Figure 7.

Role of the LTCC NTD in high K⁺-induced CREB Ser¹³³ phosphorylation. A, Ca²⁺ imaging showing that nimodipine largely prevents the high K⁺-induced increase of somatic Ca²⁺. Neurons were incubated with Tyrode's solution containing 5 mm KCl (5K) for 30 s and switched to Tyrode's solution containing 40 mm KCl for 2 min in the absence (40K control, n = 193) or presence (40K + NIM, n = 215) of 10 μm nimodipine. The black arrow indicates the buffer switch, and data were plotted as mean ± S.E. A total of five dishes from two independent cultures were analyzed per group. B, depolarization of cultured hippocampal neurons induced LTCC-dependent Ser¹³³ phosphorylation of CREB. In the columns from left to right, neurons were incubated with 5K Tyrode's solution and then switched to 5K, 40K control, or 40K + NIM Tyrode's solution, respectively, for 90 s. Neurons were then fixed and stained for DAPI, CREB Ser¹³³ phosphorylation (pCREB), and CaMKIIα. Incubation with 40 mm KCl induced an increase of pCREB that was blocked by the LTCC antagonist nimodipine. C, deletion of the CaMKII-binding domain in the Ca_V1.3 NTD disrupts nuclear signaling. Expression of a nimodipine-resistant Ca_V1.3-T1033Y mutant rescues the nimodipine blockade of pCREB induction by 40 mm KCl. However, deletion of the CaMKII-binding domain (Δ69–93) prevents the rescue of pCREB signaling by Ca_V1.3-T1033Y. Each data point represents analysis of a single cell collected from 3–4 independent neuronal cultures/transfections. Pooled data were analyzed by one-way ANOVA followed by Tukey's multiple-comparison test. ***, p < 0.001; ns, not significant (p > 0.05). All confocal images show a 40 × 40-μm area. Error bars, S.E.