Figure 9.

Overexpression of apo-CaM/CaM₁₂₃₄ (Ca²⁺-binding deficient CaM) causes a hyperpolarizing shift in voltage sensitivities and faster channel kinetics in Ca_v3.2 channels only in the presence of an intact gating brake in Ca_v3.2. A and B, Ca_v3.2 channels with and without co-expressed CaM₁₂₃₄ were measured for their current-voltage relationships and τ for mono-exponential fits of the kinetics of activation (A) and inactivation (B). Representative current traces are shown. Results are presented as mean ± S.E., and n = the number of cells. Statistical analyses were first performed with the Student's t test or with one-way ANOVA combined with a Tukey post-test for multiple comparisons (*, p < 0.05; **, p < 0.01; ***, p < 0.001). C, scatter plot (left) with mean ± S.E. (right) values illustrate the 50% activation and inactivation values taken from Boltzmann fits of activation and steady-state inactivation curves of Ca_v3.2 channels alone (−), and Ca_v3.2 channels with co-expressed apo-calmodulin (CaM₁₂₃₄) or calmodulin (CaM). Voltage responses to co-expressed apo-calmodulin (CaM₁₂₃₄) are also illustrated for Ca_v3.2 deletion mutants spanning sequences 429–539 or 453–491, lacking the 111 or 39 amino acids, respectively, flanking the gating brake in the I–II linker. The region of deletion mutants spanning the gating brake of Ca_v3.2 channels are illustrated in Fig. 2B. The statistical significance of CaM₁₂₃₄ overexpression compared with wild-type conditions was p = 0.0028 (***) and p = 0.0308 (*) for V_50% activation and V_50% inactivation, respectively, using a Kruskal-Wallis test followed by a Dunn's multiple comparisons test. The effect of CaM₁₂₃₄ overexpression compared with wild-type conditions for Ca_v3.2 deletion mutants (D^429–539 or D^453–491) were non-significant using a Mann-Whitney test.