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

Abstract

Long QT syndrome (LQTS) is a human inherited heart condition that can cause life-threatening arrhythmia including sudden cardiac death. Mutations in the ubiquitous Ca²⁺-sensing protein calmodulin (CaM) are associated with LQTS, but the molecular mechanism by which these mutations lead to irregular heartbeats is not fully understood. Here, we use a multidisciplinary approach including protein biophysics, structural biology, confocal imaging, and patch-clamp electrophysiology to determine the effect of the disease-associated CaM mutation E140G on CaM structure and function. We present novel data showing that mutant-regulated CaMKIIδ kinase activity is impaired with a significant reduction in enzyme autophosphorylation rate. We report the first high-resolution crystal structure of a LQTS-associated CaM variant in complex with the CaMKIIδ peptide, which shows significant structural differences, compared to the WT complex. Furthermore, we demonstrate that the E140G mutation significantly disrupted Ca_v1.2 Ca²⁺/CaM-dependent inactivation, while cardiac ryanodine receptor (RyR2) activity remained unaffected. In addition, we show that the LQTS-associated mutation alters CaM's Ca²⁺-binding characteristics, secondary structure content, and interaction with key partners involved in excitation-contraction coupling (CaMKIIδ, Ca_v1.2, RyR2). In conclusion, LQTS-associated CaM mutation E140G severely impacts the structure-function relationship of CaM and its regulation of CaMKIIδ and Ca_v1.2. This provides a crucial insight into the molecular factors contributing to CaM-mediated arrhythmias with a central role for CaMKIIδ.

Keywords: Ca(2+)/calmodulin-dependent protein kinase II; Ca(v)1.2; CaMKIIδ; L-type voltage-gated Ca(2+) channel; LQTS; calmodulin; cardiac arrhythmia; long QT syndrome.

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.

Figure 2

E140G mutation disrupts interaction of CaM with CaMKIIδ.A, cartoon representation of the crystal structure of CaM-WT (yellow) in complex with CaMKIIδ_294-315 (orange). B, cartoon representation of the crystal structure of CaM-E140G (gray) in complex with CaMKIIδ_294-315 (blue). C, structural superimposition of CaM-WT and CaM-E140G in complex with CaMKIIδ_294-315. D and E, H-bond interactions between Ca²⁺ and CaM residues at the EF hand 4 in the (D) Ca²⁺/CaM-WT–CaMKIIδ_294-315 and (E) Ca²⁺/CaM-E140G–CaMKIIδ_294-315 complex structure. F, superimposition of EF-hand 4 region of Ca²⁺/CaM-WT–CaMKIIδ_294-315 and Ca²⁺/CaM-E140G–CaMKIIδ_294-315 complex structures. The Glu140:OE2 atom is replaced by a water molecule in the Ca²⁺/CaM-E140G–CaMKIIδ_294-315 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 CaMKIIδ_294-315. H, affinity of the binding of Ca²⁺/CaM with CaMKIIδ_294-315 obtained by fitting to a one-site–binding model. Differences between groups were determined using two-tailed unpaired Student t test. I, thermodynamic profile of binding between Ca²⁺/CaM and CaMKIIδ_294-315. Data were processed using the MicroCal PEAQ-ITC software. K_d, 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). ITC experiments were performed in the presence of 5 mM CaCl₂ at 25 °C. Data are mean±s.e.m. Differences between groups were determined using a two-way ANOVA with Sidak’s multiple comparisons test. p-values are represented by stars with ∗∗p < 0.01 and ∗∗∗∗p < 0.0001. The ANOVA parameters are shown in Table S3. CaM, calmodulin; DP, differential power; ITC, isothermal titration calorimetry.

Figure 3

LQTS-associated CaM variant E140G does not affect Ca_v1.2 voltage-dependent activation characteristics.A, representative traces from HEK-Ca_v1.2 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, G_max, 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 are shown in Tables S5 and S6. CaM, calmodulin; LQTS, Long QT syndrome.

Figure 4

LQTS-associated CaM variant E140G impairs Ca_v1.2 Ca²⁺-dependent inactivation.A, representative Ca²⁺ (black) and Ba²⁺ (gray) current traces from HEK-Ca_v1.2 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 (r300_Ca), at +10 mV. (C-right panel), mean (±s.e.m.) residual Ba²⁺ current at the end of a 300 ms pulse (r300_Ba), 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 syndrome.

Figure 5

LQTS-associated E140G mutation alters interaction of CaM with Ca_v1.2-binding domains.A and B, representative ITC titration curves (upper panels) and binding isotherms (lower panels) for CaM interaction with (A) Ca_v1.2-NSCaTE_51-67 and (B) Ca_v1.2-IQ_1665-1685 peptides in the presence of Ca²⁺. C, affinity of the binding of Ca²⁺/CaM proteins with Ca_v1.2 peptides. D, thermodynamic profile of binding between Ca²⁺/CaM proteins and Ca_v1.2-NSCaTE_51-67 (left panel), Ca_v1.2-IQ_1665-1685 (right panel). (E-left panel), representative ITC titration curves (upper panels) and binding isotherms (lower panels) for pre-associated CaM–Ca_v1.2-IQ_1665-1685 titrated with Ca_v1.2-NSCaTE_51-67 in the presence of Ca²⁺. (E-middle panel), affinity of binding and (E-right panel) thermodynamic profile for Ca²⁺/CaM–Ca_v1.2-IQ_1665-1685 interaction with Ca_v1.2-NSCaTE_51-67. Data were processed using the MicroCal PEAQ-ITC software. K_d, 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 °C. Data are mean ± s.e.m. For comparison of affinity values, differences between groups were determined using two-tailed unpaired Student t test. For comparison of thermodynamic profiles, differences between groups were determined using a two-way ANOVA with Sidak’s multiple comparisons test. p-values are represented by stars with ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001. The ANOVA parameters are shown in Tables S9 and S10. DP, differential power; LQTS, Long QT syndrome; CaM, calmodulin; ITC, isothermal titration calorimetry.

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 (λ_ex = 277 nm and λ_em = 307 nm) are normalized to F₀ of 0 and F_max 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 < 0.01 and ∗∗∗∗p < 0.0001. The ANOVA parameters are shown in Table S11. CaM, calmodulin.

Figure 7

Proposed regulatory mechanism for LQTS-associated CaM variant E140G. A-Normal. At rest, CaM is bound to the intracellular IQ region of Ca_v1.2, 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 Ca_v1.2 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 Ca_v1.2 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 Ca_v1.2 remains open (Time 2–4 is longer). Additionally, at elevated intracellular [Ca²⁺], CaM-E140G has a higher affinity for Ca_v1.2-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 activity, which will reduce phosphorylation of Ca_v1.2. As CaMKIIδ-mediated channel phosphorylation is required for CaM to bind to Ca_v1.2, reduced CaMKIIδ activity will also affect CaM-dependent CDI. Altogether, via a complex mechanism involving impaired Ca²⁺ sensing, CaMKIIδ activation and Ca_v1.2 CDI, CaM-E140G would promote dysregulation of Ca²⁺ homeostasis and the prolonged AP duration characteristic of LQTS. AP, action potential; CaM, calmodulin; CDI, Ca²⁺-dependent inactivation; LQTS, Long QT syndrome.