Calmodulin variant E140G associated with long QT syndrome impairs CaMKIIδ autophosphorylation and L-type calcium channel inactivation

Figures

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  Figure 1. Arrhythmic variant CaM-E140G decreases kinase activity and autophosphorylation of CaMKIIδ.A, quantification of phosphorylation activity of CaMKIIδ using Amplite universal fluorimetric kinase assay kit. (B-left panel), measurement of the relative levels of CaMKIIδ Thr287 autophosphorylation. GST-CaMKIIδ was incubated with CaM variants and ATP for 0 min, 5 min, 15 min, 30 min, and 60 min at room temperature. CaM-WT or CaM-E140G recombinant proteins were used as CaMKIIδ activators. The reaction was terminated using SDS-containing solution, and samples were analyzed by Western blotting and densitometry analysis. (B-right panel), representative blots for CaM-WT and CaM-E140G samples. Phosphorylated proteins (phospho-Thr287 antibody) were normalized to total CaMKIIδ protein (GST antibody). Experiments were performed at least in triplicates. Data are expressed as mean ± s.e.m. Differences between groups were determined using a two-tailed unpaired Student t test. p-values are represented by stars with ∗∗p < 0.01 and ∗∗∗∗p < 0.0001. CaM, calmodulin.

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  Figure 2. E140G mutation disrupts interaction of CaM with CaMKIIδ.A, cartoon representation of the crystal structure of CaM-WT (yellow) in complex with CaMKIIδ294-315 (orange). B, cartoon representation of the crystal structure of CaM-E140G (gray) in complex with CaMKIIδ294-315 (blue). C, structural superimposition of CaM-WT and CaM-E140G in complex with CaMKIIδ294-315. D and E, H-bond interactions between Ca2+ and CaM residues at the EF hand 4 in the (D) Ca2+/CaM-WT–CaMKIIδ294-315 and (E) Ca2+/CaM-E140G–CaMKIIδ294-315 complex structure. F, superimposition of EF-hand 4 region of Ca2+/CaM-WT–CaMKIIδ294-315 and Ca2+/CaM-E140G–CaMKIIδ294-315 complex structures. The Glu140:OE2 atom is replaced by a water molecule in the Ca2+/CaM-E140G–CaMKIIδ294-315 complex mutant structure to coordinate the Ca2+. Ca2+ is shown in green sphere and water molecule in cyan sphere. H-bond is represented in black dashed lines. G140 is shown in magenta. Images were created with UCSF Chimera (112). G, representative ITC titration curves (upper panels) and binding isotherms (lower panels) for Ca2+–CaM interaction with CaMKIIδ294-315. H, affinity of the binding of Ca2+/CaM with CaMKIIδ294-315 obtained by fitting to a…

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  Figure 3. LQTS-associated CaM variant E140G does not affect Cav1.2 voltage-dependent activation characteristics.A, representative traces from HEK-Cav1.2 cells–transfected CaM-WT or CaM-E140G and (B) I/V relationships of untransfected cells (endogenous) or cells transfected with CaM-WT or E140G. Differences between groups were determined using a two-way ANOVA with Tukey’s multiple comparisons tests. The ANOVA parameters are shown in Table S4. C, voltage-dependent activation characteristics of untransfected cells or cells transfected with CaM-WT or E140G. D, voltage-dependent inactivation characteristics of untransfected cells or cells transfected with CaM-WT or E140G. (C and D-left panel) mean (±s.e.m.) channel conductances, G, normalized to peak conductance, Gmax, to give mean activation/activation curves. (C, and D-right panel) mean (±s.e.m.) half maximal activation/inactivation voltages, V50, calculated from individual curves fitted using the Boltzmann equation. Experiments were performed in 0.5 mM EGTA (internal solution) and 2 mM CaCl2 (external solution). Differences between groups were determined using a one-way ANOVA with Dunnett’s multiple comparisons tests. The ANOVA parameters…

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  Figure 4. LQTS-associated CaM variant E140G impairs Cav1.2 Ca2+-dependent inactivation.A, representative Ca2+ (black) and Ba2+ (gray) current traces from HEK-Cav1.2 cells either transfected with CaM-WT or E140G, in response to a 300 ms pulse to +10 mV, normalized to their respective peak currents. B, mean (±s.e.m.) fractional residual Ca2+ and Ba2+ current at the end of the 300 ms pulse (r300), at test potentials ranging from −40 to +20 mV. C and D, Ca2+-dependent and Ca2+-independent inactivation characteristics. (C-left panel), mean (±s.e.m.) residual Ca2+ current at the end of a 300 ms pulse (r300Ca), at +10 mV. (C-right panel), mean (±s.e.m.) residual Ba2+ current at the end of a 300 ms pulse (r300Ba), at +10 mV. d, mean (±s.e.m.) proportion of inactivation due to CDI (f300), at +10 mV. Experiments were performed in 0.5 mM EGTA (internal solution) and 2 mM of either CaCl2 or BaCl2 (external solution). Differences between groups were determined using using a one-way ANOVA with Dunnett’s multiple comparisons test. p-values are represented by stars with ∗∗∗∗p < 0.0001. The ANOVA parameters are shown in Tables S7 and S8. CaM, calmodulin; CDI, Ca2+-dependent inactivation; LQTS, Long QT…

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  Figure 5. LQTS-associated E140G mutation alters interaction of CaM with Cav1.2-binding domains.A and B, representative ITC titration curves (upper panels) and binding isotherms (lower panels) for CaM interaction with (A) Cav1.2-NSCaTE51-67 and (B) Cav1.2-IQ1665-1685 peptides in the presence of Ca2+. C, affinity of the binding of Ca2+/CaM proteins with Cav1.2 peptides. D, thermodynamic profile of binding between Ca2+/CaM proteins and Cav1.2-NSCaTE51-67 (left panel), Cav1.2-IQ1665-1685 (right panel). (E-left panel), representative ITC titration curves (upper panels) and binding isotherms (lower panels) for pre-associated CaM–Cav1.2-IQ1665-1685 titrated with Cav1.2-NSCaTE51-67 in the presence of Ca2+. (E-middle panel), affinity of binding and (E-right panel) thermodynamic profile for Ca2+/CaM–Cav1.2-IQ1665-1685 interaction with Cav1.2-NSCaTE51-67. Data were processed using the MicroCal PEAQ-ITC software. Kd, binding affinity; N, stoichiometry; n, number of experimental replicates. The sum of the change in enthalpy (ΔH) and the change in entropy (ΔS) multiplied by the absolute temperature (T) gives the change in free energy (ΔG). Experiments were performed in the presence of 5 mM CaCl2 at 25…

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  Figure 6. E140G mutation decrease Ca2+-binding affinity and alter CaM secondary structure content.A, equilibrium Ca2+-binding titrations for CaM-WT and E140G measured by intrinsic tyrosine fluorescence (C-lobe specific) at 20 °C. Proteins (6 μM) were titrated with increasing free Ca2+ concentrations. Fluorescence changes (λex = 277 nm and λem = 307 nm) are normalized to F0 of 0 and Fmax of 1 and fitted to the Hill equation. Fitted curves are represented by solid lines overlaying the data points. Titrations were performed in five replicates and represented as mean ± s.e.m. B, circular dichroism spectra were obtained in the presence of 1 mM EGTA (left panel) or 1 mM CaCl2 (right panel). Data displayed are average traces for CaM-WT (n = 5) and CaM-E140G (n = 6). C, protein secondary structure content estimated using the CDSSTR algorithm (DichroWeb, reference set 7) in the presence of 1 mM EGTA (left panel) or 1 mM CaCl2 (right panel). Data are mean ± s.e.m. Experiments were performed at 20 °C, CaM-WT (n = 5) and CaM-E140G (n = 6). Differences between groups were determined using a two-way ANOVA with Sidak’s multiple comparisons test. p-values are represented by stars with ∗p < 0.05, ∗∗p <…

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  Figure 7. Proposed regulatory mechanism for LQTS-associated CaM variant E140G. A-Normal. At rest, CaM is bound to the intracellular IQ region of Cav1.2, close to the opening of the channel pore, where it is ideally placed to monitor Ca2+ influx into the cytosol (Time 1). Upon stimulation (action potential), the Cav1.2 channel opens and Ca2+ enters into the cardiomyocyte (Time 2–4). CaM can sense Ca2+ influx and when intracellular [Ca2+] increases, CaM binds to the Cav1.2 NSCaTE domain to initiate the inactivation of the channel (Time 4–5). This prevents further Ca2+ influx and protect the cell from Ca2+ overload. B-LQTS. In LQTS, CaM-E140G affinity for Ca2+ is reduced, therefore intracellular [Ca2+] changes are not sensed appropriately and Cav1.2 remains open (Time 2–4 is longer). Additionally, at elevated intracellular [Ca2+], CaM-E140G has a higher affinity for Cav1.2-IQ, suggesting it may outcompete CaM-WT on the C-terminal domain of the channel (Time 3). This is particularly important as CaM-E140G has a reduced binding affinity for the NSCaTE domain, which will impair CaM-dependent CDI (Time 4–5 longer). In addition, CaMKIIδ bound to CaM-E140G shows significantly reduced kinase…