Fig 2: α2δ-1 is essential for the increased prevalence of CP-AMPARs in spinal cord synapses in neuropathic pain(A) Effect of intrathecal injection of 10 and 20 μg of IEM-1460 (IEM) on the withdrawal thresholds in rats treated with a lentiviral Cacna2d1 (α2δ-1) vector (n = 9 rats) or a control (Cont) vector (n = 8 rats). *p < 0.05, **p < 0.01, ***p < 0.001 versus baseline. One-way ANOVA followed by Dunnett test.(B–D) α2δ-1 overexpression induces a switch from CI-AMPARs to CP-AMPARs in spinal dorsal horn neurons. Original traces (B), I-V plots (C), and rectification index (D) of AMPAR-EPSCs of neurons in rats 5 weeks after injection of a lentiviral Cacna2d1 vector (n = 15 neurons) or a control vector (n = 11 neurons). *p < 0.05 versus the control vector (two-tailed Student’s t test).(E–G) Nerve injury has no effect on AMPARs in spinal dorsal horn neurons of Cacna2d1 knockout (KO) mice. Original traces (E), I-V plots (F), and rectification index (G) of AMPAR-EPSCs of lamina II neurons in wild-type (WT) and Cacna2d1 KO mice subjected to spared nerve injury (SNI; n = 15 neurons in WT, n = 13 neurons in Cacna2d1 KO) or sham surgery (n = 14 neurons in WT, n = 13 neurons in KO). *p < 0.05 versus WT sham group. One-way ANOVA followed by Tukey test.

Fig 3: CaM promotes the cell surface localization of CaV1.2 WT but not CaV1.2 G449R.A, HEKT cells were transiently transfected with CaV1.2 WT + CaVa2d1 + CaVß2a (left) and CaV1.2 G449R + CaVa2d1 + CaVß2a (right) in the presence of native/endogenous CaM. B, HEKT cells were transiently transfected with CaV1.2 WT + CaVa2d1 + CaVß2a + CaM WT (left) and CaV1.2 G449R + CaVa2d1 + CaVß2a + CaM WT (right). Two days after transfection, the cells were lysed, and cell fractions were obtained through preparative ultracentrifugation as described in the Experimental procedures section. Western blotting was carried out for the four protein fractions found in lanes 1 to 4; lane 1: total proteins; lane 2: cytoplasmic proteins; lane 3: total membrane proteins; and lane 4: plasma membrane proteins. The proteins were probed with the following antibodies: CaV1.2 (Alomone; catalog no.: ACC003, 1:250 dilution) with anti-rabbit (1:10,000 dilution); CaVa2d1 (Alomone; catalog no.: ACC015, 1:1000 dilution) with anti-rabbit (1:10,000 dilution); CaVß2a (Alomone; catalog no.: ACC105, 1:1000 dilution) with anti-rabbit (1:10,000 dilution); CaM (Millipore; catalog no.: 05-193, 1:1000 dilution) with anti-mouse (1:10,000 dilution); His (Invitrogen; catalog no.: 71700, 1:1000 dilution) with anti-mouse (1:10,000 dilution); and cadherin (Pan-cadherin; Thermo Fisher; catalog no.: 71-7100, 1:1000 dilution) with anti-rabbit (1:10,000). Cadherin was used as a marker for the plasma membrane. The membrane was cut at 115 and 28 kDa to probe first CaV1.2, CaVß2a, and CaM. Membranes were then stripped and reprobed with antibodies against the proteins: CaVa2d1, cadherin, and housekeeping GAPDH (Sigma; catalog no.: G9545, 1:10,000 dilution with anti-rabbit [1:10,000 dilution]). Each lane was loaded with 20 µg proteins. The lines to the left of the blots indicate the position of the molecular markers, and the value is provided in kilodalton. The molecular masses were estimated by linear regression and interpolation from the molecular mass markers using the Image Lab software, version 5.2 (Bio-Rad). As seen in A, in the presence of endogenous CaM, the signal for CaV1.2 WT was stronger in the total membrane protein fraction (lane 3) than in the cell surface protein fraction (lane 4). Under the same conditions, the signal for CaV1.2 G449R was stronger in the cell surface protein fraction. B, demonstrates that under conditions where CaM was overexpressed, the signal for CaV1.2 WT and CaV1.2 G449R is stronger in the cell surface protein fraction. Along with CaV1.2, CaVa2d1 (as previously reported (4)) and CaVß2a are found in the cell surface fraction but not CaM and GAPDH. This observation suggests that the interaction of CaM with the pore-forming subunit is very robust during the cell surface export and is compatible with the binding and unbinding kinetics of CaM in other cell types (42). No significant signal was found in the cytoplasmic fraction for CaVa2d1 and the membrane-anchored CaVß2a. This result was successfully obtained from two independent transfections carried out over the course of 2 months. CaM, calmodulin; HEKT, human embryonic kidney 293T cell line.

Fig 4: G449R interacts with CaVß proteins. HEKT cells were transiently transfected with CaV1.2 WT (WT) or CaV1.2 G449R (GR) with cMyc-tagged CaVß3 or cMyc-tagged CaVß2a. CaVa2d1 was present throughout. Cell lysates were immunoprecipitated overnight with anti-cMyc magnetic beads (Pierce Anti-c-Myc Magnetic Beads; catalog no.: 88842, Thermo Fisher Scientific) to capture the CaVß, eluted in a 2× Laemmli buffer and fractionated by 8% SDS-PAGE gels. A, immunoblotting was carried out on total proteins (20 µg) collected from the cell lysates before the immunoprecipitation assay (total proteins). The signal for the housekeeping protein GAPDH is shown below each blot. B, immunoblotting of “IP-proteins” was carried out after eluting the protein complexes from the beads. All immunoblots were carried out in parallel under the same transfection and extraction conditions. Western blotting was carried out with either anti-CaVß3 (Alomone; catalog no.: ACC008, 1:10,000 dilution), anti-CaVß2a (Alomone; catalog no.: ACC105, 1:1000 dilution), anti-CaV1.2 directed against CaVa1C (Alomone; catalog no.: ACC003, 1:250 dilution), anti-CaVa2d1 (Alomone; catalog no.: ACC015, 1:1000 dilution), and GAPDH (Sigma; 1:10,000 dilution) with an anti-rabbit as secondary antibody (Jackson ImmunoResearch; 1:10,000 dilution). Signals were detected with the enhanced chemiluminescence substrate. Blots were visualized with the ChemiDoc Touch system (Bio-Rad). Molecular weights were estimated using Image Lab software, version 5.2 (Bio-Rad) by linear regression of standard molecular weight markers. GAPDH, CaVß3, CaVß2a, CaVa2d1, and CaV1.2 proteins migrated (in kilodalton) at 35, 60, 80, 175, and 250 kDa, respectively. From left to right in A and B: lane 1: CaV1.2 WT + CaVa2d1 + CaVß2a; lane 2: CaV1.2 G449R + CaVa2d1 + CaVß2a; lane 3: CaV1.2 WT + CaVa2d1 + CaVß3; and lane 4: CaV1.2 G449R + CaVa2d1 + CaVß3. HEKT, human embryonic kidney 293T cell line; IP, immunoprecipitation.