Arrhythmogenic Calmodulin Mutations Affect the Activation and Termination of Cardiac Ryanodine Receptor-mediated Ca²⁺ Release

Background: Mutations in the Ca²⁺ sensing protein calmodulin (CaM) cause lethal cardiac arrhythmias.

Results: CaM mutations impair the activation and termination of store overload-induced Ca²⁺ release via the cardiac ryanodine receptor (RyR2).

Conclusion: CaM mutations alter RyR2-CaM interaction, thereby affecting RyR2-mediated Ca²⁺ release.

Significance: Aberrant regulation of RyR2 store Ca²⁺ sensing is a potential component of calmodulin-mediated cardiac arrhythmias.

Abstract

The intracellular Ca²⁺ sensor calmodulin (CaM) regulates the cardiac Ca²⁺ release channel/ryanodine receptor 2 (RyR2), and mutations in CaM cause arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and long QT syndrome. Here, we investigated the effect of CaM mutations causing CPVT (N53I), long QT syndrome (D95V and D129G), or both (CaM N97S) on RyR2-mediated Ca²⁺ release. All mutations increased Ca²⁺ release and rendered RyR2 more susceptible to store overload-induced Ca²⁺ release (SOICR) by lowering the threshold of store Ca²⁺ content at which SOICR occurred and the threshold at which SOICR terminated. To obtain mechanistic insights, we investigated the Ca²⁺ binding of the N- and C-terminal domains (N- and C-domain) of CaM in the presence of a peptide corresponding to the CaM-binding domain of RyR2. The N53I mutation decreased the affinity of Ca²⁺ binding to the N-domain of CaM, relative to CaM WT, but did not affect the C-domain. Conversely, mutations N97S, D95V, and D129G had little or no effect on Ca²⁺ binding to the N-domain but markedly decreased the affinity of the C-domain for Ca²⁺. These results suggest that mutations D95V, N97S, and D129G alter the interaction between CaM and the CaMBD and thus RyR2 regulation. Because the N53I mutation minimally affected Ca²⁺ binding to the C-domain, it must cause aberrant regulation via a different mechanism. These results support aberrant RyR2 regulation as the disease mechanism for CPVT associated with CaM mutations and shows that CaM mutations not associated with CPVT can also affect RyR2. A model for the CaM-RyR2 interaction, where the Ca²⁺-saturated C-domain is constitutively bound to RyR2 and the N-domain senses increases in Ca²⁺ concentration, is proposed.

Introduction

During cardiac excitation, Ca²⁺ entry into the cytoplasm of cardiomyocytes through sarcolemmal voltage-gated Ca²⁺ channels (Ca_V1.2) activates RyR2² channels in the SR, giving rise to the so-called Ca²⁺-induced Ca²⁺ release (1–3). Ca²⁺ released from the SR eventually leads to increases in cytosolic free Ca²⁺ ([Ca²⁺]_cyt) throughout the cardiomyocyte, where binding of Ca²⁺ to myofilaments results in contraction (4). RyR2 channels are, however, not only sensitive to [Ca²⁺]_cyt but also the SR luminal free Ca²⁺ ([Ca²⁺]_SR), and both calcium concentrations modulate the activation and termination of Ca²⁺ release (1, 5–9).

In the SR membrane, RyR2s arrange as homotetrameric channels extending into the cytosol, where the interaction with numerous proteins and ligands regulate the Ca²⁺ release activity of the channel (4, 10). Among these RyR2 modulators, CaM is a cytosolic inhibitor of Ca²⁺ release both at diastolic and systolic [Ca²⁺]_cyt and may also serve additional regulatory purposes (11–14). CaM is a ubiquitous Ca²⁺ sensing protein in vertebrates and confers its sensing of intracellular Ca²⁺ signals onto a multitude of protein targets, including ion channels and pumps responsible for excitation and contraction in cardiomyocytes (15). Although a cytosolic protein, CaM does affect the response of the RyR2 channel to [Ca²⁺]_SR, most likely as part of an extensive allosteric regulation of RyR2 (14).

CaM consists of two domains (C- and N-domains, respectively) with a flexible linker between them that enables independent and correlated functions. Each domain is comprised of two structurally integrated EF hand motifs that each binds one Ca²⁺ ion (Fig. 1). Although highly homologous with extensive sequence identity, the two domains of CaM display distinct Ca²⁺ binding properties and both independent and correlated interactions with protein targets. CaM binds to RyR2 with a stoichiometry of four per channel, primarily via the CaM binding domain (CaMBD) (Arg³⁵⁸¹–Pro³⁶⁰⁷, human RyR2) (15–18). In the Ca²⁺ saturated form, the CaM C-domain appears to bind RyR2 around Trp³⁵⁸⁷ in the CaMBD, and the CaM N-domain appears to bind RyR2 in the vicinity of Phe³⁶⁰³, although the CaM N-domain interaction is more promiscuous, especially at low free Ca²⁺ concentrations ([Ca²⁺]_free) (18–20). Even in the absence of Ca²⁺, CaM can bind to the RyR2 CaMBD, most likely via the C-domain (18, 19). Also, an engineered CaM mutant (CaM E31A/E67A/E104A/E140A, CaM₁₂₃₄) defective in Ca²⁺ binding is a competitive inhibitor of the native regulation of RyR2 by the CaM WT (21). Hence, CaM binding to RyR2 and Ca²⁺ binding to CaM are each critical determinants of RyR2 regulation by CaM.

FIGURE 1.

Representative structure of CaM. Ca²⁺-saturated CaM is shown binding a 27-residue peptide corresponding to part of the CaMBD in RyR1(Lys³⁶¹⁴–Pro³⁶⁴⁰) (PDB ID: 2BCX), differing in one residue from the CaMBD in RyR2 (RyR1 Thr³⁶³⁹). Protein and peptide secondary structures are shown as cartoon representation, the N-domain of CaM (Ala¹–Thr⁷⁹) is indicated in blue, and the C-domain (Asp⁸⁰–Lys¹⁴⁸) is in orange, and the peptide is in gray. Sites of CaM mutations (N53I, N97S, D95V, and D129G) are highlighted as green stick representations (nonmutated residues), and Ca²⁺ ions are shown as black spheres. The RyR1 CaMBD residues (Trp³⁶²⁰, Leu³⁶²⁴, and Phe³⁶³⁶) corresponding to RyR2 CaMBD residues Trp³⁵⁸⁷, Leu³⁵⁹¹, and Phe³⁶⁰³ are highlighted as magenta stick representations. Numbering is according to human RyR2 and mature CaM without initiating Met residue.

Three genes (CALM1–3) in the human genome encode the exact same CaM protein, and we previously found that two separate mutations in the CALM1 gene (N53I and N97S, mature CaM numbering) each lead to dominantly inherited CPVT (22). Subsequently, Crotti et al. (23) identified a CaM mutation in CALM2 (D95V) and two in CALM1 (D129G and F141L) each dominantly causing long QT syndrome (LQTS). More recently, Makita et al. (24) found another five mis-sense mutations in CALM2, three in individuals with severe LQTS (N97I, D133H, and, interestingly, N97S) and two in individuals showing features of both LQTS and CPVT (D131E and Q135P). In addition, a CaM F89L mutation was identified in a family with idiopathic ventricular fibrillation (25).

Both CPVT and LQTS lead to perturbations of excitation-contraction cycles in cardiomyocytes, which in turn can lead to ventricular fibrillation and sudden cardiac death. CPVT is characterized by syncope or sudden cardiac death following exercise or acute emotion, whereas LQTS also affects the resting heart and with increasing effect upon adrenergic stimulation (25, 26). Despite the universal function of CaM, all mutations identified so far confer arrhythmia phenotypes with few or no other symptoms observed in carriers (1, 22–24, 27).

The RyR2 channel accounts for the bulk of Ca²⁺ introduced into the cardiomyocyte cytosol during contraction, and mutations in RyR2 and auxiliary proteins cause CPVT (28–30). Thus, the aberrant regulation of RyR2 by CaM is a likely convergence point for the CaM N53I and N97S disease mechanisms causing CPVT (1, 15). This is also consistent with the observation that these CaM mutations increase spontaneous Ca²⁺ release in permeabilized cardiomyocytes (15, 31). Conversely, LQTS is mainly associated with dysfunction of sarcolemmal voltage-gated Na⁺, Ca²⁺, or K⁺ channels in control of the action potential. Notably the activities of a majority of these channels are regulated by CaM, for example, CaM mutations N97S, D95V, F141L and D129G all confer reduced Ca²⁺-dependent inhibition of the cardiac L-type voltage-gated Ca²⁺ channel (Ca_V1.2) (15, 26, 32).

In this study, we investigated the impact of CaM mutations linked to CPVT (N53I), LQTS (D95V and D129G), or both (N97S) on the CaM-dependent regulation of RyR2 channels. To this end, we monitored the endoplasmic reticulum (ER) Ca²⁺ dynamics in RyR2-expressing HEK293 cells transfected with CaM WT or mutants. The RyR2-mediated Ca²⁺ release in this system is triggered by increasing the Ca²⁺ load in the ER, which mimics the store overload-induced Ca²⁺ release (SOICR) model for CPVT (14, 33). Furthermore, we investigated domain-specific interactions of CaM with Ca²⁺ in the presence of a peptide corresponding to the CaMBD of RyR2. Our results demonstrate that both CPVT and LQTS-associated CaM mutations alter RyR2-mediated Ca²⁺ release.

Experimental Procedures

Plasmid Constructs

Plasmid constructs based on the pMAL vector (New England Biolabs) for recombinant expression and purification of native, full-length CaM were prepared as described previously (1, 22). For expression of CaM variants in HEK293 cells, CaM coding sequences from the pMAL vectors were PCR-amplified and the products ligated into pcDNA3.1 vectors (Invitrogen). Chemically competent Escherichia coli DH5a cells (in-house stock) were transformed with pcDNA3.1 vectors, and overnight cultures were used to prepare purified plasmid preparations using the Qiagen plasmid maxi kit. pcDNA3.1 vectors were eluted in double-distilled water, and Sanger sequencing verified the sequence of CaM encoding inserts in all plasmids.

Model Fitting and Statistical Analysis

All fitting of data to mathematical models and statistical analyses was done using GraphPad Prism 6 for Mac (version 6.0f). Models and statistical method details are described below.

Endoplasmic Reticulum Luminal Ca²⁺ Imaging of HEK293 Cells Expressing RyR2 during Store Overload-induced Ca²⁺ Release

Stable expression of murine RyR2 in HEK293 cells co-transfected with plasmids encoding CaM, and the D1ER Ca²⁺ probe was done as previously described (14). Briefly, D1ER FRET signals reflecting ER luminal [Ca²⁺]_free were monitored for individual cells in an epifluorescent microscope setting with perfusion (6, 14) (Fig. 2A). Each FRET signal trace was used to measure the Ca²⁺ release properties of the RyR2 channels relative to the ER Ca²⁺ content: the activation and termination thresholds (Fig. 2A) and their difference, the fractional Ca²⁺ release. The ER Ca²⁺ store capacities were calculated as the difference between maximum and minimum FRET signal (F_max − F_min). Experiments were also done with HEK293 cells expressing a RyR2 mutant with the CaMBD deleted (RyR2 ΔCaMBD, murine RyR2 ΔLys³⁵⁸³–Phe³⁶⁰³). Measured parameters were compared using one-way analysis of variance with Holm-Sidak multiple comparison test for all possible combinations and with p < 0.01 (against the CaM WT values) chosen as a conservative measure of significance.

FIGURE 2.

Store overload-induced Ca²⁺ release from RyR2-expressing cells transfected with CaM WT and CPVT-linked CaM mutations. A–C, the FRET signal from the ER luminal D1ER [Ca²⁺]_free indicator oscillates as Ca²⁺ is released through RyR2. Rectangles and labels indicate the concentrations of Ca²⁺, tetracaine (RyR2 channel inhibitor), and caffeine (activator) in the perfusion solution. Stepwise increase in Ca²⁺ concentration elicited RyR2 SOICR oscillations, tetracaine blocked Ca²⁺ release filling ER to maximum [Ca²⁺]_free, and finally caffeine opened RyR2 channels depleting ER Ca²⁺. Tetracaine and caffeine were used to establish maximum and minimum ER [Ca²⁺]_free as measured using D1ER (F_max and F_min), respectively. The activation and termination thresholds were calculated relative to F_max and F_min. Example traces for transfection with CaM WT (A), N53I (B), and N97S (C) are shown. D–G, the activation threshold (D), termination threshold (E), fractional release (F), and store capacity (G) averaged from multiple traces are shown as bar graphs. The error bars show S.D., and asterisks indicate significant changes compared with CaM WT (p < 0.01).

Protein Expression and Purification

CaM was expressed from the pMAL (CaM N53I and N97S) or the pET15b (D95V and D129G) vector in E. coli Rosetta B cells (EMD Chemicals) or E. coli BL21 (DE3) cells (Novagen), respectively, and purified as previously described (1, 22, 23). The identity, purity, and integrity of each protein preparation were confirmed by SDS-PAGE and MALDI-TOF mass spectrometry of trypsin-digested proteins.

Peptides Corresponding to the CaM Binding Domain of RyR2

A peptide corresponding to the CaMBD of RyR2 (RyR2(Arg³⁵⁸¹–Leu³⁶¹¹), human RyR2 ³⁵⁸¹RSKKAVWHKLLSKQRKRAVVACFRMAPLYNL³⁶¹¹) was purchased from Peptide 2.0 Inc. (Chantilly, VA) at >98% purity.

Titration Buffers and Verification of Free Ca²⁺ Concentrations

Titration experiments were performed by mixing different volumes of pH- and Ca²⁺-buffered solution (50 mm HEPES, 100 mm KCl, 0.5 mm EGTA, and 2 mm nitrilotriacetic acid at pH 7.2 (25 °C)) with the same buffer spiked to 3, 7, or 22 mm CaCl₂ to reach precalculated [Ca²⁺]_free levels (5). In practice, ×1.5 concentrated buffer stocks were prepared and proteins, peptide, Ca²⁺ probes (final concentration, 0.75 μm), and reducing agent (tris(2-carboxyethyl)phosphine; final concentration, 16.5 μm) were added to the double distilled water used for diluting concentrated buffers. The calculated buffer ionic strength, which affects Ca²⁺ binding to CaM, was stable at 0.15 m. For all titration experiments, the [Ca²⁺]_free was followed by including the Ca²⁺ probe Fura-2 or Fura-6F in solutions (Invitrogen) and indirectly via measuring of Ca²⁺ binding to CaM WT (1). Based on these measurements, a 15% error for the [Ca²⁺]_free was included throughout data sets and fitting procedures.

Titrations of CaM/ RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) and Free CaM with Ca²⁺

15 μm CaM in the presence of 16.5 μm RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) was titrated with Ca²⁺ as previously described (1, 5). Briefly, intrinsic protein fluorescence from the N- and C-domain of CaM were measured as partial Phe and Tyr emission spectra, respectively (Table 1), using a spectrofluorometer (HORIBA Jobin Yvon, FluoroMax®-4P) (1, 34, 35). In addition, the Trp fluorescence from the RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) peptide was also measured (Table 1). Titrations of free CaM WT, D95V, and D129G with Ca²⁺ were done by mixing discontinuous titration points in an automated liquid handler (Hamilton, Microlab STARlet) and serially transferring these to a 2-mm cuvette for emission spectra recording. Measurements were done in triplicate with 10 μm CaM. Each of the fractional saturations (Y) for the N- and C-domains of CaM were fitted to the raw fluorescence intensity (FI) signals from the partial Phe and Tyr spectra at 280 and 320 nm, respectively, according to the following,

where the constants b and a indicate the initial FI and the span in FI from low to high [Ca²⁺]_free, respectively. Y is the fractional saturation of the monitored CaM domain binding to two Ca²⁺ as described by a two-site Adair model (36, 37),

where K₁ is the sum of the microscopic equilibrium constants, and K₂ is the equilibrium constant for the particular domain binding to two Ca²⁺. The apparent dissociation constants for either domain in the free CaM or in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) (appK_D^free and appK_D^bound_, respectively) were calculated as the reciprocal square root of K₂. When CaM binds to RyR2(Arg³⁵⁸¹–Leu³⁶¹¹), the peptide Trp fluorescence shows a peak shift from ∼350 to 340 nm, and furthermore the FI increases markedly upon Ca²⁺ binding to CaM (22). The raw FI for Trp fluorescence at 340 nm was also fitted to the model described above. Titration curves were normalized using the fitted a and b parameters for figure plotting purposes only. Statistical significances of differences in K₂ were evaluated via nonoverlapping 95% confidence intervals or one-way analysis of variance with Dunnett's post hoc test against values measured for the CaM WT and p < 0.05 considered significant. Furthermore, the K₂ values were also used to calculate the mutation-induced change in Gibb's free energy of Ca²⁺ binding to the domains of CaM (ΔΔG^o_free and ΔΔG^o_bound respectively) according to the following,

using standard conditions of 1 m and 298.15 K (25 °C).

TABLE 1.

Fluorescence spectrophotometry measurements

Acquisition settings for measuring Trp, Phe, and Tyr fluorescence spectra are shown. Integration time was 0.2 s for all recordings. Exc and Em are excitations and emission wavelengths, respectively.

Spectra	Exc	Em	Bandwidths	Spectra averaged	Increments
	nm	nm	nm		nm
Phe	250	264–284	7/7	3	2
Tyr	270	310–330	3/3	2	3
Trp	295	336–370	3/3	2	2

Results

Arrhythmogenic CaM Mutations Decrease the Activation and Termination Thresholds for Spontaneous Ca²⁺ Release

Spontaneous Ca²⁺ release can occur in cardiomyocytes during increased Ca²⁺ load in the SR lumen. This spontaneous SOICR is arrhythmogenic, because it may lead to delayed afterdepolarizations and triggered activity, a hallmark of CPVT (25, 38). To determine whether CaM mutations affect this arrhythmogenic SOICR, we transfected RyR2-expressing HEK293 cells with CaM and monitored the ER Ca²⁺ dynamics using a FRET-based ER luminal Ca²⁺ probe D1ER (14). Perfusion of these transfected cells with increasing extracellular Ca²⁺ concentrations induced SOICR in the form of ER Ca²⁺ oscillations (Fig. 2A), as reported previously (14, 39). The oscillating D1ER FRET signal was then used to calculate the ER Ca²⁺ level required for activating SOICR, the activation threshold, and the ER Ca²⁺ depletion required for terminating SOICR, the termination threshold (Fig. 2A and “Experimental Procedures”). Perfusion with the RyR2 inhibitor tetracaine and then the agonist caffeine established maximum (F_max) and minimum (F_min) ER Ca²⁺ content. The ER capacity for storing Ca²⁺, the store capacity, was calculated as F_max − F_min.

In the SOICR experiments, the CPVT-linked CaM mutation, N53I, lowered the SOICR activation threshold (5%) relative to CaM WT, and so did the CPVT- and LQTS-linked mutation CaM N97S (4%) (Fig. 2, B–D, and Table 2). Similarly, the two LQTS-linked CaM mutations, D95V and D129G, also lowered the SOICR activation threshold by 6 and 4% (Fig. 3, B–D, and Table 2), respectively. The effects on the activation thresholds are modest, however, in cardiomyocytes SR Ca²⁺ release increases with increasing SR luminal Ca²⁺ concentrations in a steep and nonlinear fashion (40). Hence, even modest effects of CaM mutations on the response of RyR2 to SR luminal Ca²⁺ may have major impacts on SR Ca²⁺ release.

TABLE 2.

Quantified change in Ca²⁺ release properties for RyR2 channels expressed in HEK293 cells transfected with CaM

Numbers in parentheses indicate the percentages of statistically significant change in the measured properties for RyR2 Ca²⁺ release relative to RyR2 expressing cells transfected with CaM WT (p < 0.01, one-way analysis of variance). Transfections of RyR2 ΔCaMBD expressing cells with CaM variants did not change the Ca²⁺ release properties (Fig. 4, A–E). For comparison to RyR2 WT expressing cells, an average for all RyR2 ΔCaMBD experiments was calculated (Average RyR2 ΔCaMBD).

CaM variant	Activation	Termination	Fractional release
	%	%	%
WT	91	56	35
N53I	86 (−5)	44 (−21)	42 (20)
N97S	87 (−4)	39 (−30)	48 (38)
D95V	85 (−6)	38 (−33)	48 (38)
D129G	87 (−4)	40 (−28)	47 (34)
(Average RyR2 ΔCaMBD)	92	34 (−38)	58 (65)

FIGURE 3.

Store overload-induced Ca²⁺ release from RyR2-expressing cells transfected with CaM WT and LQTS-linked CaM mutations. A–C, example traces of the D1ER FRET signal for cells transfected with CaM WT (A, same as Fig. 2A), D95V (B), and D129G (C). D–G, the activation threshold (D), termination threshold (E), fractional release (F), and store capacity (G) averaged from multiple traces are shown as bar graphs. Error bars show S.D., and asterisks indicate significant changes compared with CaM WT (p < 0.01).

More strikingly, all of these CaM mutations markedly affected the ER Ca²⁺ level at which SOICR terminated. The CaM mutation N53I decreased the termination threshold by 21% relative to CaM WT (Fig. 2, B and E), and, likewise, the CaM mutations N97S, D95V and D129G also decreased the termination threshold by 30, 33, and 28% (Figs. 2, C and E, and 3, B, C, and E), respectively.

The amount of Ca²⁺ released from the ER during SOICR was calculated by subtracting the termination threshold from the activation threshold. As shown in Figs. 2 (B–E) and 3 (B–E), the disease-causing CaM mutations decreased the termination thresholds proportionally more than the activation thresholds and hence increased ER fractional Ca²⁺ release (Figs. 2F and 3F). CaM N53I increased the ER fractional Ca²⁺ release by 20% relative to the CaM WT, and CaM D95V, D129G, and N97S increased this release by 38, 38, and 34%, respectively (Table 2). Interestingly, the effect of CaM-N53I, located in the N-domain of CaM, on the ER fractional release (20%) was significantly less than that of the three CaM mutations located in the C-domain (average 33%), (p < 0.001 for each multiple comparison).

Notably, none of these CaM mutations affected the SOICR activation or termination thresholds in HEK293 cells expressing a RyR2 mutant with a deletion of the CaMBD (Fig. 4, A–D). This observation demonstrates that the CaMBD in RyR2, and not secondary effects of CaM overexpression (e.g. altered Ca²⁺ buffering), mediate the observed effects (Fig. 4, C–E, all individual data sets not shown). Furthermore, the deletion of the CaMBD in RyR2 increased the ER fractional Ca²⁺ release by 65% relative to that of WT RyR2 (Table 2), whereas the maximum increase in the ER fractional Ca²⁺ release caused by the CaM mutations was less than 40% (Figs. 2F and 3F and Table 2). It is noteworthy that this comparison clearly shows that the arrhythmogenic CaM mutations did not suppress CaM-mediated inhibition of Ca²⁺ release to the same extent as ablating the interaction between CaM and RyR2 by deleting the CaMBD in RyR2. In other words, the arrhythmogenic CaMs did interact with and inhibit RyR2, only not to the same extent as the CaM WT.

FIGURE 4.

Store overload-induced Ca²⁺ release from RyR2 ΔCaMBD-expressing cells transfected with CaM WT or D129G. A and B, example traces of the D1ER FRET signal for cells transfected with CaM WT (A) and D129G (B). Transfections with all CaM variants showed no significant changes to the properties of RyR2 ΔCaMBD Ca²⁺ release when comparing to CaM WT transfection. C–E, the activation threshold (C), termination threshold (D), and store capacity (E) averaged for multiple traces are shown as bar graphs. Because there were no significant changes to activation or termination thresholds, the bar graph for fractional Ca²⁺ release was omitted but averaged 58% (Table 2). The error bars show S.D.

Ca²⁺ homeostasis in HEK293 cells differs from that in cardiomyocytes. The [Ca²⁺]_cyt in HEK293 cells before RyR2 Ca²⁺ release (60 nm) is similar to that of diastolic cardiomyocytes (100 nm), but ER Ca²⁺ release is unlikely to increase [Ca²⁺]_cyt to the same peak levels as within the cardiomyocyte dyadic clefts during systole (>100 μm) (41, 42). High Ca²⁺ concentrations mitigate the effects of mutations in the CaM C-domain (see below), and therefore their effect on RyR2 Ca²⁺ release termination may be less at peak systole [Ca²⁺]_cyt in cardiomyocytes than in the HEK293 cells. However, the CaM mutations will on average lead to a suppressed inhibition of RyR2 by CaM and consequently increase the fractional Ca²⁺ release by RyR2 in vivo (32). No significant differences in the HEK293 cell ER Ca²⁺ store capacities were observed (Figs. 2–4), and Ca²⁺ release in cardiomyocytes terminates at ∼60% of diastole [Ca²⁺]_SR, similar to the RyR2 termination thresholds in HEK293 cells (Table 2), which supports similar ER and SR luminal [Ca²⁺]_free (7, 8).

Taken together, each of the CaM mutations N53I, N97S, D95V, and D129G conferred a slightly increased propensity for SOICR and reduced the capability for terminating SOICR. The difference in termination threshold and ER fractional Ca²⁺ release between CaM N53I and the other CaM mutations may support a mechanistically distinct effect of the CaM N53I mutation.

Arrhythmogenic Calmodulin Mutations Affect Binding of Ca²⁺ to Calmodulin in the Presence of the RyR2 CaM Binding Domain

Regulation of RyR2 by CaM critically depends on (a) CaM binding to the CaMBD in RyR2 and (b) Ca²⁺ binding to CaM (43, 44). Furthermore, this regulation of RyR2 by CaM is dependent on the characteristics of binding of Ca²⁺ to each of the CaM domains (11, 14, 16, 21). Hence, we investigated whether the mutations would affect Ca²⁺ binding to CaM in the presence of a RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) peptide corresponding to part of the CaMBD in RyR2. Ca²⁺ binding was investigated by monitoring changes in CaM protein fluorescence (Phe for the N-domain and Tyr for the C-domain) as Ca²⁺ was titrated to the CaM-peptide complex (34). Although available from a previous study, Ca²⁺ binding to CaM in the absence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) was measured for D95V and D129G as reported for CaM N53I and N97S to ensure the reliability of comparisons made here (1, 23). Average [Ca²⁺]_cyt varies between ∼0.1 and 1 μm in cardiomyocytes during each heartbeat, but there is a large spatial heterogeneity of systolic [Ca²⁺]_cyt. For example systolic [Ca²⁺]_cyt can exceed 100 μΜ in the dyadic cleft in vicinity of RyR2 channels (41, 45). Thus, a wide range of Ca²⁺ concentrations (1 nm to 2 mΜ) was used in our Ca²⁺ titration experiments with RyR2(Arg³⁵⁸¹–Leu³⁶¹¹).

The presence of the RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) increased the apparent Ca²⁺ affinity of the CaM N-domain and CaM C-domain ∼20- and ∼80-fold, respectively (Fig. 5A and Table 3). These increases clearly demonstrate the thermodynamic coupling between the Ca²⁺-CaM and the CaM-RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) binding events (Table 3) (46–48). Importantly, the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) increased the binding affinities of N-domain and C-domain for Ca²⁺ differentially, such that the Ca²⁺-CaM interaction for both domains occurred in the range 0.001–6 μm [Ca²⁺]_free. In the absence of peptide, both CaM domains bind Ca²⁺ within the range of 0.1–100 μm [Ca²⁺]_free (Fig. 5A). Ca²⁺ binding to the N-domain of CaM in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) occurred in the range 0.1–6 μm [Ca²⁺]_free with an appK_D^bound of 0.8 μm. This is ∼8-fold higher than the 0.1 μm [Ca²⁺]_cyt at diastole in cardiomyocytes (Fig. 5A and Table 3) and within [Ca²⁺]_cyt range of the diastole to systole cycling. On the other hand, Ca²⁺ binding to the C-domain in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) showed a much higher binding affinity with an appK_D^bound of 0.03 μm, i.e. 3-fold lower than the diastolic [Ca²⁺]_cyt. This suggests that the C-domain will be nearly Ca²⁺-saturated (>90%) at 0.1 μm [Ca²⁺]_free. In other words, these results imply that the C-domain of CaM in complex with RyR2 is inherently Ca²⁺-loaded at diastolic levels of [Ca²⁺]_cyt. Conversely, the N-domain of CaM in the same protein is poised for sensing increases in [Ca²⁺]_cyt. Hence, the N-domain but not the C-domain will switch between apo and Ca²⁺-loaded states during diastole to systole cycles and alter its interactions with RyR2.

FIGURE 5.

Domain-specific titration curves for Ca²⁺ binding to CaM in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹). A–E, CaM/RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) (black) Ca²⁺ titration points for CaM N-domain (circles) and C-domain (squares) were fitted with a two-site Adair model (dashed and solid, respectively), and normalized Y is plotted as a function of [Ca²⁺]_free. The dotted vertical line indicates the cardiomyocyte diastolic equivalent 0.1 μm [Ca²⁺]_free. Also in A, Ca²⁺ titration of free CaM WT is shown in gray (1), illustrating the thermodynamic coupling of CaM binding Ca²⁺ and RyR2(Arg³⁵⁸¹–Leu³⁶¹¹). In B–E, gray lines represents the Adair fits for CaM WT/RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) binding Ca²⁺ to illustrate the effects of CaM mutations. F shows the quantified mutation effect (ΔΔG^o_bound) on Ca²⁺ binding to CaM in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹). Error bars indicate S.D., and asterisks indicate statistical significant differences (p < 0.05).

TABLE 3.

Measured thermodynamic parameters for Ca²⁺ binding to CaM

Apparent Ca²⁺ dissociation constants for RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) bound and free CaM (appK_D^bound and appK_D^free, respectively) were measured using CaM intrinsic protein Phe (N-domain) and Tyr (C-domain) fluorescence. Mutation-imposed changes in Gibb's free energy of Ca²⁺ binding to CaM (ΔΔG^o_bound and ΔΔG^o_free, respectively) were calculated relative to each CaM WT value. appK_D^free and ΔΔG^o_free values for free CaM WT, N53I, and N97S are from a previous study (1). Dissociations constants and changes in Gibb's free energy are given in μm and kJ/mol, respectively. Bold font highlights significant changes versus CaM WT values (p < 0.05, one-way analysis of variance).

	appKDbound	appKDfree	ΔGobound	ΔΔGofree
N-domain
WT	0.78	16
N53I	1.21	19	2.1	1.1
N97S	0.71	16	−0.5	0.1
D95V	0.48	5.2	−2.5	−6.4
D129G	0.22	13	−6.4	−1.9
C-domain
WT	0.03	2.5
N53I	0.03	2.3	−0.3	−0.3
N97S	0.15	10.4	8.4	7.2
D95V	0.14	31	8.3	13.2
D129G	4.0	84	25.3	18.2

An additional observation was made during the titrations of the CaM-peptide complex with Ca²⁺. During the titrations, we also monitored the change in RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) Trp fluorescence (data not shown), which coincided with the change in Tyr fluorescence from the CaM C-domain, but not with the change in Phe fluorescence from the CaM N-domain. This strongly supports that the C-domain of CaM WT binds around RyR2 Trp³⁵⁸⁷ and its Ca²⁺ binding induces a structural shift of the C-domain position on RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) as is also hinted by previous studies (18, 19, 22).

The CPVT- and/or LQTS-linked CaM mutations differentially perturbed this domain-specific interaction of Ca²⁺ with CaM. The CPVT-linked CaM-N53I mutation slightly decreased (appK_D^bound 1.2 μm) the Ca²⁺ affinity of the N-domain in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) and showed no measurable effect on Ca²⁺ binding to the C-domain (Fig. 5B and Table 3). Conversely, the CaM-N97S mutation linked to both CPVT- and LQTS, had no measurable effect on Ca²⁺ binding to the N-domain, but markedly decreased (appK_D^bound 0.15 μm) the Ca²⁺ affinity of the C-domain (Fig. 5C and Table 3).

The LQTS-linked CaM mutations, D95V and D129G, both increased (appK_D^bound 0.48 and 0.22 μm) the Ca²⁺ affinity of the N-domain in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) and both also markedly decreased the Ca²⁺ affinity of the C-domain (appK_D^bound 0.14 and 4 μm) (Fig. 4, D and E, and Table 3). The effect of the D129G mutation was so marked that the Ca²⁺ affinity of the C-domain was weaker than that of the N-domain. This suggests that the N-domain of CaM D129G may bind the CaMBD in RyR2 at a lower Ca²⁺ concentration than the C-domain. Our data support the notion that mutations in the CaM C-domain (D95V, N97S, and D129G) all markedly perturb the interaction between Ca²⁺ and CaM bound to the CaMBD in RyR2. Because CaM will bind to the CaMBD even in the absence of Ca²⁺, the reduction in C-domain Ca²⁺ affinity for the mutants may result in binding to the CaMBD in RyR2 in a partially saturated or even Ca²⁺-unbound form (18). The small increase in the Ca²⁺ affinity of the N-domain in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹), as conferred by the LQTS-linked CaM mutations D95V and D129G, suggests that the N-domain CaM mutations will still respond to an increase in [Ca²⁺]_cyt from diastole to systole. In contrast to the marked perturbations of Ca²⁺ binding by the CaM C-domain mutations, the CPVT-linked CaM N53I mutation showed only a small decrease in the CaM N-domain affinity for Ca²⁺ in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹), yet a substantial effect on the interaction with RyR2 is expected because there is a substantial effect on SOICR.

Discussion

The domain-specific affinities of CaM for Ca²⁺ in the presence of the RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) offer novel insight into the distinct roles of the two domains of CaM in regulating the Ca²⁺ release from RyR2 (Fig. 6, A–C). Our results support an interaction between CaM and the CaMBD in RyR2 where the Ca²⁺-saturated C-domain of CaM is constitutively tethered around RyR2 Trp³⁵⁸⁷ even at the low diastole level of 0.1 μm Ca²⁺_free (Fig. 6B) and thus not releasing Ca²⁺ during cardiac contraction cycles (Fig. 6, B and C). This implies a critical role for Ca²⁺ binding to the C-domain in inhibiting Ca²⁺ release from RyR2. In line with this view, Tian et al. (14) showed that a CaM double mutant (D93A/D129A) with loss of C-domain Ca²⁺ binding is defective in regulating the termination of RyR2 SOICR. Both the work of Tian et al. and this study also demonstrate that the interaction between the Ca²⁺-unbound CaM C-domain with the CaMBD in RyR2 inhibits Ca²⁺ release (Fig. 6A), but not to nearly the same extent as the Ca²⁺-saturated C-domain. Thus, the novel observation in this study is that the CaM C-domain under physiologically relevant conditions binds to the RyR2 CaMBD in a Ca²⁺-saturated form.

FIGURE 6.

Schematic model for the proposed Ca²⁺-dependent interaction between domains of CaM and the CaMBD in RyR2. RyR2 CaMBD is shown as an α-helical representation of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) with Trp³⁵⁸⁷, Leu³⁵⁹¹, and Phe³⁶⁰³ highlighted as magenta sticks. The apoCaM C-domain (orange) binds in the vicinity of Trp³⁵⁸⁷ and likely involves Leu³⁵⁹¹ (A) (16), and this interaction changes conformation to fully encompass W3587 upon Ca²⁺ binding to CaM (B). Both the apoCaM and Ca₂CaM interactions with the CaMBD (A and B) inhibit RyR2 Ca²⁺ release, with the latter interaction being a markedly stronger inhibitor. The N-domain of CaM (blue) interacts with another region of RyR2, indicated by the black α-helix, in the Ca²⁺-unbound form (A and B) and clamps down on Phe³⁶⁰³ upon Ca²⁺ binding (C). appK_D^bound for the CaM WT C-domain and N-domain, respectively, are indicated above equilibrium symbols, and black circles indicate Ca²⁺ bound to domains of CaM. The physiologically relevant interactions between CaM and the RyR2 CaMBD, in our model, are those shown in B and C. In our model, mutations conferring a reduced affinity of the CaM C-domain for binding to Ca²⁺, shift the physiological state of the C-domain at diastole (B) and systole (C) toward the pathological apoCaM interaction (A).

The pivotal role of the tethered CaM C-domain for inhibiting RyR2 Ca²⁺ release is consistent with studies showing that >70% of RyR2 channels in intact cardiomyocytes have CaM bound and that dissociation of CaM leads to excessive Ca²⁺ release through RyR2 channels (13, 43, 49, 50). In addition, several studies show that single mutations in the binding site for the CaM C-domain in the CaMBD of RyR2 (W3587A or L3591D) decrease the affinity for CaM to RyR2 at 0.4 and 1 μm [Ca²⁺]_free, and that this decrease also leads to impaired inhibition of RyR2-mediated Ca²⁺ release (14, 16, 44, 51).

With the region of the RyR2 CaMBD around Trp³⁵⁸⁷ occupied by the C-domain of CaM, the binding site for the N-domain would be adjacent and likely covering RyR2 Phe³⁶⁰³, similar to the structure of CaM binding to the CaMBD in RyR1 (Fig. 1) (20). In contrast to the C-domain, our results suggest that the N-domain is Ca²⁺-unbound at the diastole level of [Ca²⁺]_free but binds Ca²⁺ upon increases in [Ca²⁺]_cyt above the diastolic level. Hence, the N-domain appears to cycle between the apo and Ca²⁺ saturated states during a heartbeat, thereby alternating between low and high affinity for binding to the RyR2 CaMBD. Accordingly, our data suggest that the CaM N-domain is a sensor of [Ca²⁺]_cyt for RyR2 channels, albeit with a so far unknown function. Conversely, the CaM C-domain may not be a Ca²⁺ sensor because of its Ca²⁺-saturated state at physiological [Ca²⁺]_cyt, when bound to the RyR2 CaMBD. Noteworthy, several Ca²⁺ binding proteins that interact with RyR2, as well as the endogenous RyR2 sensor of [Ca²⁺]_cyt, all contribute to the response of the RyR2 macro complex to [Ca²⁺]_cyt in vivo (52, 53). We further propose that the Ca²⁺-unbound N-domain interacts with regions of RyR2 outside the canonical CaMBD and then binds in the vicinity of Phe³⁶⁰³ upon cardiomyocyte excitation (Fig. 6, B and C) (18, 20). This proposed interaction would be consistent with the CaMBD in RyR2 not being the only RyR2 region reported to intact with CaM (11, 18, 54, 55). Moreover, at low [Ca²⁺]_free, the N-domain may interact with a region in the skeletal muscle SR Ca²⁺ release channel, ryanodine receptor (RyR1) that is noncontiguous with the RyR1 CaMBD (18–20, 56). On the other hand, cryo-electron microscopy models of RyR2 channels indicate that CaM does not change position on RyR2 when comparing samples prepared in low and high Ca²⁺ (55, 57). However, because activation of the RyR2 channel involves substantial movements in the channel structure, it cannot be ruled out that the N-domain may bind different RyR2 regions in the open and closed channel, respectively, without CaM markedly shifting position.

In summary, we propose that the interaction between CaM and RyR2 involves constitutive tethering of the Ca²⁺-saturated CaM C-domain to the CaMBD in RyR2, which serves to inhibit Ca²⁺ release by stabilizing the RyR2 channel closed state throughout Ca²⁺ release (14, 21, 43, 58). In our model, N-domain binding to RyR2 is a Ca²⁺ sensing step that responds to increased [Ca²⁺]_cyt upon cardiomyocyte excitation. However, determining the exact function of Ca²⁺ binding to CaM N-domain in regulating RyR2 Ca²⁺ release will require further investigation.

Ca²⁺ binding to the CaM N-domain does not appear to affect the SOICR activation and termination thresholds of RyR2 Ca²⁺ release, because no effect was detected in experiments using a CaM mutant with dramatically decreased N-domain Ca²⁺ affinity (CaM D20A/D56A) (14). Also, in the current study only minor effects of the CaM N53I mutation on the binding of Ca²⁺ to the N-domain in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹) were observed (see below). The binding of Ca²⁺ to the N-domain of CaM, when CaM is tethered to RyR2, may instead be part of inhibiting Ca²⁺-induced Ca²⁺ release during the high systolic [Ca²⁺]_cyt. Alternatively, Ca²⁺ binding to the N-domain could be part of the triggering mechanism for the activation of RyR2 channel Ca²⁺ release during Ca²⁺-induced Ca²⁺ release. Both of these functions would be in combination with endogenous cytosolic and SR luminal Ca²⁺ sensors in RyR2 (52, 59). An inhibiting or facilitating function of CaM on Ca²⁺-induced Ca²⁺ release would not be detected in the SOICR experiments performed in this study (6, 14).

Our results support the notion that arrhythmogenic CaM mutations adversely affect the native interaction between CaM and RyR2. We show that the C-domain mutations (D95V, N97S, and D129G) perturb the interactions between CaM, Ca²⁺ and the CaMBD in RyR2. Notably, this perturbation occurs within physiologic relevant Ca²⁺ concentrations. We suggest that at diastole [Ca²⁺]_cyt, CaM with either of these C-domain mutations will bind to the RyR2 CaMBD with the CaM C-domain in a partially saturated or Ca²⁺-unbound form as opposed to the native Ca²⁺-saturated state (transition from Figs. 6B to 6A). This in turn leads to an insufficient inhibition of the RyR2 Ca²⁺ release during SOICR in intact cells, similar to the aberrant regulation of RyR2 by CaM₁₂₃₄ and CaM D93A/D129G mutants (14, 21). In vivo CaM D95V, N97S, and D129G are likely to still tether to RyR2 because each mutated CaM retains a significant affinity for intact RyR2 channels, despite reduced affinity for Ca²⁺ (31).

Interestingly, the N53I mutation in the N-domain of CaM adversely affected the regulation of RyR2-mediated Ca²⁺ release during SOICR, without any changes to the affinity of the CaM N53I C-domain for binding Ca²⁺ in the presence of RyR2(Arg³⁵⁸¹–Leu³⁶¹¹). This strongly suggests that the integrity of the CaM N-domain is important in inhibiting RyR2 Ca²⁺ release but not dependent on Ca²⁺ binding to the N-domain, as discussed above (14). In strong support of this hypothesis is that CaM D20A/D56A, with markedly decreased N-domain Ca²⁺ affinity, does not affect activation or termination thresholds for RyR2-mediated SOICR, whereas CaM N53I does. A likely explanation is that the N53I mutation affects an interaction of the Ca²⁺-unbound N-domain of CaM with a region of RyR2 that is outside the CaMBD. The significantly different impact of the CaM N53I mutation on RyR2 Ca²⁺ release, compared with the C-domain mutations, also supports this notion. Furthermore, we previously found divergent effects of the N53I and N97S mutations on protein properties and the binding of Ca²⁺ to CaM, which supports different molecular disease mechanisms for the two mutations (1).

Overexpression of CaM in HEK293 cells could theoretically lead to buffering of [Ca²⁺]_cyt, which in turn may affect RyR2 Ca²⁺ release through mechanisms independent of the interaction between CaM and RyR2, and lead to differences between the CaM mutations caused by differences in Ca²⁺ buffering capacities. This was, however, not the case in the SOICR experiments as judged from three observations: (a) CaM N53I had marked effects on RyR2-mediated Ca²⁺ release, although it displayed Ca²⁺ binding properties highly similar to those of CaM WT both in the RyR2-bound and free form (Fig. 2, A and B, and Table 3); (b) CaM WT and CaM D129G had no effect on Ca²⁺ release from the RyR2 ΔCaMBD mutant, although they exhibited widely different Ca²⁺ binding properties (Fig. 4, A and B) (1, 14); and (c) because the Ca²⁺ affinity (appK_D^free) of the CaM C-domain not bound to a protein target is ∼2.5 μm, the buffering capacity of free CaM is highly limited at the ∼60 nm [Ca²⁺]_cyt in HEK293 cells.

The combined results of this study show that both CPVT- and LQTS-linked CaM mutations can lead to excessive Ca²⁺ release from RyR2 channels, primarily from insufficient termination of RyR2-mediated Ca²⁺ release. Also, the same CaM mutations lower the activation thresholds for SOICR, which would increase the propensity for SOICR in vivo during conditions with increased SR Ca²⁺ load. Excessive Ca²⁺ release and leaky RyR2 channels are a well documented hallmark of CPVT-linked RyR2 mutations. It follows that dysregulation of RyR2 likely underlies the disease mechanisms of CaM N53I and N97S mutation-associated CPVT (38). Taken together, we propose that the regulation of RyR2 Ca²⁺ release is highly sensitive to CaM and that aberrant regulation of RyR2 may be a common component of both CPVT and LQTS arrhythmias caused by CaM mutations.

Author Contributions

M. T. S., S. R. W. C., and M. T. O. designed the research; M. T. S., X. T., Y. L., and R. W. performed research; M. T. S., X. T., Y. L., S. R. W. C., and M. T. O. analyzed data; and M. T. S., W. J. C., S. R. W. C., and M. T. O. wrote the paper.