Fig. 1: Identification and validation of Cav2.3 as substrate of CDKL5 kinase.

a Volcano plot of differential phosphoprotein levels between WT and Cdkl5 KO primary neurons, obtained using SILAC based quantitative proteomics (3 embryos/genotype). Each point represents one peptide; those with CDKL5 consensus motif RPXS/T are highlighted in magenta. The x axis shows log2 transformed fold change in phosphopeptide levels between genotypes and the y axis shows significance as -log10 transformed p value (one sample t test). Significance was established as indicated by the dotted lines (p < 0.01, fold change 1.5, one sample t test). b Top: Schematic depiction of Cav2.3 formed of α1E channel pore subunit and associated proteins Cavβ and α2δ. CDKL5 phosphorylation site at α1E S15/14 is highlighted in green. Bottom: Alignment of proximal N-terminus of mouse and human Cav2.3 and related human Cav2.1 and 2.2 channel proteins. *S is the site of CDKL5 phosphorylation. c Western blot of HEK293 cells stably expressing human β3/α2δ1 subunits and transiently co-transfected with human HA-α1E (Cav2.3: WT or S14A mutant) and HA-CDKL5 kinase ___domain (kd CDKL5: WT or K42A mutant). S14 phosphorylation (pS14 Cav2.3) was detected with a custom made phosphoantibody. Example blots derive from two gels run and processed in parallel. d Western blot of P20 cortices from WT and Cdkl5 KO mice. Example blots derive from three gels: phospho and total Cav were run and processed in parallel; control α-tubulin was obtained from a separate experiment using the same samples and loading volume. e Western blot of iPSC-derived forebrain neurons from three CDD patients and related controls (parents) at 6 weeks of differentiation. Gender and CDKL5 mutation are specified⁹⁰. Example blots derive from two gels run and processed in parallel. f Quantification for immunoblot in (c) (p < 0.0001 and p = 0.0006, Kruskal-Wallis ANOVA & Dunn’s test, n = 11 for each condition: 5 transfections, 2–3 technical replicates). g Quantification for immunoblot in (d) (total Cav: p = 0.25, n = 23, 6 blots, 6 mice/genotype; pCav: p < 0.0001, two tailed unpaired t test, n = 9, 3 blots, 6 mice/genotype; no technical replicates). h Quantification for WB in (e) (p = 0.0051, two tailed paired t test, n = 6, 3 control/patient pairs, 4 technical replicates). For antibodies used see Methods. Data is presented as mean ± S.E.M. All source data is provided in a Source Data File.