Fig. 3: The S165F mutation-induced specific autoinhibition in JPH2 MORN-Helix.

a Summary of ITC-derived dissociation constants, showing that S165F and S165Y mutations of JPH2 significantly weakened the binding between CaV1.2 JBM and JPH2 NT. Results corresponding to the S165F and S165Y mutants are highlighted in red. Black asterisks indicate that the value represents the mean of three independent experiments, expressed as Mean ± SD. b Representative pull-down experiments showing interactions between Trx-Strep-CaV1.2 JBM and MBP-JPH2 NT WT and mutants. c Quantification of the percentages of MBP-JPH2 in pellets relative to WT from the assay shown in (b), considering only intact protein bands. Values are expressed as means ± SD, n = 3 independent experiments, and analyzed using an independent-sample t-test. n.s. not significant, p > 0.05; ***p < 0.001; SD standard deviation. d Sequence alignment of JPH2 Joining region S165F (human and zebrafish) and reported CaV1.1/1.2 JBMs. Critical residues are highlighted. e Structural superposition of the AlphaFold3-predicted JPH2/Joining region S165F (aa 154-173) and the reported Homo_JPH2/CaV1.1 JBM complex (PDB ID: 7RXQ). Detailed interactions between JPH2 MORN and Joining region S165F (f) or CaV1.1 (g). Key residues are shown as stick models. h Summary of ITC-derived dissociation constants showing that R160E S165F (DM) restores the binding to CaV1.2 JBM, and L163E S165F binds to CaV1.2 JBM more strongly than the S165F mutant alone. JR-JBM chimera weakened the binding significantly. Results corresponding to the DM mutant are highlighted in red. Black asterisks indicate that the value represents the mean of three independent experiments, expressed as Mean ± SD. i Co-IP assay of HA-JPH2 WT/S165F/DM fragments with EGFP-CaV1.2 full length. CaV1.2 binds to JPH2 WT and DM, but the interaction is significantly weakened by JPH2 S165F. j Quantification of the percentage of CaV1.2 in pellets relative to WT from the assay shown in (i). Values are expressed as means ± SD from n = 3 independent experiments and analyzed using an independent-sample t-test; n.s. not significant, p > 0.05; ***p < 0.001; SD standard deviation.