit (A) Mouse whole brain lysates were immunoprecipitated with an anti-BK channel antibody and blotted with anti-Cavα2δ-1. (B) The BK channel N terminus fragments (BK fragment) were fused with mKate2 through the transmembrane region (TM) of CD4, with signal peptide (SP) from cellular prion protein linked at N-terminal, and expressed in tsA-201 cells. Whole-cell lysates from transfected cells were immunoprecipitated with anti-mKate2 and blotted with anti-Cavα2δ-1. (C) Current-voltage relationships for Cav2.2 (α1B + Cavβ2a, without Cavα2δ-1) in the presence of either empty vector (peak current was −53.71 ± 4.47 pA/pF at +20 mV; n = 10) or full-length BK channel (−55.36 ± 3.44 pA/pF at +20 mV; n = 10; p = 0.772). (D) Current-voltage relationships for Cav2.1 (α1A + Cavβ1b, without Cavα2δ-1) after coexpression with either empty vector (−158.85 ± 7.27 pA/pF at −5 mV; n = 10) or full-length BK channel (−157.20 ± 9.44 pA/pF at −5 mV; n = 10; p = 0.892). (E) Current-voltage relationships for Cav1.2 (α1C + Cavβ1b, without Cavα2δ-1) in the presence of either empty vector (−46.10 ± 4.41 pA/pF at +15 mV; n = 10) or full-length BK channel (−46.77 ± 3.22 pA/pF at +15 mV; n = 10; p = 0.904). (F) Current-voltage relationships for Cav2.2(NITNKS, w/o α2δ1) (α1B(NITNKS) + Cavβ2a) (−190.70 ± 14.32 pA/pF at 0 mV; n = 15) or Cav2.2(NITNKS) (α1B(NITNKS) + Cavβ2a + Cavα2δ-1) (−184.39 ± 11.95 pA/pF at +10 mV; n = 15; p = 0.738 versus Cav2.2(NITNKS, w/o α2δ1)), and Cav2.2(NITNKS) + BK (−189.08 ± 11.53 pA/pF at +5 mV; n = 15; p = 0.780 versus Cav2.2(NITNKS)). (G) Representative confocal images of tsA-201 cells expressing Cav2.2 (α1B + Cavβ2a + GFP-Cavα2δ-1) in the presence of either empty vector (left) or BK (right). Scale bar, 10 μm. (H) Statistical analysis of plasma membrane expression of GFP-Cavα2δ-1 (plasma membrane fluorescence to total fluorescence) in tsA-201 cells expressing α1B + Cavβ1b + GFP-Cavα2δ-1 in the presence of either empty vector (0.620 ± 0.018; n = 79) or full-length BK (0.647 ± 0.019; n = 76; p = 0.286). Cell Reports 2018 22, 1956-1964DOI: (10.1016/j.celrep.2018.01.073) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 The BK Channel N Terminus Inhibits Neuropathic and Inflammatory Pain (A) Representative confocal image of cultured DRG neurons infected with AAV9-N3A (1 × 109 vector genomes [vg]/mL) 2 weeks after virus application. The arrow indicates soma and arrow head indicates neurite. Scale bar, 100 μm. The BK channel N terminus (BK(1-86)(N3A)) was GPI anchored to the plasma membrane, with signal peptide (SP) from cellular prion protein linked at the N-terminal. Viral expression was visualized via the mKate2 fluorescence, which is separately expressed by an IRES2. (B) Current-voltage relationships for Cav currents recorded from cultured DRG neurons 2 weeks after infection with either control AAV9 (AAV9-GFP; 1 × 109 vg/mL) (peak current was −96.25 ± 8.48 pA/pF at −10 mV; n = 10) or AAV9-N3A (1 × 109 vg/mL) (−39.30 ± 3.32 pA/pF at −5 mV; n = 10; p < 0.001 versus control), or AAV9-N3D (1 × 109 vg/mL) (−90.80 ± 5.92 pA/pF at −10 mV; n = 10; p = 0.605 versus control). (C and D) Mechanical (C) and thermal (D) hyperalgesia of mice injected prophylactically with either AAV9-GFP (1 × 1011 vg/mouse; n = 8) or AAV9-N3A (1 × 1011 vg/mouse; n = 11), or AAV9-N3D (1 × 1011 vg/mouse; n = 12) during neuropathic pain. AAV9-N3A injection prevented the development of both mechanical (p < 0.001 versus AAV9-GFP, two-way ANOVA) and thermal (p < 0.001 versus AAV9-GFP, two-way ANOVA) hyperalgesia induced by PSNI. AAV9-N3D injection was ineffective (p = 0.193 for mechanical and p = 0.398 for thermal, versus AAV9-GFP, two-way ANOVA). The hashtag (#) indicates the difference between injured animals (AAV9-GFP) and the sham group (p < 0.001). (E and F) Mechanical (E) and thermal (F) hyperalgesia of mice injected therapeutically with either AAV9-GFP (1 × 1011 vg/mouse; n = 8) or AAV9-N3A (1 × 1011 vg/mouse; n = 7) during neuropathic pain. AAV9-N3A injection inhibited hyperalgesia of neuropathic mice (p < 0.001 for mechanical and p = 0.014 for thermal, versus AAV9-GFP, two-way ANOVA). The hashtag (#) indicates the difference between injured mice (AAV9-GFP) and the sham group (p < 0.001). (G and H) Mechanical (G) and thermal (H) hyperalgesia of mice under persistent inflammatory pain induced by intraplantar CFA injection, treated with either AAV9-GFP (1 × 1011 vg/mouse; n = 5 for mechanical and 8 for thermal) or AAV9-N3A (1 × 1011 vg/mouse; n = 5 for mechanical and 9 for thermal). The withdrawal thresholds of mice injected with AAV9-N3A are different than those treated with AAV9-GFP (p = 0.004 for mechanical and p = 0.0012 for thermal, Student’s t test). The hashtag (#) indicates the difference between inflammatory mice (AAV9-GFP) and the sham group (intraplantar injection of PBS) (p < 0.001 for mechanical and p = 0.011 for thermal, Student’s t test). Note that the baseline groups in (G) and (H) reflect the same animals as those in the treatment groups, but were assessed prior to virus and CFA injections. Cell Reports 2018 22, 1956-1964DOI: (10.1016/j.celrep.2018.01.073) Copyright © 2018 The Author(s) Terms and Conditions