Enhanced Basal Activity of a Cardiac Ca2+ Release Channel (Ryanodine Receptor) Mutant Associated With Ventricular Tachycardia and Sudden Death
Mutations in the human cardiac Ca2+ release channel (ryanodine receptor, RyR2) gene have recently been shown to cause effort-induced ventricular arrhythmias. However, the consequences of these disease-causing mutations in RyR2 channel function are unknown. In the present study, we characterized the properties of mutation R4496C of mouse RyR2, which is equivalent to a disease-causing human RyR2 mutation R4497C, by heterologous expression of the mutant in HEK293 cells. [3H]ryanodine binding studies revealed that the R4496C mutation resulted in an increase in RyR2 channel activity in particular at low Ca2+ concentrations. This increased basal channel activity remained sensitive to modulation by caffeine, ATP, Mg2+, and ruthenium red. In addition, the R4496C mutation enhanced the sensitivity of RyR2 to activation by Ca2+ and by caffeine. Single-channel analysis showed that single R4496C mutant channels exhibited considerable channel openings at low Ca2+ concentrations. HEK293 cells transfected with mutant R4496C displayed spontaneous Ca2+ oscillations more frequently than cells transfected with wild-type RyR2. Substitution of a negatively charged glutamate for the positively charged R4496 (R4496E) further enhanced the basal channel activity, whereas replacement of R4496 by a positively charged lysine (R4496K) had no significant effect on the basal activity. These observations indicate that the charge and polarity at residue 4496 plays an essential role in RyR2 channel gating. Enhanced basal activity of RyR2 may underlie an arrhythmogenic mechanism for effort-induced ventricular tachycardia.
An increasing body of evidence indicates that defects in cardiac ion channels can lead to ventricular arrhythmias, the major cause of sudden death.1 To date, 6 ion channel genes have been linked to cardiac arrhythmias. These include genes encoding the potassium channels, KVLQT1, HERG, minK, and MiRP1, the sodium channel, SCN5A, and the cardiac Ca2+ release channel (ryanodine receptor, RyR2). Mutations in the potassium channels can cause long-QT syndrome, whereas mutations in the sodium channel can lead to both long-QT syndrome and familial ventricular fibrillation.1
Mutations in the human RyR2 gene have recently been shown to cause different forms of cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia (PMVT) and arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2).2–4⇓⇓ However, the molecular and cellular mechanisms underlying these forms of ventricular arrhythmias are not clear. Catecholaminergic PMVT is an autosomal-dominant, genetic arrhythmogenic disorder associated with syncope and sudden death.5 This disorder is characterized by stress-, emotion-, or physical exercise-induced bidirectional ventricular tachycardia. Patients with catecholaminergic PMVT apparently have functionally and structurally normal hearts and display normal QT intervals at rest and during exercise. Interestingly, the ECG pattern of catecholaminergic PMVT resembles that of digitalis-induced arrhythmia.6 Based on these observations, it has been proposed that catecholaminergic PMVT and digitalis-induced arrhythmia may share a similar arrhythmogenic mechanism, which is believed to be Ca2+ overload-induced delayed afterdepolarizations.2,7⇓ How mutations in the RyR2 gene could lead to Ca2+ overload-induced delayed afterdepolarizations is, however, unclear.
RyR2 is a key component involved in excitation-contraction (EC) coupling that is thought to occur in cardiac muscle via a mechanism known as Ca2+-induced Ca2+ release.8,9⇓ In this process, the RyR2 Ca2+ release channel located in the sarcoplasmic reticulum (SR) is activated by a small influx of Ca2+ through the voltage-dependent L-type Ca2+ channel, leading to a large Ca2+ release from the SR. In addition to this stimulated Ca2+ release during normal EC coupling, Ca2+ release from SR can also occur spontaneously as a result of spontaneous openings of the RyR2 channels. Under normal Ca2+ loading conditions, spontaneous Ca2+ release can be detected as localized Ca2+ transients known as “Ca2+ sparks.”10,11⇓ On the other hand, when cardiac cells are overloaded with Ca2+, Ca2+ sparks lead to propagating Ca2+ waves.12 It has been well established that propagating Ca2+ waves cause delayed afterdepolarizations via Ca2+-activated inward currents, which in turn could lead to triggered arrhythmias.13–16⇓⇓⇓ Alterations in RyR2 channel function are likely to influence the properties of spontaneous Ca2+ release and propagating Ca2+ waves, and thus the occurrence of triggered arrhythmias.
In the present study, we have expressed a mouse RyR2 mutant R4496C, corresponding to an arrhythmogenic human RyR2 mutant R4497C, in HEK293 cells and characterized the mutant by using [3H]ryanodine binding assay, single-channel analysis, and single-cell Ca2+ imaging. Our results show, for the first time, that mutation R4496C enhances the basal channel activity and the propensity for spontaneous Ca2+ release. These observations shed important insights into the molecular basis of catecholaminergic PMVT.
Materials and Methods
Site-Directed Mutagenesis and DNA Transfection
Point mutations were generated by the overlap extension method using the polymerase chain reaction as described previously.17,18⇓ HEK293 cells grown on 100-mm tissue culture dishes were transfected with wild-type or mutant RyR cDNA using Ca2+ phosphate precipitation.19
Preparation of Cell Lysate
Cell lysate was prepared as described previously.18
Equilibrium [3H]ryanodine binding to cell lysate (30 μL, 3 to 5 μg/μL) was performed as described previously.18
Single-Cell Ca2+ Imaging
Ca2+ transients in transfected HEK293 cells were measured using single-cell Ca2+ imaging and the fluorescence Ca2+ indicator dye fura-2-AM.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Mutation R4496C Enhances the Basal Level of [3H]Ryanodine Binding
To investigate the impacts of PMVT mutations on RyR2 channel function, we have generated a mutation, R4496C, in mouse RyR2 equivalent to the disease-causing mutation R4497C in human RyR2.2 The mouse R4496C mutant RyR2 was expressed in HEK293 cells and characterized by [3H]ryanodine binding. Figure 1A shows [3H]ryanodine binding to wild-type (wt) and R4496C mutant mouse RyR2 in the presence of a wide range of Ca2+ concentrations. Analysis of the Ca2+ dependence of [3H]ryanodine binding to the wt channels by the Hill equation yielded an EC50 of 0.26±0.04 μmol/L and a Hill coefficient of 2.41±0.15 (n=4), similar to those reported previously.18 Different from the wt channels, the R4496C mutant channels were activated by Ca2+ with a decreased EC50 of 0.19±0.02 μmol/L (P<0.03) and a decreased Hill coefficient of 1.82±0.21 (n=5). In addition to the slightly leftward shift in Ca2+ dependence, the R4496C mutant channels exhibited an elevated level of [3H]ryanodine binding particularly at low Ca2+ concentrations. For instance, at Ca2+ concentrations between 0.2 to 20 nmol/L, a considerable level of [3H]ryanodine binding (10.6±1.4% maximum binding; n=4) was detected in mutant R4496C, significantly higher than the 2.11±0.74% (n=4) observed in the wt channels (P<0.001) (Figure 1A). These results indicate that mutation R4496C increases the basal level and decreases the threshold for Ca2+ activation of [3H]ryanodine binding to RyR2.
Mutation R4496C Does Not Alter the Affinity of [3H]Ryanodine Binding to RyR2
The increased basal [3H]ryanodine binding activity observed in mutant R4496C may result from alterations in the properties of [3H]ryanodine binding. To test this possibility, we carried out [3H]ryanodine binding to the R4496C mutant and wt RyR2 in the presence of different concentrations of [3H]ryanodine. Scatchard analysis of the binding data revealed that mutant R4496C exhibited a binding affinity (Kd) of 2.5±0.1 nmol/L and a maximum binding capacity (Bmax) of 0.98±0.04 pmol/mg (n=3) (Figure 1B). The [3H]ryanodine binding properties of mutant R4496C were very similar to those of the wt RyR2 (Kd=2.8 nmol/L, Bmax=1.01 pmol/mg) (Figure 1B).18 Therefore, mutation R4496C does not appear to alter the intrinsic properties of [3H]ryanodine binding to RyR2.
Increased Basal [3H]Ryanodine Binding Activity Is Sensitive to Modulation
It is also possible that the increased basal [3H]ryanodine binding activity of mutant R4496C may be due to nonspecific binding. Accordingly, we examined the effects of various channel modulators on [3H]ryanodine binding to mutant R4496C. As shown in Figure 2a, at ≈3 nmol/L Ca2+ concentration (pCa=8.49), mutant R4496C showed ≈10% maximum [3H]ryanodine binding. This basal activity was activated by caffeine and ATP and was inhibited by Mg2+ and ruthenium red (Figure 2a). Furthermore, the responses of mutant R4496C to various modulators at this Ca2+ concentration were similar to those observed at a higher Ca2+ concentration (≈100 nmol/L, pCa=7.02) (Figure 2b). Thus, the basal [3H]ryanodine binding activity observed in mutant R4496C is sensitive to regulation by various channel modulators and does not result from nonspecific [3H]ryanodine binding.
Single R4496C Mutant Channels Exhibit Enhanced Channel Activity at Nanomolar Ca2+ Concentrations
[3H]ryanodine binding is thought to reflect channel openings of RyR, as ryanodine binds to only the open state of the channel.21 Our observation that mutant R4496C displayed increased [3H]ryanodine binding at nanomolar Ca2+ concentrations (Figure 1A) suggests that mutant R4489C may exhibit considerable channel openings in the near absence of Ca2+. To examine this possibility directly, we incorporated single wt and R4496C mutant channels into planar lipid bilayers and examined their channel activities at low Ca2+ concentrations. As shown in Figure 3A, a single wt channel was almost completely inhibited by the addition of 0.1 mmol/L EGTA, which would reduce the free Ca2+ concentration to less than 5 nmol/L (assuming that the contaminated level of Ca2+ in the recording solution is less than 5 μmol/L). The average open probability (Po) of single wt channels under this condition was 1.6×10−5±4.7×10−6 (n=30). In contrast, under the same condition, considerable opening events were detected in single R4496C mutant channels (Figure 3b). The average Po of the EGTA-inhibited single R4496C mutant channels was 1.1×10−4±3.3×10−5 (n=28), significantly greater than that observed with the wt channels (P<0.005). These observations are consistent with the view that mutant R4496C displays increased basal channel activity.
[3H]ryanodine binding assay is very sensitive for monitoring RyR channel activities when Po of the channel is relatively low (less than ≈0.05). However, when Po is greater than ≈0.3, [3H]ryanodine binding becomes saturated and relatively insensitive to changes in channel activities.21 In order to characterize the overall Ca2+ dependence of the R4496C mutant channels, we determined the Po of single R4496C mutant channels at a wide range of Ca2+ concentrations. As shown in Figure 3C, single R4496C mutant channels were activated by ≈100 nmol/L Ca2+ and was inhibited by ≈10 mmol/L Ca2+ (Figures 3C-a through 3C-e). At Ca2+ concentrations between ≈1 μmol/L and 1000 μmol/L, the R4496C mutant channels were maximally activated with Po near unity. This bell-shaped Ca2+ response of single R4496C mutant channels (n=14) could be described by an EC50 of 0.46 μmol/L and a Hill coefficient of 2.8 for Ca2+ activation, and an IC50 of 6.3 mmol/L and a Hill coefficient of 0.9 for Ca2+ inactivation. Under the same conditions, single wt RyR2 channels (n=7) were activated by Ca2+ with an EC50 of 0.42 μmol/L and a Hill coefficient of 2.2, and were inactivated by Ca2+ with an IC50 of 7.0 mmol/L and a Hill coefficient of 1.1. These analyses indicate that the overall Ca2+ response of single R4496C mutant channels is similar to that of single wt channels. Taken together, it appears that mutation R4496C affects RyR2 channel gating mainly at low Ca2+ concentrations.
Mutation R4496C Increases the Sensitivity of RyR2 to Activation by Caffeine
To examine whether mutation R4496C affects the caffeine sensitivity of RyR2, we determined [3H]ryanodine binding to the R4496C mutant and wt RyR2 at various concentrations of caffeine. As shown in Figure 4, the wt RyR2 channels were activated by caffeine with an EC50 of 2.64±0.05 mmol/L and a Hill coefficient of 1.99±0.11 (n=3). On the other hand, under the same condition, the R4496C mutant channels were activated by caffeine with an EC50 of 1.44±0.01 mmol/L, significantly lower than that of the wt (P<0.0001) (n=3), and a Hill coefficient of 1.66±0.31. Therefore, mutation R4496C enhances the sensitivity of RyR2 to activation by caffeine.
Effects of Mutations R4496E, R4496A, and R4496K on [3H]Ryanodine Binding
To investigate whether removal of the positive charge at residue 4496 accounts for the increased basal channel activity seen in mutant R4496C, we mutated R4496 to a negatively charged glutamate (R4496E), an uncharged nonpolar alanine (R4496A), and a positively charged lysine (R4496K). The effects of these mutations on the Ca2+ dependence of activation were determined by [3H]ryanodine binding. As shown in Figure 5, the Ca2+ dependence of [3H]ryanodine binding to mutant R4496E displayed a considerable leftward shift as compared with that of the wt channels. The R4496E mutant channels were activated with an EC50 of 0.12±0.01 μmol/L and a Hill coefficient of 1.70±0.38 (n=4). On the other hand, the Ca2+ responses of [3H]ryanodine binding to mutants R4496A and R4496K were found to be similar to that of the wt. The values of EC50 and Hill coefficient were 0.24±0.03 μmol/L and 2.41±0.44 (n=3) for mutant R4496A, and 0.33±0.01 μmol/L and 2.91±0.37 (n=3) for mutant R4496K, respectively.
The mutations also affect the basal level of [3H]ryanodine binding to different extents. At nanomolar Ca2+ concentrations (0.2 to 20 nmol/L), mutant R4496K exhibited a basal level of [3H]ryanodine binding of 1.43±0.65% maximum binding (n=3), similar to the 2.11±0.74% observed with the wt, whereas mutant R4496A showed a basal level of [3H]ryanodine binding of 5.30±1.55% (n=3), which is slightly higher than that of the wt. On the other hand, mutant R4496E displayed a basal level of [3H]ryanodine binding of 30.0±3.84% (n=4), much higher than that of the wt. Thus, these results indicate that the charge and polarity at residue 4496 have a strong influence on RyR2 channel gating, particularly at low Ca2+ concentrations. It should be noted that the marked increase in basal [3H]ryanodine binding activity observed in mutant R4496E does not result from nonspecific [3H]ryanodine binding. [3H]ryanodine binding to mutant R4496E at both low (≈3 nmol/L, pCa=8.49) (Figure 6a) and high (≈100 nmol/L, pCa=7.02) (Figure 6b) Ca2+ concentrations was inhibited by Mg2+ and ruthenium red, and was activated by caffeine and ATP, similar to the ligand responses seen with the R4496C mutant channel (Figure 2).
HEK293 Cells Transfected With Mutant R4496C Exhibit Frequent Spontaneous Ca2+ Oscillations
To test the possibility that mutation R4496C may lead to an increased propensity for spontaneous Ca2+ release, we measured intracellular Ca2+ transients in intact transfected HEK293 cells using fluorescent Ca2+ indicator fura-2-AM and single-cell Ca2+ imaging. These measurements revealed that HEK293 cells transfected with mutant R4496C displayed spontaneous Ca2+ oscillations much more frequently than HEK293 cells transfected with wt RyR2. In the absence of caffeine, about 16% (27 out of 169 cells from 4 separate experiments) caffeine (2 mmol/L)-sensitive R4496C-transfected HEK293 cells showed spontaneous Ca2+ oscillations (Figure 7A), whereas under the same conditions, only about 2% (3/177) caffeine (2 mmol/L)-sensitive wt RyR2-transfected cells exhibited spontaneous Ca2+ oscillations (Figure 7D). Addition of low concentrations of caffeine increased the number of oscillating cells. About 25% (43/169) caffeine-sensitive R4496C-transfected cells displayed Ca2+ oscillations in the presence of 0.1 mmol/L caffeine (Figures 7A and 7B), and about 30% (51/169) in the presence of 0.2 mmol/L caffeine (Figures 7A through 7C). On the other hand, about 6% (10/177) of caffeine-sensitive wt transfected cells showed Ca2+ oscillations in the presence of 0.1 mmol/L caffeine (Figures 7D and 7E), and about 11% (20/177) in the presence of 0.2 mmol/L caffeine (Figures 7D through 7F). It should be noted that HEK293 cells (>190) transfected with the expression plasmid pCDNA3 did not respond to caffeine, but responded to ATP (5 mmol/L) (Figure 7G). These data demonstrate that mutation R4496C increases the propensity of RyR2 for spontaneous Ca2+ release when expressed in HEK293 cells.
RyRs are a family of intracellular Ca2+ channels that govern the release of Ca2+ from the sarco(endo)plasmic reticulum of muscle or nonmuscle cells.22,23⇓ Given the essential roles of RyRs in excitation-contraction coupling in muscles and Ca2+ signaling in a variety of cells,24 one would expect that alterations in RyR function would lead to various disease states. Indeed, mutations in the skeletal muscle RyR (RyR1) have been shown to cause two skeletal muscle diseases, malignant hyperthermia (MH) and central core disease (CCD).25,26⇓ Consistent with this view, mutations in RyR2 have also been shown recently to cause PMVT and ARVD2.2–4⇓⇓
Genetic studies and mutational analyses have identified a number of MH and CCD mutations in RyR1. Almost all the MH/CCD mutations are clustered in 3 conserved regions of RyR1: the NH2 terminal region, a region near the center (2162 to 2458), and the COOH terminal region.25,26⇓ Interestingly, all arrhythmogenic mutations found to date are located in the corresponding regions in RyR2.2–4⇓⇓ Expression studies have shown that MH/CCD mutants exhibit an increased sensitivity to caffeine and halothane.27 In addition, HEK293 cells, myocytes, or lymphocytes expressing the MH/CCD mutants showed reduced sizes of intracellular Ca2+ stores. These observations have led to the conclusion that MH/CCD mutations result in spontaneous Ca2+ leaks.28–30⇓⇓ The consequences of PMVT and ARVD2 mutations in RyR2 channel function, however, are unclear. Based largely on their similar locations as the MH/CCD mutations, it has been hypothesized that the disease-associated RyR2 mutations are likely to cause the channel to open spontaneously4 or to increase the sensitivity of the channel to activation by Ca2+.2
Consistent with these hypotheses, the results of our present study demonstrate directly that mutation R4496C enhances the basal channel activity as assessed by [3H]ryanodine binding, single-channel recordings, and single-cell Ca2+ imaging. Mutant R4496C shows a considerable level of [3H]ryanodine binding (Figure 1) and single-channel openings (Figure 3) at nanomolar Ca2+ concentrations. HEK293 cells expressing the R4496C mutant exhibit frequent spontaneous Ca2+ oscillations (Figure 7). Like most of the MH/CCD mutants, the R4496C mutant also shows an increased sensitivity to activation by caffeine (Figures 4). Interestingly, the overall Ca2+ dependence of single R4496C mutant channels is indistinguishable from that of single wt channels (Figure 3), although a slight increase in the sensitivity to Ca2+ activation was detected by [3H]ryanodine binding assay (Figure 1). The increased Ca2+ sensitivity was, however, observed mainly at low Ca2+ concentrations. Hence, it is most likely that enhanced basal activity is the main manifestation of mutation R4496C.
The mechanism by which mutation R4496C leads to increased basal channel activity is unclear. Replacing the positively charged arginine (4496) with lysine, a relatively smaller but still positively charged amino acid, did not have significant impact on basal activity. On the other hand, replacement with a neutral amino acid, alanine, led to an increase in basal activity. Mutation R4496C, in which a positive charge was replaced with an uncharged but negatively polar group, resulted in a further increase in basal activity. A more dramatic increase in basal activity was observed when R4496 was replaced by a negatively charged glutamate (Figure 5). Thus, a positive charge at this position appears to be important for stabilizing the closed state of the channel at low Ca2+ concentrations, suggesting that electrostatic interactions may be involved in channel gating.
The molecular basis of how an increased basal channel activity of RyR2 could lead to catecholaminergic PMVT is also unclear. In cardiac muscle, normal Ca2+ release is triggered by the Ca2+ current through the L-type Ca2+ channel via the Ca2+-induced Ca2+ release mechanism.8,9⇓ The amount of Ca2+ released from the SR is controlled by the level of Ca2+ entry, the activity of RyR2, and the level of Ca2+ content in the SR.31 It has been shown that moderate activation of RyR2 by channel activators such as caffeine or moderate inhibition by inhibitors such as tetracaine did not cause maintained effect on the resting cytoplasmic Ca2+ level or the stimulated Ca2+ release in cardiac myocytes.32 This is because small changes in RyR2 channel activity could be compensated by the SR Ca2+ content. For instance, a small increase in RyR2 activity due to activation by low concentrations of caffeine would result in an increase in Ca2+ leak and a decreased SR Ca2+ content, which in turn would reduce the channel activity as a result of luminal Ca2+ regulation of RyR2.33,34⇓ Thus, in analogy, an enhanced basal channel activity observed in the R4496C mutant channel would likely be compensated by a reduced SR Ca2+ content and would be unlikely to result in maintained changes in Ca2+ transients and excitation-contraction coupling. This explanation appears to be consistent with the observation that patients with catecholaminergic PMVT show structurally and functionally normal heart and normal electrocardiograms.5,6⇓
Although moderate alterations in RyR2 activity may not have maintained effects on stimulated Ca2+ release, they can influence the properties of spontaneous Ca2+ release and propagating Ca2+ waves induced by Ca2+ overload. It has been shown that an increase in RyR2 activity (for instance by low concentrations of caffeine) lowered the critical SR Ca2+ concentration at which spontaneous Ca2+ release and propagating Ca2+ waves occur, and increased the frequency of propagating Ca2+ waves.34 It is of interest to know that the effects on channel function of mutation R4496C resemble those of low concentrations of caffeine, and that HEK293 cells expressing the R4496C mutation show frequent spontaneous Ca2+ oscillations. However, whether cardiac myocytes expressing the R4496C mutation or other PMVT and ARVD2 mutations are prone to spontaneous Ca2+ oscillations has yet to be determined. It has long been recognized that spontaneous Ca2+ release can induce inward currents and delayed afterdepolarization, which could lead to triggered arrhythmia.13–16⇓⇓⇓ An increased propensity for spontaneous Ca2+ release and propagating Ca2+ waves under the condition of Ca2+ overload may underlie an arrhythmogenic mechanism of catecholaminergic PMVT.
It has now become clear that alterations in RyR2 channel function as a result of genetic defects can cause ventricular arrhythmias.2–4⇓⇓ These findings raise a question as to whether acquired dysfunction of RyR2 could lead to arrhythmia susceptibility. Ventricular arrhythmias are common in various cardiac conditions such as heart failure,35 hypertrophy,36 and ischemic heart disease.37 Cardiac myocytes or muscles of failing or hypertrophied hearts are known to be vulnerable to Ca2+ overload-induced delayed afterdepolarizations and triggered activity. It has been suggested that abnormal intracellular Ca2+ handling may give rise to arrhythmias in these settings.38 In particular, alterations in RyR2 function may be involved. It has been shown that cardiac muscle of congestive heart failure exhibited enhanced spontaneous Ca2+ release,39 and that RyR2 from failing hearts displayed an increased sensitivity to Ca2+ activation.40 Further genetic and molecular studies of RyR2-associated arrhythmias should provide a better understanding of the role of RyR2 in cardiac arrhythmias.
This work was supported by research grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, NWT, and Nunavut to S.R.W.C. D.J. was the recipient of Alex W. Church Graduate Student Award. S.R.W.C. is a Senior Scholar of the Alberta Heritage Foundation for Medical Research. The authors sincerely appreciate Dr Jonathan Lytton and Jeremy Dunn’s suggestions, discussions, and assistance in performing the single-cell Ca2+ imaging experiments and data analysis. The authors would like to thank Dr Wayne R. Giles and the Ion Channels and Transporters Group for continued support and encouragement, Dr Henk E.D.J. ter Keurs for helpful discussions, and Jeff Bolstad for critical reading of the manuscript.
Original received February 7, 2002; revision received June 21, 2002; accepted June 24, 2002.
- ↵Priori SG, Napolitano C, Tiso N, Memmi M, Vignati G, Bloise R, Sorrentino VV, Danieli GA. Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia. Circulation. 2001; 103: 196–200.
- ↵Laitinen PJ, Brown KM, Piippo K, Swan H, Devaney JM, Brahmbhatt B, Donarum EA, Marino M, Tiso N, Viitasalo M, Toivonen L, Stephan DA, Kontula K. Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation. 2001; 103: 485–490.
- ↵Tiso N, Stephan DA, Nava A, Bagattin A, Devaney JM, Stanchi F, Larderet G, Brahmbhatt B, Brown K, Bauce B, Muriago M, Basso C, Thiene G, Danieli GA, Rampazzo A. Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2). Hum Mol Genet. 2001; 10: 189–194.
- ↵Leenhardt A, Lucet V, Denjoy I, Grau F, Ngoc DD, Coumel P. Catecholaminergic polymorphic ventricular tachycardia in children: a 7-year follow-up of 21 patients. Circulation. 1995; 91: 1512–1519.
- ↵Fabiato A. Time and calcium dependence of activation and inactivation of calcium- induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. J Gen Physiol. 1985; 85: 247–289.
- ↵Bers DM. Excitation-Contraction Coupling, and Cardiac Contractile Force. Dordrecht, The Netherlands: Kluwer Academic Publisher; 1991.
- ↵Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993; 262: 740–744.
- ↵Li P, Chen SR. Molecular basis of Ca2+ activation of the mouse cardiac Ca2+ release channel (ryanodine receptor). J Gen Physiol. 2001; 118: 33–44.
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- ↵Fabiato A, Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris). 1979; 75: 463–505.
- ↵Tanna B, Welch W, Ruest L, Sutko JL, Williams AJ. Interactions of a reversible ryanoid (21-amino-9alpha-hydroxy-ryanodine) with single sheep cardiac ryanodine receptor channels. J Gen Physiol. 1998; 112: 55–69.
- ↵Franzini-Armstrong C, Protasi F. Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions. Physiol Rev. 1997; 77: 699–729.
- ↵Tong J, Oyamada H, Demaurex N, Grinstein S, McCarthy TV, MacLennan DH. Caffeine and halothane sensitivity of intracellular Ca2+ release is altered by 15 calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease. J Biol Chem. 1997; 272: 26332–26339.
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- ↵Avila G, Dirksen RT. Functional effects of central core disease mutations in the cytoplasmic region of the skeletal muscle ryanodine receptor. J Gen Physiol. 2001; 118: 277–290.
- ↵Tilgen N, Zorzato F, Halliger-Keller B, Muntoni F, Sewry C, Palmucci LM, Schneider C, Hauser E, Lehmann-Horn F, Muller CR, Treves S. Identification of four novel mutations in the C-terminal membrane spanning domain of the ryanodine receptor 1: association with central core disease and alteration of calcium homeostasis. Hum Mol Genet. 2001; 10: 2879–2887.
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