Figure 2. Biophysical and Pharmacologic Characterization of p.(G1169D)-Containing Human Ca_V1.3 Ca²⁺ Channels.

Whole-cell patch-clamp recordings in tsA201 cells of transiently transfected wild-type (WT, black) or p.(G1169D) (grey/G1169D) Ca_V1.3 Ca²⁺ channel complexes (coexpressed with β3 and α2δ1, 15 mM Ca²⁺). Data were measured using a holding potential of −109.3 mV (A–E) and are given as mean ± SEM for the indicated number of recordings. Parameters and statistics are provided in eTables 1–3. (A) The p.(G1169D) variant shifted the voltage dependence of activation (G-V curve, circles) and steady-state inactivation (squares) by ∼35 mV toward more negative potentials. (B) This enabled a significantly increased constant background Ca²⁺ influx (“window current”) at subthreshold potentials. Two-way ANOVA (interaction, potential, genotype: p < 0.001) with the Šídák multiple comparison post hoc test as indicated. (C) During a prolonged (5 s) depolarization to the voltage of maximal activation (V_max), the p.(G1169D) variant displayed slower inactivation kinetics. Averaged current traces (mean ± SEM) are shown. (D–E) Deactivation kinetics of the p.(G1169D) variant were significantly slower, as shown by representative traces of tail currents elicited by 40-ms long repolarization from the reversal potential (V_rev) to −59.3 mV or −39.3 mV (D) and quantification of the normalized tail current area for all tested repolarization potentials (details on normalization in Methods). Two-way ANOVA (interaction, repolarization potential, genotype: p < 0.001) with the Šídák multiple comparison post hoc test as indicated. (F) Sensitivity to the L-type Ca²⁺ channel inhibitor isradipine (30 and 100 nM) was evaluated during 100-ms long depolarizations to the V_max (holding potential −89.3 mV, 0.1 Hz, 3 independent transfections). Normalized representative current traces for 30 nM and subsequent full block with 3-µM isradipine are shown (left, tail currents were cut). Inhibition was corrected for current run-down (details in Methods) and is presented as means ± SEM (right). No statistically significant difference in isradipine inhibition was observed between WT and p.(G1169D) Ca_V1.3 Ca²⁺ channels (two-way ANOVA). This provides class IV evidence. It is an observational study without controls.