Journal: bioRxiv
Article Title: Epilepsy-linked kinase CDKL5 phosphorylates voltage-gated calcium channel Cav2.3, altering inactivation kinetics and neuronal excitability
doi: 10.1101/2022.11.24.517538
Figure Lengend Snippet: A) Representative depolarization-evoked current responses in HEK293 cells stably expressing human β1/α2δ1 subunits and co-transfected with human Cav2.3 (WT α1E or S14A α1E mutant) and FLAG-CDKL5 full length (WT CDKL5 or K42R*+DFG-AFG kinase dead CDKL5 mutant). Construct combinations are presented in different colours. Traces show steps from -10 to +20mV from a holding potential of -100mV and Ca 2+ as charge carrier. B) Open channel inactivation tau (τ inact ) for Cav2.3 with (WT/WT, n=28-32) and without (S14A/WT, n=34-37; WT/K45R, n=25-26) CDKL5 phosphorylation. WT/WT vs. S14A/WT: *p<0.05, **p<0.01 as indicated; WT/WT vs. WT/K42R*, p<0.01 at +40mV, p<0.05 at -10,10,20,30mV, TW ANOVA, Fisher’s LSD. Some of the cells included in this plot co-expressed M3 receptors; this was per se not found to affect inactivation kinetics. C) Individual data points for τ inact at +10mV (p<0.05, unpaired t tests). D) Current-voltage relationship for experiments in A (WT/WT, n=15; S14A/WT n=15-17; WT/K42R* n=12-15) and current density at +10mV (inset, p>0.05, one way ANOVA). Data was acquired with 100ms voltage steps from -100mV in +10mV increments every 10s. E) Normalised Cav2.3 conductance and voltage dependence of inactivation for the same three transfection conditions. Activation V 1/2 , n: WT/WT -3±1 mV, 14; S14A/WT -3±1 mV,17; WT/K42R* -2±1 mV, 14; Inactivation V 1/2 , n: WT/WT -66±2 mV,14; S14A/WT -63±1 mV,14; WT/K42R* -65±2 mV,11 (p>0.05, one way ANOVA). For inactivation protocol see Methods. Solid lines are Boltzman fits to the average data points. F) Inactivation recovery time using a double pulse protocol with variable inter-pulse recovery time (inset). Recovery tau, n: WT/WT 265±21 ms, 4; S14A/WT 263±21 ms, 5; WT/K42R* 290±15 ms, 4, p>0.05 Kruskall-Wallis, Dunn’s test). G) Example time course of Cav2.3 current recording at 0mV in the β1/α2δ1-stable cell line co-transfected with α1E WT, CDKL5 and muscarinic receptor type 3 (M3). PKC activation with PMA did not occlude CCh enhancing effect. H) Relative change in current amplitude for same experiment as G) with either M1 or M3 receptors. No muscarinic inhibition was observed. I) Representative Cav2.3 current traces at +10mV in β1/α2δ1 stable cell line co-transfected with α1E (WT or S14A), CDKL5 (WT) and dopamine receptor type 2 (D2). Quinpirole 100nM (QP) application resulted in reliable and reversible inhibition of WT and S14A currents. J) Quantified quinpirole inhibition of Cav2.3 with (n=7) or without pS14 (n=12-13) (p>0.05 TW ANOVA, Fisher’s LSD). Data for α1E S14A phosphomutant and K42R* CDKL5 conditions (no phosphorylation) were pooled together.
Article Snippet: For dopamine modulation experiments, human α1E-HA, FLAG-CDKL5 and dopamine D2 receptor (gfp-DRD2, Addgene) were co-transfected at a ratio of 2:0.75:1.
Techniques: Stable Transfection, Expressing, Transfection, Mutagenesis, Construct, Phospho-proteomics, Activation Assay, Inhibition
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