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Peptide Probe Study of the Critical Regulatory Domain of the Cardiac Ryanodine Receptor
Biochemical and Biophysical Research Communications 291, 1102–1108 (2002) doi:10.1006/bbrc.2002.6569, available online at http://www.idealibrary.com on
Peptide Probe Study of the Critical Regulatory Domain of the Cardiac Ryanodine Receptor 1 Takeshi Yamamoto* and Noriaki Ikemoto* ,† ,2 *Boston Biomedical Research Institute, Watertown, Massachusetts 02472; and †Harvard Medical School, Boston, Massachusetts 02115
Received February 4, 2002
The recently devised domain peptide probe technique was used to identify and characterize critical domains of the cardiac ryanodine receptor (RyR2). A synthetic peptide corresponding to the Gly 2460-Pro 2495 domain of the RyR2, designated DPc10, enhanced the ryanodine binding activity and increased the sensitivity of the RyR2 to activating Ca 2ⴙ: the effects that resemble the typical phenotypes of cardiac diseases. A single Arg-to-Ser mutation made in DPc10, mimicking the recently reported Arg 2474-to-Ser 2474 human mutation, abolished all of these effects that would have been produced by DPc10. On the basis of the principle of the domain peptide probe approach (see Model 1), these results indicate that the in vivo RyR2 domain corresponding to DPc10 plays a key role in the cardiac channel regulation and in the pathogenic mechanism. This domain peptide approach opens the new possibility in the studies of the regulatory and pathogenic mechanisms of the cardiac Ca 2ⴙ channel. © 2002 Elsevier Science (USA)
Key Words: cardiac ryanodine receptor; calcium channel regulation; pathogenesis of cardiac disease; point mutations; domain peptide approach.
The skeletal and cardiac ryanodine receptor (RyR) isoforms (skeletal, RyR1; cardiac, RyR2) appear to share some common features in the arrangement of the critical domains involved in Ca 2⫹ channel regulation and pathogenesis as described below. In the search of the putative domains of the RyR1 involved in the Ca 2⫹ Abbreviations used: RyR, ryanodine receptor; MH, malignant hyperthermia; CCD, central core disease; PMSF, phenylmethanesulfonyl fluoride; MES, 2-(N-morpholino) ethanesulfonic acid; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid; MOPS, 3-(N-morpholino) propanesulfonic acid; VT, ventricular tachycardia; ARVD, arrhythmogenic right ventricular dysplasia. 1 This work was supported by National Institutes of Health Grant AR 16922. 2 To whom correspondence should be addressed at Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472. Fax: 617-972-1761. E-mail: [email protected]. 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
channel regulation, a particular attention has been paid to the fact that many of the reported mutations in the RyR1 of MH and CCD patients (1–3) are localized in the two cytoplasmic domains of the RyR1. These domains must play a key role in the channel regulation, because mutations taken place in either of these domains cause abnormal modes of the Ca 2⫹ channel regulation, generally characterized as hyper-activation and hyper-sensitization effects (2– 4). In our recent publications (5– 8), we designated these domains N-terminal domain and central domain as shown in Diagram 1. The cardiac sequence corresponding to the skeletal N-terminal and central domains is relatively well conserved. Furthermore, seven out of eleven mutations of the RyR2, which have recently been reported in the cardiac disease patients (9 –11), are located in the regions corresponding to the skeletal N-terminal and central domains (see Diagram 1), suggesting that in the cardiac system too these domains play an important role in the channel regulation. In our recent studies on the RyR1 (5– 8, 12, 13), the peptide corresponding to the Leu 2442-Pro 2477 region of the central domain (DP4) was found to bind to the N-terminal region of the RyR1, and produced MH/ CCD-like hyper-activation/hyper-sensitization effects. These effects of DP4 were abolished by mutating one residue of DP4 as if it would have happened in an MH patient. These findings led us to the hypothesis (Model 1) that a close contact between the domain X (e.g., a part of the N-terminal domain) and the domain Y (a part of the central domain), namely zipping of the X-Y domain pair (X 䡠 Y), stabilizes the closed state of the Ca 2⫹ channel; while, agonists of the RyR unzip the X 䡠 Y domain pair to X ⫹ Y and opens the Ca 2⫹ channel (Model 1 (a)). A mutation taken place in either domain (e.g., mutation occurred in domain Y, making domain Y⬘) weakens the domain-domain interaction, causing unzipping (X ⫹ Y⬘), leading to the aforementioned hyper-activation and hyper-sensitization of the channel (Model 1 (b)). Domain peptide corresponding to domain Y, i.e., domain peptide y, binds to domain X in
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region weakens the inter-domain interaction and causes channel-activation effects, as in the case of skeletal muscle disease such as MH and CCD. MATERIALS AND METHODS
MODEL 1. Hypothetical model how opening and closing of the ryanodine receptor Ca 2⫹ channel are controlled by the inter-domain interactions. The model also illustrates why an appropriate domain peptide and its mutant serve as useful probes to identify and characterize the key domains of the ryanodine receptor.
competition with domain Y, resulting in the unzipped state (X 䡠 y ⫹ Y), which induces MH-like hyperactivation/hyper-sensitization effects (Model 1 (c)), as we have seen with DP4 (6). Mutation made in the domain peptide y (making domain peptide y⬘) reduces the affinity of its binding to domain X, and abolishes the activating function of domain peptide y (Model 1 (d)), as we have seen with the mutant of DP4 (6, 13). Thus, if appropriate domain peptides and their mutants are found that have the properties as shown in Model 1, the in vivo domains of the RyR2 corresponding to these domain peptides are the domains playing critical roles in the channel regulation and pathogenesis. The aim of the present study is to identify and characterize, by using the above strategy, the critical domains involved in the cardiac channel regulation and in the pathogenesis of RyR2-linked cardiac disease. For this purpose, we synthesized the peptide corresponding to the Gly 2460-Pro 2495 region (rabbit sequence), designated DPc10 (or DPc 2460 –2495), containing a potential cardiac disease-mutation site (Table I, cf. Diagram 1). We also synthesized DPc10-mut (DPc 2460 –2495-mut), in which Arg 2475-to-Ser 2475 (rabbit sequence) mutation was made mimicking the corresponding Arg 2474-to-Ser 2474 human mutation in the polymorphic VT patient (11). As shown here, DPc10 considerably enhanced the ryanodine binding activity of the RyR2 in a concentrationdependent manner particularly in a physiological range of Ca 2⫹ concentration. DPc10 also increased the sensitivity of the Ca 2⫹ channel to the RyR-agonist polylysine. DPc10-mut lost all of such effects. These results suggest that the Gly 2460-Pro 2495 region of the RyR2 represents one of the key regulatory domains and the mutation in this
Preparation. Cardiac muscle microsomes were prepared from the dog ventricles (frozen tissue purchased from Pel-Freez Biologicals) by a method of differential centrifugation as described previously (5, 14). Microsomes from the final centrifugation were homogenized in a sample solution containing 0.3 M sucrose, 0.15 M K gluconate, proteolytic enzyme inhibitors (0.1 mM PMSF, 1 g/ml leupeptin, 2.0 g/ml soybean trypsin inhibitor), 20 mM MES, pH 6.8 to a final concentration of 20 –30 mg/ml, frozen immediately in liquid N 2 and stored at ⫺78°C. Peptides used and peptide synthesis. We used the two domain peptides listed in Table I, DP10c and DP10c-mut. Peptides were synthesized on an Applied Biosystems model 431A synthesizer employing Fmoc (N-(9-fluorenyl)methoxycarbonyl) as the ␣-amino protecting group. The peptides were cleaved and de-protected with 95% trifluoroacetic acid and purified by reversed-phase high-pressure liquid chromatography. [ 3H]Ryanodine binding assay. The microsomes (1.0 –2.0 mg/ml) were incubated in 0.1 ml of a reaction solution containing 10 nM [ 3H]ryanodine (68.4 Ci/ml, Perkin Elemer Life Science), 0.15 M KCl, 1 mM BAPTA and various concentrations of CaCl 2 (to create various levels of well-defined Ca 2⫹ concentration), 20 mM MOPS, pH 7.2 for 2 h at 36°C in the presence of various concentrations of peptides and/or modulators. Samples were filtered onto glass fiber filters (Whatman GF/A) and washed twice with 7 ml (in each washing step) distilled water. Filters were then placed in scintillation vials containing 10 ml scintillation cocktail Ecoscint A and counted in a Beckman LS 3801 counter. Specific binding was calculated as the difference between the binding in the absence (total binding) and in the presence (nonspecific binding) of 10 M non-radioactive ryanodine. The assays were carried out in duplicate and each datum point was obtained by averaging the duplicates.
RESULTS Figure 1 depicts the data of ryanodine binding to the SR vesicles isolated from the dog ventricular muscle in the presence of various concentrations of DPc10 at two different Ca 2⫹ concentrations in the assay solution: A, 10 M; B, 0.2 M. As seen, DPc10 produced a significant enhancement of the ryanodine binding activity in a peptide concentration-dependent manner. The AC 50 (concentration at half-maximal activation) for the peptide activation was 70 M at 10 M Ca 2⫹ and was 100 M or higher at 0.2 M Ca 2⫹. The magnitude of ryanodine binding enhancement at a maximally activating concentration of the peptide was much larger at 0.2 M Ca 2⫹ (7.2-fold) than that at 20 M Ca 2⫹ (1.9-fold). The magnitude of activation by DPc10 at the lower Ca 2⫹
TABLE I
Amino Acid Sequences of the Cardiac Domain Peptide and Its Mutant Used in This Study DPc10 (DPc 2460–2495): DPc10-mut (DPc 2460–2495-mut):
2460 2460
GFCPDHKAAMVLFLDRVYGIEVQDFLLHLLEVGFLP 2495 GFCPDHKAAMVLFLDSគ VYGIEVQDFLLHLLEVGFLP 2495
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DIAGRAM 1. Comparison of the locations of ARVD and polymorphic VT mutation sites in the RyR2 with skeletal MH and CCD mutation sites in the RyR1.
concentration (7.2-fold) was significantly higher than the corresponding values obtained with other RyR2 agonists; e.g., 2.0 with DPc1–2 (5) and 2.1 with polylysine (15). Thus, DPc10 is the most potent activator of the RyR2 we have examined so far, although its apparent affinity is rather low. In order to test the specificity and the physiological relevance of the observed activating function of DPc10, we replaced the Arg residue of DPc10 corresponding to Arg 2475 of rabbit RyR2 with Ser, mimicking the Arg 2474to-Ser 2474 human mutation that was reported recently in the patient with polymorphic VT (11), making DPc10-mut. Importantly, this single mutation abolished the activating function that would have been present in the wild-type DPc10 (Figs. 1A and 1B). Figure 2A shows the Ca 2⫹ concentration-dependence of ryanodine binding activity of the RyR2 in the absence of added peptide (i.e., no DP), and in the presence of 100 M DPc10 or 100 M DPc10-mut. As seen in the control, no DP, ryanodine binding activity was very low in the range of Ca 2⫹ concentration below 0.2 M, but it increased sharply upon increasing the Ca 2⫹ concentration above 0.3 M, consistent with the idea that the threshold Ca 2⫹ concentration for contraction lies in the range between 0.2 M and 0.3 M (cf. Ref. 16). In the presence of 100 M DPc10, the ryanodine binding activity was much higher than the control at all Ca 2⫹ concentrations examined. However, the magnitude of peptide activation was much higher in the lower concentration range of Ca 2⫹ as we have already seen from the comparison between Figs. 1A (10 M Ca 2⫹) and 1B (0.2 M Ca 2⫹) (see Fig. 2B). Interestingly, there was a rather abrupt change in the magnitude of peptide activation above the threshold concentration of Ca 2⫹ mentioned above. As shown in Fig. 2A (cross mark), the Ca 2⫹ concentration at a half-maximum activation
(AC 50) was 0.373 M in the absence of added DPc10 and 0.347 M in the presence of 100 M DPc10, indicating that DPc10 produced hyper-sensitization effect as well as hyper-activation effect. In the presence of DPc10-mut instead of DPc10, the Ca 2⫹ concentration dependence pattern of the ryanodine binding activity was essentially identical with that of the control in the sub-threshold Ca 2⫹ concentration range (Fig. 2B). In the experiments shown in Figs. 3A and 3B, we investigated the concentration-dependence of activation of the RyR2 by polylysine (4 kDa) in the absence of added domain peptides and in the presence of 100 M DPc10 or 100 M DPc10-mut at two different Ca 2⫹ concentrations: A, 10 M; B, 0.2 M. As seen, there was a small but significant decrease in the AC 50 for polylysine-activation in the presence of DPc10 as indicated in the figures, whereas there was virtually no such change in the presence of DPc10-mut. Summarizing the above results, DPc10 produced hyper-activation effect on the RyR2 and also increased its sensitivity to activating Ca2⫹ and also to the agonist polylysine (hyper-sensitization effect). The hyperactivation/hyper-sensitization effects induced by DPc10 on the RyR2 are quite similar to the MH/CCD-like effects produced by DP4 on the RyR1 (6). Furthermore, there is an important common feature between DPc10 and DP4 in that the disease-linked mutation taken place in the peptide completely abolished these effects. These data suggest that the in vivo RyR2 domain corresponding to DPc10 plays a key role in the cardiac channel regulation and in the pathogenic mechanism. DISCUSSION Several mutations in the RyR2 have recently been found in the patients with inherited cardiac diseases,
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are involved also in the pathogenesis of the RyR-linked skeletal and cardiac muscle diseases. An earlier study by Zorzato et al. with a domainspecific anti-RyR1 antibody (17) and the oxidationinduced inter-domain cross-linking study by Hamilton and her associates (18) suggested that inter-domain interactions within the RyR molecule play an important role in the mechanism of channel regulation. Our recent domain peptide probe studies with the peptides corresponding to the portions of the aforementioned N-terminal and central domains of the RyR1 led us to
FIG. 1. (A, B) Effects of the cardiac domain peptide DPc10 (DPc 2460 –2495) and its mutant on the ryanodine binding activity of the RyR2 at 10 M Ca 2⫹ (A) and at 0.2 M Ca 2⫹ (B). DPc10 enhances ryanodine binding to the RyR2 in a concentration-dependent manner. DPc10-mut, in which Arg 2475-to-Ser 2475 (rabbit sequence) mutation was made in the domain peptide mimicking the Arg 2474-to-Ser 2474 (human sequence) mutation, abolished activating function that would have been present in DPc10. The whole set of the experiment shown in this figure was repeated at least five times for the reproducible results. Error bar represents mean ⫾ S.E.
as summarized in Diagram 1. As seen in the diagram, these mutation sites are distributed in three distinguishable regions of the RyR2. Importantly, the first and second regions from the N-terminus correspond approximately to the N-terminal and central domains of the RyR1, respectively, where most of the known MH and CCD-linked mutation sites are located (1–3). This immediately suggests that these two regions represent the domains that are critical for the regulation of both RyR1 and RyR2 Ca 2⫹ channels, and that these domains
FIG. 2. (A) The [Ca 2⫹]-dependence of the ryanodine binding activity of the RyR2 in the absence of added domain peptide (solid circle), and in the presence of 100 M DPc10 (open circle) or 100 M DPc10-mutant (triangle). Data were fitted by an equation: y ⫽ B maxK nx n/(1 ⫹ K nx n), and the AC 50 values were calculated as 1/K. The satisfactory fitting could be achieved with the n value of 6. (B) Relative activation of the ryanodine binding activity (% control) as a function of the Ca 2⫹ concentration. The whole set of the experiment shown in this figure was repeated at least five times for the reproducible results. Error bar represents mean ⫾ S.E.
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FIG. 3. (A, B) The concentration-dependence of polylysineinduced activation of the RyR2 was shifted to the left in the presence of 100 M DPc10, but not in the presence of the equivalent concentration of DPc10-mut. The extent of the left-hand shift of the concentration-dependence curve is about the same at 10 M Ca 2⫹ (A) and at 0.2 M Ca 2⫹ (B). The whole set of the experiment shown in this figure was repeated at least five times for the reproducible results. Error bar represents mean ⫾ S.E.
the hypothesis that the mode of interaction between the two domains controls the functional state of the channel. According to the hypothesis (cf. Model 1, ‘Introduction’), a tight zipping of the interacting domain (expressed in a general term as X 䡠 Y) closes the channel; upon arrival of the activation signal the domain pair is unzipped (X ⫹ Y), making the channel open (see Model 1a). A mutation in either domain weakens the inter-domain interaction increasing the tendency of being unzipped, which causes activation and leakiness of the Ca 2⫹ channel (Model 1b). This hypothesis could be tested by appropriate domain peptides. For in-
stance, a central domain peptide DP4 was found to bind to the N-terminal region of the RyR1 (8) and produce MH/CCD-like activation effects (6), consistent with the prediction from the hypothesis that domain peptide y will bind to domain X and unzip the X 䡠 Y domain pair to X 䡠 y ⫹ Y and will cause channel activation (Model 1c). Furthermore, the activation effect of DP4 was diminished by making an MH-like mutation in the peptide (6, 13), again consistent with the prediction that the mutation in the domain peptide y, as it happened in domain Y, will reduce the affinity of its binding to domain X, resulting in the loss of activating function of domain peptide y. The most important aspect of the present study is the finding that a new cardiac domain peptide (DPc10 or DPc 2460 –2495) corresponding to the Gly 2460-Pro 2495 region of the rabbit RyR2 (equivalent to the Gly 2459Pro 2494 region of the human RyR2) produced significant activation of the RyR2 Ca 2⫹ channel. As shown in Figs. 1 and 2, the magnitude of activation is quite significant especially at sub-threshold (relaxing) Ca 2⫹ concentrations. This indicates that the activation effect of the peptide has a quite significant impact on the cardiac channel regulation especially in the Ca 2⫹ concentration range, where the muscle is supposed to relax. Another interesting finding in this study is that DPc10 produced an appreciable decrease in the AC 50 for the activation of the RyR2 by polylysine (Fig. 3). In most of the MH and CCD muscle function tests caffeine has been used as the RyR-agonist. However, we regard polylysine as the better agonist, because it binds specifically to the RyR moiety of the SR with a very high affinity (19) and activates both RyR1 and RyR2 channels (15). The Arg-to-Ser mutation made in the peptide mimicking the Arg 2474-to-Ser 2474 human polymorphic VT mutation completely abolished both of these hyperactivation and hyper-sensitization effects seen with the DPc10. According to the hypothesis shown in Model 1, these findings suggest that the in vivo domain of the RyR2 corresponding to DPc10 (i.e., the Gly 2460-Pro 2495 region of the RyR2) plays an important role in the cardiac channel regulation, and that the mutation occurring in this domain will produce hyper-activation and hyper-sensitization effects on the channel particularly at relaxing concentrations of Ca 2⫹. Based upon the present data and hypothesis, we predict that the mutation occurred in the Gly 2460-Pro 2495 domain of the RyR2 not only makes the Ca 2⫹ channel leaky but also increases its sensitivity to various pharmacological agonists, which lead to the cytoplasmic Ca 2⫹ overload as widely seen in cardiac diseases. These predicted properties of the diseased RyR2 Ca 2⫹ channel are exactly identical to the phenotypes reported in the RyR1 Ca 2⫹ channel of the MH/CCD patients. In this context it is interesting to point out another common feature existing in the MH/CCD and the ARVD/polymorphic VT accompanied with RyR2 mutation; that is, the ef-
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fects of the RyR-linked mutations are unseen in normal conditions, but they become manifested upon anesthesia (MH/CCD) or exercise (ARVD/polymorphic VT, Refs. 9 –11). In order to firmly establish the cause-result relationship between the mutation and the channel dysfunction, however, it will be necessary to express the mutated RyR2 in an appropriate expression system and to examine whether the Arg2474-to-Ser 2474 human mutation in fact produces hyper-activation/hyper-sensitization effects on the RyR2 Ca 2⫹ channel. Such expression studies were already done in the case of skeletal muscle disease model. Tong et al. (20) expressed fifteen human MH/CCD mutations and the wild-type in the HEK-293 cells and demonstrated that those mutations in fact cause an increased response of the RyR Ca 2⫹ channel to caffeine. Until similar expression studies are carried out in the cardiac disease model, the domain peptide probe technique is the sole method that permits us to test the physiological role of the designated RyR2 domain and the pathogenic impact of the potential mutation in this domain. The domain peptide probe approach presented here has an additional and unique merit of its own as follows. In the case of cardiac disease, really critical mutations will tend to remain uncovered, because the survival rate of the patients with such mutations would be extremely low. Presumably this is the major reason why fewer disease-linked cardiac mutations have been found to the date. Then, the unique advantage with the peptide probe study is that the method will permit us to identify such ‘critical-butundetectable’ mutation sites and domains. For example, some of amino acid residues of the RyR2 corresponding to the skeletal MH/CCD mutation sites might represent the critical-but-undetectable mutation sites for some fatal cardiac diseases. It would be possible to reveal such sites and domains by making the corresponding cardiac domain peptides and by investigating the effect of the mutations made in these peptides. The results obtained by the peptide probe studies would then be tested by the appropriate expression studies as mentioned above. In conclusion, the present data suggest that the in vivo RyR2 domain corresponding to DPc10 plays a key role in the cardiac channel regulation and in the pathogenic mechanism. The extension of this approach to a larger number of selected domains (e.g., those domains containing the reported cardiac disease mutations and the RyR2 domains corresponding to the critical domains of the RyR1 described above) should permit the better understanding of the regulatory and pathogenic mechanisms of the cardiac Ca 2⫹ channel regulation. The present study suggests that one of the most promising strategies to resolve the mechanism of cardiac Ca 2⫹ channel regulation and the pathogenic mechanism of the RyR2-linked cardiac diseases will be first to
identify and characterize the critical domains by the domain peptide probes and then to test physiologic impacts of mutations taken place in these domains in an appropriate expression system. ACKNOWLEDGMENTS We thank Dr. Renne C. Lu, Dr. Paul Leavis, Ming-Jen Tsay, and Elizabeth Gowell for their help in the synthesis and purification of the peptides.
REFERENCES 1. McCarthy, T. V., Quane, K. A., and Lynch, P. J. (2000) Ryanodine receptor mutations in malignant hyperthermia and central core disease. Hum. Mutat. 15, 410 – 417. 2. Loke, J., and MacLennan, D. H. (1998) Malignant hyperthermia and central core disease: Disorders of Ca 2⫹ release channels. Am. J. Med. 104, 470 – 486. 3. Jurkat-Rott, K., McCarthy, T., and Lehmann-Horn, F. (2000) Genetics and pathogenesis of malignant hyperthermia. Muscle Nerve 23, 4 –17. 4. Mickelson, J. R., and Louis, C. F. (1996) Malignant hyperthermia: Excitation-contraction coupling, Ca 2⫹ release channel, and cell Ca 2⫹ regulation defects. Physiological Reviews 76, 537–592. 5. El-Hayek, R., Saiki, Y., Yamamoto, T., and Ikemoto, N. (1999) Identification of calcium release-triggering and blocking regions of the II–III loop of the skeletal muscle dihydropyridine receptor. J. Biol. Chem. 274, 33341–33347. 6. Yamamoto, T., El-Hayek, R., and Ikemoto, N. (2000) Postulated role of interdomain interaction within the ryanodine receptor in Ca 2⫹ channel regulation. J. Biol. Chem. 275, 11618 –11625. 7. Ikemoto, N., and Yamamoto, T. (2000) Postulated role of interdomain interaction within the ryanodine receptor in Ca 2⫹ channel regulation. Trends. Cardiovasc. Med. 10, 310 –316. 8. Yamamoto, T., and Ikemoto, N. (2002) Spectroscopic monitoring of local conformational changes during the intramolecular domain-domain interaction of the ryanodine receptor. Biochemistry 41, 1492–1501. 9. Laitinen, P. J., Brown, K. M., Piippo, K., Swan, H., Devaney, J. M., Brahmbhatt, B., Donarum, E. A., Marino, M., Tiso, N., Viitasalo, M., Toivonen, L., Stephan, D. A., and Kontula, K. (2001) Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic ventricular tachycardia. Circulation 103, 485– 490. 10. Tiso, N., Stephan, D. A., Nava, A., Bagattin, A., Devaney, J. M., Stanchi, F., Larderet, G., Brahmbhatt, B., Brown, K., Bauce, B., Muriago, M., Basso, C., Thiene, G., Danieli, G. A., and Rampazzo, A. (2001) Identification of mutations in the cardiac ryanodine receptor gene in families affected with arrhythmogenic right ventricular cardiomyopathy type 2 (ARVD2). Hum. Mol. Genet. 10, 189 –194. 11. Priori, S. G., Napolitano, C., Tiso, N., Memmi, M., Vignati, G., Bloise, R., Sorrentino, V. V., and Danieli, G. A. (2001) Mutations in the cardiac ryanodine receptor gene (hRyR2) underlie catecholaminergic polymorphic ventricular tachycardia. Circulation 103, 196 –200. 12. Lamb, G. D., Posterino, G. S., Yamamoto, T., and Ikemoto, N. (2001) Effects of a domain peptide of the ryanodine receptor on Ca 2⫹ release in skinned skeletal muscle fibers. Am. J. Physiol. Cell Physiol. 281, C207–C214. 13. Shtifman, A., Ward, C. W., Yamamoto, T., Wang, J., Olbinsky, B., Valdivia, H. H., Ikemoto, N., and Schneider, M. F. (2002) Interdomain interactions within ryanodine receptors regulate
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of malignant hyperthermia domain in the regulation of Ca 2⫹ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J. Biol. Chem. 271, 22759 –22763. 18. Wu, Y., Aghdasi, B., Dou, S. J., Zhang, J. Z., Liu, S. Q., and Hamilton, S. L. (1997) Functional interactions between cytoplasmic domains of the skeletal muscle Ca 2⫹ release channel. J. Biol. Chem. 272, 25051–25061. 19. Cifuentes, M. E., Ronjat, M., and Ikemoto, N. (1989) Polylysine induces a rapid Ca 2⫹ release from sarcoplasmic reticulum vesicles by mediation of its binding to the foot protein. Arch. Biochem. Biophys. 273, 554 –561. 20. Tong, J., McCarthy, T. V., and MacLennan, D. H. (1999) HEK293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca 2⫹ store that is responsive to stop-flow medium changes and insensitive to caffeine and ryanodine. J. Biol. Chem. 274, 693–702.
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Peptide Probe Study of the Critical Regulatory Domain of the Cardiac Ryanodine Receptor 1 Takeshi Yamamoto* and Noriaki Ikemoto* ,† ,2 *Boston Biomedical Research Institute, Watertown, Massachusetts 02472; and †Harvard Medical School, Boston, Massachusetts 02115
Received February 4, 2002
The recently devised domain peptide probe technique was used to identify and characterize critical domains of the cardiac ryanodine receptor (RyR2). A synthetic peptide corresponding to the Gly 2460-Pro 2495 domain of the RyR2, designated DPc10, enhanced the ryanodine binding activity and increased the sensitivity of the RyR2 to activating Ca 2ⴙ: the effects that resemble the typical phenotypes of cardiac diseases. A single Arg-to-Ser mutation made in DPc10, mimicking the recently reported Arg 2474-to-Ser 2474 human mutation, abolished all of these effects that would have been produced by DPc10. On the basis of the principle of the domain peptide probe approach (see Model 1), these results indicate that the in vivo RyR2 domain corresponding to DPc10 plays a key role in the cardiac channel regulation and in the pathogenic mechanism. This domain peptide approach opens the new possibility in the studies of the regulatory and pathogenic mechanisms of the cardiac Ca 2ⴙ channel. © 2002 Elsevier Science (USA)
Key Words: cardiac ryanodine receptor; calcium channel regulation; pathogenesis of cardiac disease; point mutations; domain peptide approach.
The skeletal and cardiac ryanodine receptor (RyR) isoforms (skeletal, RyR1; cardiac, RyR2) appear to share some common features in the arrangement of the critical domains involved in Ca 2⫹ channel regulation and pathogenesis as described below. In the search of the putative domains of the RyR1 involved in the Ca 2⫹ Abbreviations used: RyR, ryanodine receptor; MH, malignant hyperthermia; CCD, central core disease; PMSF, phenylmethanesulfonyl fluoride; MES, 2-(N-morpholino) ethanesulfonic acid; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid; MOPS, 3-(N-morpholino) propanesulfonic acid; VT, ventricular tachycardia; ARVD, arrhythmogenic right ventricular dysplasia. 1 This work was supported by National Institutes of Health Grant AR 16922. 2 To whom correspondence should be addressed at Boston Biomedical Research Institute, 64 Grove St., Watertown, MA 02472. Fax: 617-972-1761. E-mail: [email protected]. 0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
channel regulation, a particular attention has been paid to the fact that many of the reported mutations in the RyR1 of MH and CCD patients (1–3) are localized in the two cytoplasmic domains of the RyR1. These domains must play a key role in the channel regulation, because mutations taken place in either of these domains cause abnormal modes of the Ca 2⫹ channel regulation, generally characterized as hyper-activation and hyper-sensitization effects (2– 4). In our recent publications (5– 8), we designated these domains N-terminal domain and central domain as shown in Diagram 1. The cardiac sequence corresponding to the skeletal N-terminal and central domains is relatively well conserved. Furthermore, seven out of eleven mutations of the RyR2, which have recently been reported in the cardiac disease patients (9 –11), are located in the regions corresponding to the skeletal N-terminal and central domains (see Diagram 1), suggesting that in the cardiac system too these domains play an important role in the channel regulation. In our recent studies on the RyR1 (5– 8, 12, 13), the peptide corresponding to the Leu 2442-Pro 2477 region of the central domain (DP4) was found to bind to the N-terminal region of the RyR1, and produced MH/ CCD-like hyper-activation/hyper-sensitization effects. These effects of DP4 were abolished by mutating one residue of DP4 as if it would have happened in an MH patient. These findings led us to the hypothesis (Model 1) that a close contact between the domain X (e.g., a part of the N-terminal domain) and the domain Y (a part of the central domain), namely zipping of the X-Y domain pair (X 䡠 Y), stabilizes the closed state of the Ca 2⫹ channel; while, agonists of the RyR unzip the X 䡠 Y domain pair to X ⫹ Y and opens the Ca 2⫹ channel (Model 1 (a)). A mutation taken place in either domain (e.g., mutation occurred in domain Y, making domain Y⬘) weakens the domain-domain interaction, causing unzipping (X ⫹ Y⬘), leading to the aforementioned hyper-activation and hyper-sensitization of the channel (Model 1 (b)). Domain peptide corresponding to domain Y, i.e., domain peptide y, binds to domain X in
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region weakens the inter-domain interaction and causes channel-activation effects, as in the case of skeletal muscle disease such as MH and CCD. MATERIALS AND METHODS
MODEL 1. Hypothetical model how opening and closing of the ryanodine receptor Ca 2⫹ channel are controlled by the inter-domain interactions. The model also illustrates why an appropriate domain peptide and its mutant serve as useful probes to identify and characterize the key domains of the ryanodine receptor.
competition with domain Y, resulting in the unzipped state (X 䡠 y ⫹ Y), which induces MH-like hyperactivation/hyper-sensitization effects (Model 1 (c)), as we have seen with DP4 (6). Mutation made in the domain peptide y (making domain peptide y⬘) reduces the affinity of its binding to domain X, and abolishes the activating function of domain peptide y (Model 1 (d)), as we have seen with the mutant of DP4 (6, 13). Thus, if appropriate domain peptides and their mutants are found that have the properties as shown in Model 1, the in vivo domains of the RyR2 corresponding to these domain peptides are the domains playing critical roles in the channel regulation and pathogenesis. The aim of the present study is to identify and characterize, by using the above strategy, the critical domains involved in the cardiac channel regulation and in the pathogenesis of RyR2-linked cardiac disease. For this purpose, we synthesized the peptide corresponding to the Gly 2460-Pro 2495 region (rabbit sequence), designated DPc10 (or DPc 2460 –2495), containing a potential cardiac disease-mutation site (Table I, cf. Diagram 1). We also synthesized DPc10-mut (DPc 2460 –2495-mut), in which Arg 2475-to-Ser 2475 (rabbit sequence) mutation was made mimicking the corresponding Arg 2474-to-Ser 2474 human mutation in the polymorphic VT patient (11). As shown here, DPc10 considerably enhanced the ryanodine binding activity of the RyR2 in a concentrationdependent manner particularly in a physiological range of Ca 2⫹ concentration. DPc10 also increased the sensitivity of the Ca 2⫹ channel to the RyR-agonist polylysine. DPc10-mut lost all of such effects. These results suggest that the Gly 2460-Pro 2495 region of the RyR2 represents one of the key regulatory domains and the mutation in this
Preparation. Cardiac muscle microsomes were prepared from the dog ventricles (frozen tissue purchased from Pel-Freez Biologicals) by a method of differential centrifugation as described previously (5, 14). Microsomes from the final centrifugation were homogenized in a sample solution containing 0.3 M sucrose, 0.15 M K gluconate, proteolytic enzyme inhibitors (0.1 mM PMSF, 1 g/ml leupeptin, 2.0 g/ml soybean trypsin inhibitor), 20 mM MES, pH 6.8 to a final concentration of 20 –30 mg/ml, frozen immediately in liquid N 2 and stored at ⫺78°C. Peptides used and peptide synthesis. We used the two domain peptides listed in Table I, DP10c and DP10c-mut. Peptides were synthesized on an Applied Biosystems model 431A synthesizer employing Fmoc (N-(9-fluorenyl)methoxycarbonyl) as the ␣-amino protecting group. The peptides were cleaved and de-protected with 95% trifluoroacetic acid and purified by reversed-phase high-pressure liquid chromatography. [ 3H]Ryanodine binding assay. The microsomes (1.0 –2.0 mg/ml) were incubated in 0.1 ml of a reaction solution containing 10 nM [ 3H]ryanodine (68.4 Ci/ml, Perkin Elemer Life Science), 0.15 M KCl, 1 mM BAPTA and various concentrations of CaCl 2 (to create various levels of well-defined Ca 2⫹ concentration), 20 mM MOPS, pH 7.2 for 2 h at 36°C in the presence of various concentrations of peptides and/or modulators. Samples were filtered onto glass fiber filters (Whatman GF/A) and washed twice with 7 ml (in each washing step) distilled water. Filters were then placed in scintillation vials containing 10 ml scintillation cocktail Ecoscint A and counted in a Beckman LS 3801 counter. Specific binding was calculated as the difference between the binding in the absence (total binding) and in the presence (nonspecific binding) of 10 M non-radioactive ryanodine. The assays were carried out in duplicate and each datum point was obtained by averaging the duplicates.
RESULTS Figure 1 depicts the data of ryanodine binding to the SR vesicles isolated from the dog ventricular muscle in the presence of various concentrations of DPc10 at two different Ca 2⫹ concentrations in the assay solution: A, 10 M; B, 0.2 M. As seen, DPc10 produced a significant enhancement of the ryanodine binding activity in a peptide concentration-dependent manner. The AC 50 (concentration at half-maximal activation) for the peptide activation was 70 M at 10 M Ca 2⫹ and was 100 M or higher at 0.2 M Ca 2⫹. The magnitude of ryanodine binding enhancement at a maximally activating concentration of the peptide was much larger at 0.2 M Ca 2⫹ (7.2-fold) than that at 20 M Ca 2⫹ (1.9-fold). The magnitude of activation by DPc10 at the lower Ca 2⫹
TABLE I
Amino Acid Sequences of the Cardiac Domain Peptide and Its Mutant Used in This Study DPc10 (DPc 2460–2495): DPc10-mut (DPc 2460–2495-mut):
2460 2460
GFCPDHKAAMVLFLDRVYGIEVQDFLLHLLEVGFLP 2495 GFCPDHKAAMVLFLDSគ VYGIEVQDFLLHLLEVGFLP 2495
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DIAGRAM 1. Comparison of the locations of ARVD and polymorphic VT mutation sites in the RyR2 with skeletal MH and CCD mutation sites in the RyR1.
concentration (7.2-fold) was significantly higher than the corresponding values obtained with other RyR2 agonists; e.g., 2.0 with DPc1–2 (5) and 2.1 with polylysine (15). Thus, DPc10 is the most potent activator of the RyR2 we have examined so far, although its apparent affinity is rather low. In order to test the specificity and the physiological relevance of the observed activating function of DPc10, we replaced the Arg residue of DPc10 corresponding to Arg 2475 of rabbit RyR2 with Ser, mimicking the Arg 2474to-Ser 2474 human mutation that was reported recently in the patient with polymorphic VT (11), making DPc10-mut. Importantly, this single mutation abolished the activating function that would have been present in the wild-type DPc10 (Figs. 1A and 1B). Figure 2A shows the Ca 2⫹ concentration-dependence of ryanodine binding activity of the RyR2 in the absence of added peptide (i.e., no DP), and in the presence of 100 M DPc10 or 100 M DPc10-mut. As seen in the control, no DP, ryanodine binding activity was very low in the range of Ca 2⫹ concentration below 0.2 M, but it increased sharply upon increasing the Ca 2⫹ concentration above 0.3 M, consistent with the idea that the threshold Ca 2⫹ concentration for contraction lies in the range between 0.2 M and 0.3 M (cf. Ref. 16). In the presence of 100 M DPc10, the ryanodine binding activity was much higher than the control at all Ca 2⫹ concentrations examined. However, the magnitude of peptide activation was much higher in the lower concentration range of Ca 2⫹ as we have already seen from the comparison between Figs. 1A (10 M Ca 2⫹) and 1B (0.2 M Ca 2⫹) (see Fig. 2B). Interestingly, there was a rather abrupt change in the magnitude of peptide activation above the threshold concentration of Ca 2⫹ mentioned above. As shown in Fig. 2A (cross mark), the Ca 2⫹ concentration at a half-maximum activation
(AC 50) was 0.373 M in the absence of added DPc10 and 0.347 M in the presence of 100 M DPc10, indicating that DPc10 produced hyper-sensitization effect as well as hyper-activation effect. In the presence of DPc10-mut instead of DPc10, the Ca 2⫹ concentration dependence pattern of the ryanodine binding activity was essentially identical with that of the control in the sub-threshold Ca 2⫹ concentration range (Fig. 2B). In the experiments shown in Figs. 3A and 3B, we investigated the concentration-dependence of activation of the RyR2 by polylysine (4 kDa) in the absence of added domain peptides and in the presence of 100 M DPc10 or 100 M DPc10-mut at two different Ca 2⫹ concentrations: A, 10 M; B, 0.2 M. As seen, there was a small but significant decrease in the AC 50 for polylysine-activation in the presence of DPc10 as indicated in the figures, whereas there was virtually no such change in the presence of DPc10-mut. Summarizing the above results, DPc10 produced hyper-activation effect on the RyR2 and also increased its sensitivity to activating Ca2⫹ and also to the agonist polylysine (hyper-sensitization effect). The hyperactivation/hyper-sensitization effects induced by DPc10 on the RyR2 are quite similar to the MH/CCD-like effects produced by DP4 on the RyR1 (6). Furthermore, there is an important common feature between DPc10 and DP4 in that the disease-linked mutation taken place in the peptide completely abolished these effects. These data suggest that the in vivo RyR2 domain corresponding to DPc10 plays a key role in the cardiac channel regulation and in the pathogenic mechanism. DISCUSSION Several mutations in the RyR2 have recently been found in the patients with inherited cardiac diseases,
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are involved also in the pathogenesis of the RyR-linked skeletal and cardiac muscle diseases. An earlier study by Zorzato et al. with a domainspecific anti-RyR1 antibody (17) and the oxidationinduced inter-domain cross-linking study by Hamilton and her associates (18) suggested that inter-domain interactions within the RyR molecule play an important role in the mechanism of channel regulation. Our recent domain peptide probe studies with the peptides corresponding to the portions of the aforementioned N-terminal and central domains of the RyR1 led us to
FIG. 1. (A, B) Effects of the cardiac domain peptide DPc10 (DPc 2460 –2495) and its mutant on the ryanodine binding activity of the RyR2 at 10 M Ca 2⫹ (A) and at 0.2 M Ca 2⫹ (B). DPc10 enhances ryanodine binding to the RyR2 in a concentration-dependent manner. DPc10-mut, in which Arg 2475-to-Ser 2475 (rabbit sequence) mutation was made in the domain peptide mimicking the Arg 2474-to-Ser 2474 (human sequence) mutation, abolished activating function that would have been present in DPc10. The whole set of the experiment shown in this figure was repeated at least five times for the reproducible results. Error bar represents mean ⫾ S.E.
as summarized in Diagram 1. As seen in the diagram, these mutation sites are distributed in three distinguishable regions of the RyR2. Importantly, the first and second regions from the N-terminus correspond approximately to the N-terminal and central domains of the RyR1, respectively, where most of the known MH and CCD-linked mutation sites are located (1–3). This immediately suggests that these two regions represent the domains that are critical for the regulation of both RyR1 and RyR2 Ca 2⫹ channels, and that these domains
FIG. 2. (A) The [Ca 2⫹]-dependence of the ryanodine binding activity of the RyR2 in the absence of added domain peptide (solid circle), and in the presence of 100 M DPc10 (open circle) or 100 M DPc10-mutant (triangle). Data were fitted by an equation: y ⫽ B maxK nx n/(1 ⫹ K nx n), and the AC 50 values were calculated as 1/K. The satisfactory fitting could be achieved with the n value of 6. (B) Relative activation of the ryanodine binding activity (% control) as a function of the Ca 2⫹ concentration. The whole set of the experiment shown in this figure was repeated at least five times for the reproducible results. Error bar represents mean ⫾ S.E.
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FIG. 3. (A, B) The concentration-dependence of polylysineinduced activation of the RyR2 was shifted to the left in the presence of 100 M DPc10, but not in the presence of the equivalent concentration of DPc10-mut. The extent of the left-hand shift of the concentration-dependence curve is about the same at 10 M Ca 2⫹ (A) and at 0.2 M Ca 2⫹ (B). The whole set of the experiment shown in this figure was repeated at least five times for the reproducible results. Error bar represents mean ⫾ S.E.
the hypothesis that the mode of interaction between the two domains controls the functional state of the channel. According to the hypothesis (cf. Model 1, ‘Introduction’), a tight zipping of the interacting domain (expressed in a general term as X 䡠 Y) closes the channel; upon arrival of the activation signal the domain pair is unzipped (X ⫹ Y), making the channel open (see Model 1a). A mutation in either domain weakens the inter-domain interaction increasing the tendency of being unzipped, which causes activation and leakiness of the Ca 2⫹ channel (Model 1b). This hypothesis could be tested by appropriate domain peptides. For in-
stance, a central domain peptide DP4 was found to bind to the N-terminal region of the RyR1 (8) and produce MH/CCD-like activation effects (6), consistent with the prediction from the hypothesis that domain peptide y will bind to domain X and unzip the X 䡠 Y domain pair to X 䡠 y ⫹ Y and will cause channel activation (Model 1c). Furthermore, the activation effect of DP4 was diminished by making an MH-like mutation in the peptide (6, 13), again consistent with the prediction that the mutation in the domain peptide y, as it happened in domain Y, will reduce the affinity of its binding to domain X, resulting in the loss of activating function of domain peptide y. The most important aspect of the present study is the finding that a new cardiac domain peptide (DPc10 or DPc 2460 –2495) corresponding to the Gly 2460-Pro 2495 region of the rabbit RyR2 (equivalent to the Gly 2459Pro 2494 region of the human RyR2) produced significant activation of the RyR2 Ca 2⫹ channel. As shown in Figs. 1 and 2, the magnitude of activation is quite significant especially at sub-threshold (relaxing) Ca 2⫹ concentrations. This indicates that the activation effect of the peptide has a quite significant impact on the cardiac channel regulation especially in the Ca 2⫹ concentration range, where the muscle is supposed to relax. Another interesting finding in this study is that DPc10 produced an appreciable decrease in the AC 50 for the activation of the RyR2 by polylysine (Fig. 3). In most of the MH and CCD muscle function tests caffeine has been used as the RyR-agonist. However, we regard polylysine as the better agonist, because it binds specifically to the RyR moiety of the SR with a very high affinity (19) and activates both RyR1 and RyR2 channels (15). The Arg-to-Ser mutation made in the peptide mimicking the Arg 2474-to-Ser 2474 human polymorphic VT mutation completely abolished both of these hyperactivation and hyper-sensitization effects seen with the DPc10. According to the hypothesis shown in Model 1, these findings suggest that the in vivo domain of the RyR2 corresponding to DPc10 (i.e., the Gly 2460-Pro 2495 region of the RyR2) plays an important role in the cardiac channel regulation, and that the mutation occurring in this domain will produce hyper-activation and hyper-sensitization effects on the channel particularly at relaxing concentrations of Ca 2⫹. Based upon the present data and hypothesis, we predict that the mutation occurred in the Gly 2460-Pro 2495 domain of the RyR2 not only makes the Ca 2⫹ channel leaky but also increases its sensitivity to various pharmacological agonists, which lead to the cytoplasmic Ca 2⫹ overload as widely seen in cardiac diseases. These predicted properties of the diseased RyR2 Ca 2⫹ channel are exactly identical to the phenotypes reported in the RyR1 Ca 2⫹ channel of the MH/CCD patients. In this context it is interesting to point out another common feature existing in the MH/CCD and the ARVD/polymorphic VT accompanied with RyR2 mutation; that is, the ef-
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fects of the RyR-linked mutations are unseen in normal conditions, but they become manifested upon anesthesia (MH/CCD) or exercise (ARVD/polymorphic VT, Refs. 9 –11). In order to firmly establish the cause-result relationship between the mutation and the channel dysfunction, however, it will be necessary to express the mutated RyR2 in an appropriate expression system and to examine whether the Arg2474-to-Ser 2474 human mutation in fact produces hyper-activation/hyper-sensitization effects on the RyR2 Ca 2⫹ channel. Such expression studies were already done in the case of skeletal muscle disease model. Tong et al. (20) expressed fifteen human MH/CCD mutations and the wild-type in the HEK-293 cells and demonstrated that those mutations in fact cause an increased response of the RyR Ca 2⫹ channel to caffeine. Until similar expression studies are carried out in the cardiac disease model, the domain peptide probe technique is the sole method that permits us to test the physiological role of the designated RyR2 domain and the pathogenic impact of the potential mutation in this domain. The domain peptide probe approach presented here has an additional and unique merit of its own as follows. In the case of cardiac disease, really critical mutations will tend to remain uncovered, because the survival rate of the patients with such mutations would be extremely low. Presumably this is the major reason why fewer disease-linked cardiac mutations have been found to the date. Then, the unique advantage with the peptide probe study is that the method will permit us to identify such ‘critical-butundetectable’ mutation sites and domains. For example, some of amino acid residues of the RyR2 corresponding to the skeletal MH/CCD mutation sites might represent the critical-but-undetectable mutation sites for some fatal cardiac diseases. It would be possible to reveal such sites and domains by making the corresponding cardiac domain peptides and by investigating the effect of the mutations made in these peptides. The results obtained by the peptide probe studies would then be tested by the appropriate expression studies as mentioned above. In conclusion, the present data suggest that the in vivo RyR2 domain corresponding to DPc10 plays a key role in the cardiac channel regulation and in the pathogenic mechanism. The extension of this approach to a larger number of selected domains (e.g., those domains containing the reported cardiac disease mutations and the RyR2 domains corresponding to the critical domains of the RyR1 described above) should permit the better understanding of the regulatory and pathogenic mechanisms of the cardiac Ca 2⫹ channel regulation. The present study suggests that one of the most promising strategies to resolve the mechanism of cardiac Ca 2⫹ channel regulation and the pathogenic mechanism of the RyR2-linked cardiac diseases will be first to
identify and characterize the critical domains by the domain peptide probes and then to test physiologic impacts of mutations taken place in these domains in an appropriate expression system. ACKNOWLEDGMENTS We thank Dr. Renne C. Lu, Dr. Paul Leavis, Ming-Jen Tsay, and Elizabeth Gowell for their help in the synthesis and purification of the peptides.
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