Figure 3.

Ca_V2.2-mediated signaling in α₂δ-1 neurons. A, Imaging of high K⁺-evoked Ca²⁺ transients in control DRG neurons pretreated for 15 min with 1 μm CTX. B, In control DRG neurons, in the absence of α₂δ-1 HA overexpression CTX application (n = 7) decreased the peak amplitude of responses with respect to untreated (n = 21) and Nif (1 μm; n = 13) samples (p < 0.0001 one-way ANOVA and Bonferroni post hoc test, #p < 0.001). C, Inhibition of N- and L-type channels did not alter the width of signals imaged in control neurons (p = 0.4, one-way ANOVA). D, Examples of Ca²⁺ responses evoked in α₂δ-1 HA overexpressing neurons in the absence (left) or presence (right) of CTX. E, CTX pretreatment (n = 12) diminished the intensity of signals in α₂δ-1 HA overexpressing DRG neurons compared with responses obtained in untreated (n = 19) or Nif-treated (n = 19) DRGs (p = 0.0001 one-way ANOVA and Bonferroni post hoc test, #p < 0.001). F, Prolonged high K⁺-evoked Ca²⁺ transients were abolished following CTX pretreatment (p = 0.02 one-way ANOVA and Bonferroni post hoc test, *p < 0.05). G, Overexpression in DRG neurons of exofacially HA-tagged Ca_V2.2, β1B subunits and either α₂δ-1 (top) or α₂δ-1 MIDAS^AAA (bottom). Surface and intracellular detection of HA Ca_V2.2 proteins was performed by staining the HA epitope in nonpermeabilizing conditions and intracellular labeling with an anti Ca_V2.2 antibody, respectively. Scale bars, 10 μm. H, Quantification of surface HA staining in HA Ca_V2.2/α₂δ-1/β1B (black bar; n = 32) or HA Ca_V2.2/α₂δ-1 MIDAS^AAA/β1B (white bar; n = 36) transfected neurons (p = 0.02, t test). I, Labeling of overexpressed channels by Ca_V2.2 polyclonal antibody. DRG neurons were transfected with Ca_V2.2YFP and β1B cDNAs. J, Ca_V2.2 antibody failed to identify native subunits in eYFP overexpressing neurons indicating that the amount of protein is a limiting factor in immunocytochemistry experiments.