The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor

FIGURE 1.. Overview of CaM and selected CaM mutations. A, the primary structure of CaM (N-domain, Met-1–Thr-80; C-domain, Asp-81–Lys-149) with EF-hands 1–4 aligned. Dashed boxes indicate α-helices and connected black boxes the Ca2+ coordinating residues. Red bars highlight individual sites for arrhythmogenic mutations (N54I, D96V, N98S, D130G, and F142L). B, representative tertiary structure of CaM. Ca2+-saturated CaM binding to a 27-residue peptide corresponding to part of the CaMBD in RyR1 (Lys-3614–Pro-3640) (Protein Data Bank code 2BCX) is shown. Protein and peptide secondary structures are represented schematically. The N-domain of CaM is indicated in blue, the C-domain in orange, and the peptide in gray. Sites of arrhythmogenic CaM mutations are highlighted as green stick representations (non-mutated residues) and Ca2+ ions as black spheres. RyR1 CaMBD residues (Trp-3620, Leu-3624, and Phe-3636) corresponding to RyR2 CaMBD residues Trp-3587, Leu-3591, and Phe-3603 are highlighted as magenta stick representations.

FIGURE 2.. CaM F142L minutely affects regulation of RyR2 Ca2+ release during SOICR. A and B, example traces for the D1ER signal during SOICR from RyR2 expressing HEK293 cells co-transfected with CaM-WT (A) or CaM-F142L (B). The FRET signal from the ER luminal D1ER [Ca2+]free indicator oscillated as Ca2+ was released through RyR2. The rectangular bars are labeled to indicate the concentrations of Ca2+, tetracaine (RyR2 channel inhibitor), and caffeine (activator) in the perfusion solution. The increase in the ER Ca2+ concentration elicited RyR2 SOICR oscillations; the tetracaine blocked Ca2+ release, filling ER to the maximum [Ca2+]free; and finally caffeine depleted ER Ca2+ to the minimum [Ca2+]free (Fmax and Fmin). C–F, the activation (C) and termination (D) thresholds and the fractional Ca2+ release (E) relative to Fmax and Fmin; their difference was the fractional ER Ca2+ release (F). The expression of CaM-WT or -F142L affected the measured termination thresholds compared with endogenous CaM (Ctrl). Error bars show S.D., and values were calculated from 90–140 single cell traces. The a and b indicate values significantly different from those for the Ctrl or CaM-WT (one-way ANOVA, p <…

FIGURE 3.. Effect of CaM-WT and mutants on single RyR2 channel properties. A–C, example of single RyR2 channel current traces without CaM (left traces) and after the addition of 1 μm CaM. D–F, RyR2 channel PO, MOT, and MCT after the addition of CaM mutants (Ctrl with no added CaM). Measurements were done at +40 mV with 1 mm free Ca2+ at the luminal face and 10 μm free Ca2+, 1 mm free Mg2+, and 1 mm ATP at the cytosolic face. The a indicates values significantly different from CaM-WT, and b indicates values significantly different from Ctrl (t test, p < 0.05). Error bars show S.E. from 6–8 channels for each CaM variant and 32 channels before or without CaM addition (Ctrl).

FIGURE 4.. Domain-specific titration curves for Ca2+ binding to CaM in the presence of RyR2(R3581-L3611) peptide. CaM-F142L/RyR2(R3581-L3611) Ca2+ titration points for CaM N-domain (A, triangles) and C-domain (B, circles) were fitted with a two-site Adair model (solid blue lines), and normalized Y (see “Experimental Procedures”) was plotted as a function of [Ca2+]free. The solid lines in either panel show the fit to the equivalent titrations of CaM-WT (black), -N54I (green), -N98S (magenta), -D96V (tangerine), and -D130G (gray) with data from Søndergaard et al. (8). Bold, highlighted appKD values were significantly different from those for CaM-WT/RyR2(R3581-L3611) (one-way ANOVA p < 0.05).

FIGURE 5.. ITC example data for the titration of the RyR2(R3581-P3607) peptide with CaM. Example ITC thermograms for apoCaM-WT and -F142L (A and B) and for CaCaM conditions (D and E). C and F, averaged integrated change in enthalpy per injection (ΔH°) comparing CaM-WT, -D96V, -N98S, and -F142L under apo (C) or CaCaM conditions (F). Without Ca2+ present, the binding of apoCaM-WT to the peptide is an endothermic reaction (positive ΔH°), whereas for the apoCaM-F142L the interaction is exothermic (A and B). With saturating Ca2+ present, both CaCaM-WT and -F142L display exothermic binding reactions (D and E). Thermograms for CaM-D96V and -N98S were not visibly different from those for the CaM-WT and therefore are not shown. DP, change in ITC instrument heat effect.

FIGURE 6.. Fitted thermodynamic parameters for the binding of CaM variants to the RyR2(R3581-P3607) peptide as calculated from ITC. Panels in the upper row summarize values for the apoCaM condition, and those in the lower row summarize values for CaCaM. A, the affinity of CaM for binding to the peptide expressed as the dissociation constant (KD). B and C, enthalpy (ΔH°) and entropy contribution (−T·ΔS°), respectively, for the binding interaction between CaM and the peptide. D, the change in Gibb's free energy (ΔG°) as conferred by the CaM mutations. Error bars show S.D. The a and b indicate values significantly different from those for CaM-WT or CaM-F142L, respectively (one-way ANOVA, p < 0.05).

FIGURE 7.. 2D [15N,1H]HSQC NMR spectra of CaM in complex with the RyR2(R3581-L3611) peptide. 15N-hydrogen HSQC NMR spectra of the RyR2(R3581-L3611) peptide with 15N-labeled Val-3586 and Phe-3603 in complex with CaM-WT, -N98S, or -F142L. The labeled residues correspond to Phe-3636 and the Val adjacent to Trp-3620 highlighted in Fig. 1. Spectra were recorded without any Ca2+ (A) and with saturating Ca2+ (B). The peaks are color-coded according to the CaM variant in the protein-peptide complex. C, amino acid sequence of the RyR2(R3581-L3611) peptide with 15N-labeled residues highlighted in bold and underlined. The CaM C-domain binds around Trp-3587 and the N-domain around Phe-3603.

FIGURE 8.. Estimation of CaM and RyR2 expression levels in HEK293 cells. A, example of Western-blotted protein bands visualized using chemiluminescence imaging. HEK293 cells were cultured with CaM-WT overexpression from a low or high expression plasmid and also without overexpression (Ctrl). B, the expression levels of CaM and RyR2 were normalized to that of β-actin. Averaged CaM expression levels were 0.18, 0.43, and 0.87 for endogenous CaM expression (Ctrl), and high and low plasmid expression, respectively. Opposite from CaM, the stable and inducible RyR2 expression levels appeared constant across all samples.