Fig. 2: Functional characterization of phospho-Ser14 Cav2.3 in HEK 293 cells.

a Depolarization-evoked current responses in HEK293 cells stably expressing human β3/α2δ1 subunits and co-transfected with human Cav2.3 (WT α1E or S14A α1E) and FLAG-CDKL5 full length (WT CDKL5). Colours denote different construct combinations. Traces show steps from −10 to +30 mV from −80 mV; Ba²⁺ was charge carrier (b) Open channel inactivation tau (τ_inact) for Cav2.3 with (WT/WT, n = 13–15 recordings from −10 to +40) and without (S14A/WT, n = 11–13 recordings from −10 to +40) CDKL5 phosphorylation (*p = 0.02, 0.03 & 0.01 at +10, +20 & +40 mV Two-Way ANOVA, Fisher’s LSD). Some voltage steps were excluded as described in Methods. Data were acquired using 100 ms voltage steps from −80 mV in +10 mV increments every 10 s. c Individual data points for τ_inact at +20 mV (WT/WT n = 14, S14A/WT n = 11; two-tailed unpaired t test). d WT or phophomutant S14A Cav2.3 inactivation in the same cell line in absence of CDKL5 (both conditions n = 7–9; p > 0.05 Two-Way ANOVA); Ca²⁺ was charge carrier. e Current voltage relationship for cells in (b). Inset: comparison of current density at 0 mV (inset, two-tailed unpaired t test). f Normalised Cav2.3 current conductance and voltage dependence of inactivation for the same transfection conditions. Activation V_1/2, n: WT/WT −6 ± 1 mV, 15; S14A/WT −6 ± 1 mV,12; Inactivation V_1/2, n: WT/WT −61 ± 3 mV,10; S14A/WT −62 ± 2 mV,11 (p > 0.05, two tailed unpaired t tests). For inactivation protocol see Methods. Solid lines are Boltzman fits to the average data points (g) Normalised Ba²⁺ currents in the β3/α2δ1-cell line or (h) Ca²⁺ currents in the β1/α2δ1-cell line, co-transfected with α1E (WT or S14A), WT CDKL5 and muscarinic type 3 receptor (M3), in presence and absence of carbachol (CCh) 30 and 10 μM, respectively. HP = −100 mV. i IV curves and (j) CCh-induced change in current amplitude for experiment in (g) (pS14 n = 10, no pS14 n = 5, Two-Way ANOVA, Fisher’s LSD). k IV curves and (l) CCh-induced change in current amplitude for experiment in (h) (pS14 n = 7, no pS14 n = 16, Two-Way ANOVA, Fisher’s LSD). m Conductance plots for experiment in (g) and n Corresponding change in activation (V_1/2: −5 ± 0.6 mV (n = 10) vs. −10 ± 1.9 mV (n = 5), two-tailed Kolmogorov-Smirnov). o Conductance plots for experiment in (h) and p corresponding change in activation (V_1/2: −4 ± 0.5 mV (n = 7) vs −9 ± 1 mV (n = 16), two-tailed Welch’s t test). From (l–p), ‘pS14’ refers to WT α1E/WT CDKL5; ‘no pS14’ refers to S14A α1E /WT CDKL5 and α1E WT/K42R* CDKL5 conditions pulled together. Data is presented as mean ± S.E.M or in box plots representing minimum, maximum, median, 25/75 percentile and mean (indicated by a marker). Source data are provided as a Source Data file.