PKA-dependent phosphorylation of Ser1928 regulates CaV1.2 gating. (A) Representative images of rat DRG neurons stimulated with solvent (Ctrl) or KCl (40 mM) for 1 min. Cultures were labeled for UCHL1 to identify the neurons, phospho-Ser1928 of CaV1.2 (pCaV1.2), and CaV1 channels (CaV1, clone L57/46). Green or red encircled objects indicate automatically selected or rejected objects, respectively. Scale bar, 100 µm. (B) Enlarged section demonstrating the modified image analysis to quantify in nuclear (orange) and cytoplasmic (blue) regions of neurons (green). (C) Cell density plots of single-cell data of pCaV1.2/CaV1-labeled neurons stimulated with solvent control (Ctrl) or KCl (40 mM) for 1 min. The Spearman’s rank correlation coefficient (ρ) and its P value are shown. (D) Depolarization (40 mM KCl) induces CaV1.2 phosphorylation, which is inhibited by verapamil (VP; 200 µM, 10 min). (E) The CaV1 intensity is unchanged after depolarization. (F and G) KCl-induced increase of pCaV1.2 intensity in cytoplasmic versus nuclear regions. (H) Effect of the PKA inhibitor H89 on the pCaV1.2 increase induced by depolarization. (I) Effect of the calcineurin inhibitor FK506 on the of pCaV1.2 increase induced by depolarization. (J) Inhibitory effect of the PKA inhibitor H89 (25 µM, 30 min) on the pRII increase induced by depolarization. (K) Dose–response of KCl (0–80 mM) in the absence or presence of H89 (25 µM, 30 min). Data in D–J represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; **, P < 0.01; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions; §, P < 0.05; §§, P < 0.01; §§§, P < 0.001 indicate significance levels between the absence and presence of an agonist/antagonist.
Figure 6.

PKA-dependent phosphorylation of Ser1928 regulates CaV1.2 gating. (A) Representative images of rat DRG neurons stimulated with solvent (Ctrl) or KCl (40 mM) for 1 min. Cultures were labeled for UCHL1 to identify the neurons, phospho-Ser1928 of CaV1.2 (pCaV1.2), and CaV1 channels (CaV1, clone L57/46). Green or red encircled objects indicate automatically selected or rejected objects, respectively. Scale bar, 100 µm. (B) Enlarged section demonstrating the modified image analysis to quantify in nuclear (orange) and cytoplasmic (blue) regions of neurons (green). (C) Cell density plots of single-cell data of pCaV1.2/CaV1-labeled neurons stimulated with solvent control (Ctrl) or KCl (40 mM) for 1 min. The Spearman’s rank correlation coefficient (ρ) and its P value are shown. (D) Depolarization (40 mM KCl) induces CaV1.2 phosphorylation, which is inhibited by verapamil (VP; 200 µM, 10 min). (E) The CaV1 intensity is unchanged after depolarization. (F and G) KCl-induced increase of pCaV1.2 intensity in cytoplasmic versus nuclear regions. (H) Effect of the PKA inhibitor H89 on the pCaV1.2 increase induced by depolarization. (I) Effect of the calcineurin inhibitor FK506 on the of pCaV1.2 increase induced by depolarization. (J) Inhibitory effect of the PKA inhibitor H89 (25 µM, 30 min) on the pRII increase induced by depolarization. (K) Dose–response of KCl (0–80 mM) in the absence or presence of H89 (25 µM, 30 min). Data in D–J represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; **, P < 0.01; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions; §, P < 0.05; §§, P < 0.01; §§§, P < 0.001 indicate significance levels between the absence and presence of an agonist/antagonist.

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