FIGURE 7.

Activation of autophosphorylation of CaMKII by binding of Ca_V2.1(1848–1964). A, autophosphorylation of CaMKII was carried out by incubation in the presence of Ca²⁺ (0.6 μm), CaM (2.2 μm), ATP (32 μm), Mg²⁺(2.3 mm), CaMKII (20 nm) and the indicated concentrations (0–113.75 nm) of Ca_v2.1 (1848–1964). Free Ca²⁺ was precisely controlled. Left to right (lanes 1–6), the concentration of Ca_V2.1(1848–1964) channel peptide ranging from 2.28 nm to 113.75 nm. Extreme right (lane 7), the control in the absence of Ca_V2.1(1848–1964). a, the blot was probed with anti-phospho-CaMKII(Thr-286). b, the same blot after stripping and re-probing using anti-CaMKII. B, quantitation of relative CaMKII autophosphorylation using anti-phospho-CaMKII(Thr-286) under the indicated conditions (mean ± S.E.; *, p < 0.05; **, p < 0.01 by Student's t test). C, CaMKII autophosphorylation in the presence of Na_V1.2(1848–1964)-GST is shown. Autophosphorylation of CaMKII was carried out by incubation in the presence of Ca²⁺ (0.6 μm), CaM (2.2 μm), ATP (32 μm), Mg²⁺(2.3 mm), CaMKII (20 nm), and the indicated concentrations (0–113.75 nm) of Na_V1.2(1848–1964). Free Ca²⁺ was precisely controlled. Left to right (lanes 1–6), concentration of Na_V1.2(1848–1964) channel peptide ranging from 0 to 113.75 nm. Extreme right (lane 7), control in the absence of Na_V1.2(1848–1964). a, the blot was probed with anti- phospho-CaMKII(Thr-286). b, the same blot after stripping and re-probing using anti-CaMKII. D, the quantification of relative CaMKII autophosphorylation using anti-phospho- CaMKII(Thr-286) under the indicated conditions (mean ± S.E.; not significant by Student's t test; n = 3).