Postulated role of interdomain interaction within the ryanodine receptor in Ca(2+) channel regulation.

Localized distribution of malignant hyperthermia (MH) and central core disease (CCD) mutations in N-terminal and central domains of the ryanodine receptor suggests that the interaction between these domains may be involved in Ca(2+) channel regulation. To test this hypothesis, we investigated the effects of a new synthetic domain peptide DP4 corresponding to the Leu(2442)-Pro(2477) region of the central domain. DP4 enhanced ryanodine binding and induced a rapid Ca(2+) release. The concentration for half-maximal activation by agonists was considerably reduced in the presence of DP4. These effects of DP4 are analogous to the functional modifications of the ryanodine receptor caused by MH/CCD mutations (viz. hyperactivation of the channel and hypersensitization of the channel to agonists). Replacement of Arg of DP4 with Cys, mimicking the in vivo Arg(2458)-to-Cys(2458) mutation, abolished the activating effects of DP4. An N-terminal domain peptide DP1 (El-Hayek, R., Saiki, Y., Yamamoto, T., and Ikemoto, N. (1999) J. Biol. Chem. 274, 33341-33347) shows similar activation/sensitization effects. The addition of both DP4 and DP1 produced mutual interference of their activating functions. We tentatively propose that contact between the two (N-terminal and central) domains closes the channel, whereas removal of the contact by these domain peptides or by MH/CCD mutations de-blocks the channel, resulting in hyperactivation/hyper-sensitization effects.

Skeletal muscle-type excitation-contraction coupling is triggered presumably by the voltage-dependent binding of the dihydropyridine receptor ␣1 subunit II-III loop to the Ca 2ϩ release channel protein of the SR, also referred as ryanodine receptor (RyR) 1 (1)(2)(3)(4)(5)(6)(7). This signal is transmitted to the transmembrane channel domain to activate the channel by a yetunidentified mechanism. The RyR Ca 2ϩ channel is also activated by a variety of types of chemical reagents and polypeptides (8 -10), most of which are presumably acting on the bulky cytoplasmic domain of the RyR. Thus, there must be intricate mechanisms, which mediate the transmission of various types of signals received at various sites of the RyR to its channel domain. However, very little information is available regarding the putative intra-molecular signal transduction mechanism.
The concept that interactions among several regions of the RyR might be involved in the intra-molecular signal transduction mechanism is suggested from several pieces of evidence as follows. First, several regulatory domains have been deduced from the locations of the putative binding sites for calmodulin (11)(12)(13), FK506-binding protein (14,15), activating Ca 2ϩ (16), the sites for phosphorylation (17), and the mutation sites occurring in MH susceptible animal and in MH and CCD-susceptible human patients (18 -26). An antibody raised against the N-terminal region altered the Ca 2ϩ -dependence of channel gating, and the construct containing the epitope to this antibody reacted with other regions of the RyR (27). Chemical modification, under conditions that would produce oxidation-induced channel activation in fact altered the intra-molecular crosslinking pattern (28,29).
We have paid a particular attention to an interesting relation existing between the distribution of the mutation sites of the RyR and the altered function of the RyR in MH and CCD. Namely, most of the reported mutations are localized in either of the two restricted regions of the RyR: one, the domain including several mutation sites in the Cys 35 -Arg 614 region (designated as N-terminal domain to facilitate discussion), and the other, the domain including the Arg 2168 -Arg 2458 region (designated as central domain). Furthermore, mutations at different positions of these domains produce the same type of functional modification characterized by two common features: (a) hyperactivation of the Ca 2ϩ channel and (b) an increase in the sensitivity of the RyR to agonists (26, 30 -34). The most feasible hypothesis to account for such unique situations would be as follows. The interdomain interactions between the N-terminal domain and the central domain (defined above) may play an important role in the channel regulation mechanism. Thus, mutations in either domain produce the same type of functional abnormality, since the interdomain interaction rather than the domain property itself is the critical factor.
To investigate the above hypothesis, we recently embarked on a series of studies using synthetic peptides corresponding to various regions of the N-terminal domain or the central domain described above. The strategy of such peptide probe studies is as follows. Suppose that x-region is interacting with y-region serving as a regulatory mechanism. Then, a synthetic peptide corresponding to the x-region, i.e. peptide x, would bind to the y-region, producing interference with the in vivo x-y domaindomain interaction. If the x-y interdomain interaction is playing a key role in channel regulation, then peptide x would produce appreciable effects on the mode of regulation of the RyR channel. Such peptide probes will not only permit us to identify the putative regulatory domains, but also permit us to characterize the mode of channel regulation. Our recent study revealed several interesting RyR-domain peptides (35). For instance, synthetic peptide corresponding to a portion of the N-terminal domain, DP1 (see Table I), activated the RyR2 significantly and the RyR1 to a lesser extent. Another N-terminal domain peptide DP3 was without effect on both RyR1 and RyR2.
In the present study, we selected two new regions from the central domain defined above. One is the Leu 2442 -Pro 2477 region of the RyR1, which contains the C-terminal region of the central domain and the putative FK506-binding protein binding segment (36), and a peptide, DP4, corresponding to this region, was synthesized. The other is the Val 2149 -Ile 2185 region, which contains an N-terminal region of the central domain, and a peptide, DP5, corresponding to this region, was synthesized. DP4 was found to exert the most conspicuous effect on the RyR1 among the domain peptides we have investigated so far. As shown here, DP4 produced a significant enhancement of ryanodine binding and induced a rapid Ca 2ϩ release from the SR. Furthermore, this peptide increased the sensitivity of the RyR to agonists such as peptide A (the activating II-III loop peptide) and polylysine (Ca 2ϩ release-inducing RyR-specific ligand). Both of these effects are essentially identical to the types of functional alterations seen in MH/CCD conditions described above (viz. hyperactivation of the channel and hypersensitization of the channel to agonists). Interestingly, replacement of Arg of DP4 with Cys, in the same way as it happens in the MH/CCD mutation (i.e. Arg 2458 -to-Cys 2458 mutation (37)), abolished the activating function of DP4. These findings suggest that the Leu 2442 -Pro 2477 region of the RyR plays a critical role in the Ca 2ϩ channel regulation and that a single amino acid residue (Arg 2458 ) mutation produces crucial effects on the function of this domain. Another central domain peptide DP5 produced no appreciable effect on the RyR if added alone. When added together with DP4, however, it produced a considerable suppression of the activating function of DP4.
In this study, we also re-investigated two N-terminal domain peptides, DP1 and DP3, described in our recent report (35). DP1, although weaker in its ability to activate the RyR1 than DP4, produced a significant increase of the sensitivity of RyR to both peptide A and polylysine in the same way as seen with peptide DP4. Thus, both of the two peptides (DP4 and DP1), corresponding to different domains (the central and N-terminal domains, respectively), mimic MH/CCD modifications. DP3 was found to produce the same type of effects as DP5; namely, it by itself produces no appreciable effect but suppresses the function of DP4. These findings, together with other pieces of evidence shown here, suggest that the interdomain interaction specified in the above hypothesis is involved in the channel regulation mechanism, and changes in this mechanism will result in the abnormal channel regulation.

Preparation
Skeletal Microsomes-Triad-enriched microsomal fractions were prepared from the rabbit back paraspinous and hind leg skeletal muscle by a method of differential centrifugation as described previously (38). Microsomes from the final centrifugation were homogenized in 0.3 M sucrose, 0.15 M potassium gluconate, proteolytic enzyme inhibitors (0.1 mM phenylmethanesulfonyl fluoride, 10 g/ml aprotinin, 0.8 g/ml antipain, 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 Ϫ78°C.
Cardiac Microsomes-Cardiac microsomes were prepared from dog ventricular cardiac muscle as described previously (35).

Domain Peptides Used and Peptide Synthesis
Six domain peptides were used in this study. The amino acid sequence and the residue numbers of the corresponding sequence of the in vivo domain are shown in Table I. The number assigned to the domain peptide (DP) represents the chronological order of the synthesis. DP4, DP4-mut, DP5, and DP6 were used for the first time in this study. Other peptides (DP1, DP1-2, and DP3) were already investigated (35), but DP1 and DP3 were used again with a new perspective in this study. DP1, which is the N-terminal half of DP1-2, retains the function of DP1-2 (35); hence DP1-2 was not used in this study.
Peptides were synthesized on an Applied Biosystems model 431 A synthesizer employing Fmoc (N-(9-fluorenyl)methoxycarbonyl) as the ␣-amino-protecting group. The peptides were cleaved and deprotected with 95% trifluoroacetic acid and purified by reversed-phase high pressure liquid chromatography.
[ 3 H]Ryanodine Binding Assay-Dog cardiac or rabbit skeletal (0.5 mg/ml) microsomes were incubated in 0.1 ml of a reaction solution containing 10 nM [ 3 H]ryanodine (68.4 Ci/ml, NEN Life Science Products), 0.3 M KCl (unless specified in the figure legends), CaCl 2 (10 M except for [Ca 2ϩ ] dependence assay), 20 mM MOPS, pH 7.2, for 16 h at 22°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 5 ml of distilled water. Filters were then placed in scintillation vials containing 10 ml of scintillation mixture 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 nonradioactive ryanodine. Assays were carried out in duplicate, and each datum point was obtained by averaging the duplicates (39).
Assays of Peptide-induced Ca 2ϩ Release-Rabbit skeletal microsomes (0.4 mg/ml) were incubated in a solution containing 0.15 M potassium gluconate, 1 mM MgATP, an ATP-regenerating system, 20 mM MES, pH 6.8 (Solution A) for 5 min to load the SR moiety with Ca 2ϩ . Then one volume of Solution A was mixed with one volume of Solution B containing 0.15 M potassium gluconate, 20 mM MES, pH 6.8, and various concentrations of peptides. The Ca 2ϩ concentration in both solutions was ϳ 0.25 M. The time course of SR Ca 2ϩ release was monitored in a stopped flow apparatus (Bio-Logic SFM-4) using 2.5 M fluo-3 as a Ca 2ϩ indicator as described previously (40). Ten to 15 traces (each representing 1,000 data points) of the fluo-3 signal were averaged for each experiment. Fig. 1 depicts the data of ryanodine binding to the SR vesicles isolated from skeletal and cardiac muscle in the presence of different concentrations of DP4. As seen, DP4 produced significant enhancement of ryan- odine binding to both RyR1 and RyR2 in a concentration-dependent manner. However, the extent of enhancement in skeletal SR (maximal enhancement: 380% control) was much larger than that in cardiac SR (240% control). The concentration for the half-maximal activation (AC 50 ) was 28 M for the RyR1 and 100 M for the RyR2. DP4 induced a rapid Ca 2ϩ release from the SR. Fig. 2 depicts the time courses of Ca 2ϩ release from skeletal muscle SR induced by various concentrations of DP4. With no added DP4, stopped-flow mixing produced negligible Ca 2ϩ release. At low concentrations (e.g. at 2 M), DP4 produced a slow Ca 2ϩ release. Upon increasing the concentration of DP4 up to 100 M, both the rate and the size of Ca 2ϩ release increased. The AC 50 of the initial rate of Ca 2ϩ release was 34.0 Ϯ 5.7 M, which is comparable to the AC 50 of RyR1 activation in ryanodine binding experiments. These results indicate that DP4 is a potent activator of both RyR1 and RyR2 and is capable of inducing a rapid Ca 2ϩ release from the skeletal muscle SR. Judging from the rapid induction of Ca 2ϩ release by high concentrations of DP4, it appears that this domain peptide is readily accessible to its target domain within the RyR.

Domain Peptide DP4 Activates Both RyR1 and RyR2 in a Concentration-dependent Manner-
Effects of DP4 on the [Ca 2ϩ ]-dependent Activation/Inhibition Profile-The general pattern of the biphasic [Ca 2ϩ ] dependence of activation in the lower [Ca 2ϩ ] range (0.01-10 M) and inhibition at higher range (10 M-3 mM) is nearly identical in the absence and the presence of 100 M DP4 (see Fig. 3), although the magnitude of ryanodine binding activity is considerably different as described above. However, there are few important differences. First, in the presence of DP4 there is a significant enhancement of ryanodine binding at 0.01 M Ca 2ϩ (Fig. 3 (Fig. 4).
DP4 Also Affects Activation Patterns of Peptide A and Polylysine-As shown in our recent studies, peptide A (a synthetic peptide corresponding to the Thr 671 -Leu 690 region of the dihydropyridine receptor ␣1 subunit II-III loop) activates the RyR1 in an isoform-specific manner and induces Ca 2ϩ release from skeletal muscle SR (41)(42). On this basis, we proposed that this peptide mimics the voltage-dependent induction of skeletal muscle-type excitation-contraction coupling (41)(42)(43). In the experiments shown in Fig. 5, we investigated the possibility that DP4 might affect the modes of activation by this peptide. As seen, peptide A alone produced a concentration-dependent enhancement of ryanodine binding, with an AC 50 of ϳ20 M. In the presence of DP4 (100 M), peptide A produced further activation in an additive fashion. Interestingly, the [peptide A] dependence of activation showed a considerable shift to the left in the presence of DP4, resulting in an apparent AC 50 of ϳ5 M. As described previously (41-43), excessively higher concentrations of peptide A becomes inhibitory, resulting in a biphasic activation/inhibition profile. The inhibitory phase was also shifted to the left in the presence of DP4. Fig. 6 shows the results of similar experiments with polylysine. Polylysine is the RyR-specific ligand that induces Ca 2ϩ release at much lower concentrations than other release-inducing reagents such as caffeine and serves as an excellent agonist in terms of its specificity and potency (40). As seen, in the presence of DP4 the AC 50 for polylysine decreased from 7 M to 4 M, and the inhibition by higher concentrations polylysine (9,42) was also shifted to the left. Thus, DP4 produced common effects on the two different types of agonists: peptide A and polylysine. Namely, (a) DP4 potentiated the peptide-induced Ca 2ϩ release in an "additive" manner, and (b) it shifted the biphasic activation/inhibition curve to the left. These effects correspond to the changes in the activity pattern in MH/CCD conditions; i.e. (a) hyperactivation of the release channel activity and (b) the increased affinity to the release triggering reagents.
An Arg-to-Cys Mutation of DP4 Abolishes Its Activating Function-As deduced from the above finding, DP4 mimics several abnormalities of the Ca 2ϩ channel seen in MH and CCD, namely the abnormalities caused by site-specific mutations. The RyR domain corresponding to DP4 contains one Arg 2458 -to-Cys 2458 (MH) mutation site (37). We made the same mutation in DP4 and synthesized a peptide DP4-mut as shown in Table I. Interestingly, this mutation completely abolished the activating effect that DP4 must have produced. Thus, as shown in Fig. 7, DP4-mut produced no appreciable effects on ryanodine binding even at high concentrations equivalent to the maximally activating concentrations of DP4. Furthermore, 100 M DP4-mut produced no appreciable effect on AC 50 for peptide A activation either, the AC 50 value for peptide A being 12 Ϯ 8 M and 12 Ϯ 10 M at 0 M and 100 M DP4-mut. This indicates that the highly specific amino acid sequence is required for the regulatory function of the in situ domain and that the corresponding synthetic peptide DP4 represents this situation.
Another Domain Peptide, DP1, Also Produces Activation/ Sensitization Effects-As described in the Introduction, another domain peptide DP1 (the peptide corresponding to the N-terminal MH/CCD domain) activated the RyR and induced Ca 2ϩ release in an RyR2-specific manner, as described in our recent report (35). In view of the new finding that DP4 has two effects (activation and sensitization to agonists), we investigated whether this is also the case with DP1. As shown in Figs. 8 and 9, 100 M DP1 produced a small but appreciable enhancement of ryanodine binding to the RyR1 (see binding in the absence of peptide A or polylysine). In the presence of DP1, the activation/inhibition curves of peptide A (Fig. 8) and polylysine ( Fig. 9) were both shifted to the left in the essentially identical manner as seen with DP4.
Counteractions among Various Domain Peptides-According to our working hypothesis (see the Introduction), the interaction between the two MH/CCD domains (the N-terminal domain and the central domain) may be involved in the channel regulation mechanism. Both DP4 (central domain peptide) and DP1 (N-terminal domain peptide) produced similar effects as described above. We investigated whether the addition of both peptides produces additive effects or some other effects. Fig. 10 depicts the ryanodine binding data obtained with various concentrations of DP1 in combination with 100 M DP4. As seen, DP1 alone produced appreciable enhancement of ryanodine binding in a concentration-dependent manner (up to 2.5-fold). DP4 alone produced about 4.5-fold enhancement (see the activation level of DP4 at 0 M DP1). Interestingly, however, upon the addition of increasing concentrations of DP1, the level of enhancement of ryanodine binding that had been produced by DP4 decreased, and reached a plateau at the level nearly identical to the level of maximal activation by DP1 alone. This indicates that the combined effects of the two activating domain peptides are not additive, but competitive. Another unique feature seen in Fig. 10 is that the IC 50 for the DP1 inhibition of DP4 (ϳ6 M) is more than one order of magnitude lower than the AC 50 for the activation by DP1 alone (ϳ100 M), suggesting that there is a unique counteraction between these domain peptides (see "Discussion"). As described in the Introduction, we synthesized another new peptide DP5 corresponding to the N-terminal region of the central domain. As shown in Fig. 11, DP5 alone produced virtually no effect on ryanodine binding in a broad concentration range investigated. However, if added together with 100 M DP4, which produced a nearly maximal activation of the RyR, increasing concentrations of the added DP5 produced progressive suppression of the activating function of DP4.
Similarly, another N-terminal MH/CCD domain peptide, DP3, had no appreciable effect on the ryanodine binding in a wide concentration range examined, as shown in Fig. 12 (cf. Ref. 35). However, when DP3 was combined with DP4, it inhibited the DP4 activation in a concentration-dependent manner. Thus, the domain peptides so far examined fall in either of the two categories: those mimicking MH/CCD-like effects (DP4 and DP1) and those removing MH/CCD-like effects (DP5 and DP3).
In the present study, we tested a control peptide DP6, which does not belong to either the N-terminal domain or the central domain (see Table I). This peptide neither mimicked MH/CCDlike effects nor removed MH/CCD-like effects (data not shown).

DISCUSSION
The RyR opens its Ca 2ϩ channel in response to various types of stimuli acting on its cytoplasmic region. Especially, the binding of the dihydropyridine receptor II-III loop to the RyR, the main physiologic stimulus in skeletal muscle-type excitationcontraction coupling (44), takes place presumably in a region of the RyR in the near vicinity of the transverse tubular system membrane. The important unsolved question is how the stimuli received at the cytoplasmic domains of the RyR control the opening of its channel.
Our current working hypothesis to approach the above question has been deduced from the literature on the unique distribution of the mutation sites in MH-susceptible animals and in MH and CCD-susceptible human patients. As described in the Introduction, most mutations occur in either of the two domains of the RyR defined as the N-terminal domain (ϳ600 residue long) and the central domain (ϳ300 residue long). Furthermore, it appears that mutations at different positions in either domain result in the same type of functional modifications: namely, hyperactivation of the Ca 2ϩ channel and hypersensitization to agonists (26, 30 -34). The most feasible hypothesis to account for these unique features would be as follows. The mutual interactions between these domains play a key role in the regulation of the RyR channel; hence, mutations occurring in different positions of either domain would produce a common effect, viz. interference with the interdomain interaction.
One of the most important aspects in this study is the finding that the newly synthesized peptide corresponding to the Cterminal portion of the central domain, DP4, showed two major effects on the RyR relevant to the hypothesis. First, it produced significant activation of the RyR (both RyR1 and RyR2), as shown by its high potency of enhancing ryanodine binding activity and inducing SR Ca 2ϩ release. Second, it increased the sensitivity (i.e. the apparent affinity) of the RyR1 to the Ca 2ϩ release-inducing peptides (peptide A and polylysine and activating Ca 2ϩ ), as shown by the appreciable left-shift of the concentration dependence of activation. Interestingly, these two effects of DP4 correspond to the two major functional modifications of the RyR seen in MH/CCD, viz. hyperactivation of the Ca 2ϩ channel and hypersensitization of the RyR to various agonists. This suggests that DP4 is mimicking a causative mechanism of the MH/CCD conditions. According to the principle of our peptide probe approach (see the Introduction), this would indicate that the in vivo domain, with the same structure as that of DP4 (i.e. domain 4, a part of the central domain), is involved in the interdomain interactions. We propose the following mechanism to account for the observed effects of DP4. The added DP4 will presumably compete with domain 4 for the binding to their common binding site(s) (i.e. the domain 4 counter domain). This will interfere with the interaction between domain 4 and its counter domain; in turn, the contact between these domains will be loosened. Since the outcome of such interference was activation of the channel as seen here, the tight interaction between these domains must have contributed to the channel closing. In the case of MH and CCD, the Arg 2458 -to-Cys 2458 mutation that has taken place in domain 4 will also interfere with the interdomain interaction, resulting in the hyperactivation of the channel.
The most important evidence supporting the above mechanism is the present finding that the same Arg-to-Cys mutation made in DP4 completely abolished both hyperactivation and hypersensitization effects that must have been produced by DP4. This would indicate that this specific mutation resulted in the loss of the ability of the peptide to bind to the counter domain, in turn resulting in the loss of the ability to interfere with the interdomain interaction. This is in accord with the above concept that the Arg 2458 -to-Cys 2458 mutation in domain 4 reduces the ability to bind to its counter domain. The result also indicates that the observed activation of the RyR by DP4 requires a very specific amino acid sequence.
In the context of this discussion, it should be noted that another domain peptide DP1, which corresponds to a part of the N-terminal domain, also produces hyperactivation and hypersensitization effects similar to those of DP4. Again, this would indicate that domain 1 is also involved in the interdomain interaction and the added DP1 competes with domain 1 for the binding to their common counter domain. In light of the interdomain interaction concept, it would be interesting to speculate that the domain 4 of the central domain interacts with the N-terminal domain; conversely, the domain 1 interacts with the central domain. Additional evidence for this hypothesis has been revealed in the present study. First, the addition of both activating domain peptides DP4 and DP1 together produced a much lower level of activation than the level achieved by DP4 alone. As a matter of fact, higher concentrations of DP1 almost completely reversed the activation by DP4. This is consistent with the view that the two peptides are competing to the common "de-blocking" mechanism mediated by the interdomain interaction (see above) but is inconsistent with the view that both peptides bind to activating sites. Second, the IC 50 for the DP1 block of DP4 was much lower than the AC 50 for DP1 activation, suggesting that the competition between the two domain peptides is highly cooperative.
Putting the above pieces of evidence altogether, we tentatively propose the following mechanism. In the normally operating RyR, contact between the N-terminal domain and the central domain would correspond to an Off position of a regulator switch of the channel. In MH/CCD conditions, this putative blocking mechanism is altered (i.e. loosening of the interdomain contact) by the mutations in either of these domains, resulting in the apparent activation and hypersensitization that actually represents a de-blocking phenomenon. The exogenously added domain peptides will bind to their designated interaction sites and interfere with the interdomain contact, resulting in functional alterations similar to those in MH/CCD. This mechanism accounts for the fact that the competition between the two domain peptides is highly cooperative, since loosening of the interdomain contact by one peptide would facilitate further loosening by the other peptide.
The sensitization of the RyR to Ca 2ϩ release-inducing peptides by DP4 shown here suggests an interesting new concept as follows. The interdomain interaction postulated here might be used as a device for the intramolecular signal transduction mechanism mentioned above. For example, binding of the activating II-III loop peptide (peptide A) or the in vivo II-III loop to the RyR would remove the interdomain contact, changing the regulator switch from the Off position (see above) to the On position. Thus, the presence of DP4 or DP1, which removes the interdomain contact and, hence, catalyzes the Off-to-On action, would facilitate the peptide A-induced signal transduction process and would increase the apparent affinity for peptide A as seen in this study.
Another important piece of information obtained in the present study is that one of the new central domain peptides investigated here, namely DP5, by itself produced no appreciable effect, but if added together with DP4, it produced almost complete suppression of the DP4 activation. Similarly, an Nterminal domain peptide DP3 (35) also produced almost complete suppression of the DP4 activation. Thus, it appears that at least four subdomains represented by these peptides (DP4, DP1, DP5, and DP3) are involved in the postulated interdomain interaction.
In conclusion, the data shown here are consistent with the following model. The mode of interaction between the two MH/ CCD domains (defined as N-terminal domain and central domain) controls the functional state of the RyR Ca 2ϩ channel: the interdomain contact produces the blocked state of Ca 2ϩ channel, and the removal of such contact produces the activated state. Synthetic peptides DP4 and DP1 corresponding to two of such domains produced both activation of the channel and hypersensitization to agonists, mimicking the functional alterations produced by MH/CCD mutations occurring in these domains. According to the above model, these are produced by de-blocking caused by the interference of the interdomain interaction by domain peptides or mutations.