Critical Amino Acid Residues Determine the Binding Affinity and the Ca2+ Release Efficacy of Maurocalcine in Skeletal Muscle Cells*

Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion Scorpio maurus palmatus. MCa and mutated analogues were chemically synthesized, and their interaction with the skeletal muscle ryanodine receptor (RyR1) was studied on purified RyR1, sarcoplasmic reticulum (SR) vesicles, and cultured myotubes. MCa strongly potentiates [3H]ryanodine binding on SR vesicles (7-fold at pCa 5) with an apparent EC50 of 12 nm. MCa decreases the sensitivity of [3H]ryanodine binding to inhibitory high Ca2+ concentrations and increases it to the stimulatory low Ca2+ concentrations. In the presence of MCa, purified RyR1 channels show long-lasting openings characterized by a conductance equivalent to 60% of the full conductance. This effect correlates with a global increase in Ca2+ efflux as demonstrated by MCa effects on Ca2+ release from SR vesicles. In addition, we show for the first time that external application of MCa to cultured myotubes produces a cytosolic Ca2+ increase due to Ca2+ release from 4-chloro-m-cresol-sensitive intracellular stores. Using various MCa mutants, we identified a critical role of Arg24 for MCa binding onto RyR1. All of the other MCa mutants are still able to modify [3H]ryanodine binding although with a decreased EC50 and a lower stimulation efficacy. All of the active mutants produce both the appearance of a subconductance state and Ca2+ release from SR vesicles. Overall, these data identify some amino acid residues of MCa that support the effect of this toxin on ryanodine binding, RyR1 biophysical properties, and Ca2+ release from SR.

In skeletal muscles, contraction is triggered by the massive release of Ca 2ϩ from sarcoplasmic reticulum (SR). 1 The chan-nel responsible for this release is the type-1 ryanodine receptor (RyR1). RyR1 has been intensively studied because of its unique structural and functional organization. It forms part of a calcium mobilization complex in which RyR1 is apposed to the L-type voltage-dependent calcium channel (dihydropyridine receptor, DHPR) along many other structural and regulatory components (1). The activation of RyR1 requires a chain of events that starts with plasma membrane depolarization inducing a change in the conformation of DHPR itself transmitted to RyR1. The entire set of events is called excitation-contraction coupling (EC coupling).
In vitro, the activity of RyR1 can be modulated by a number of different effectors such as Ca 2ϩ , ryanodine, ATP, caffeine, and 4-chloro-m-cresol (CMC) (2,3). Among these effectors, only few present high selectivity and affinity for RyR1. More specific pharmacological agents for RyR1 have been discovered in scorpion venoms (4,5). One such peptide has been isolated from the venom of the chactid scorpion Scorpio maurus palmatus and has been termed maurocalcine (MCa). It is a 33-mer basic peptide reticulated by three disulfide bridges. MCa can be chemically synthesized without any loss of activity (5). It is potently active on RyR1 as it alters channel properties in the low nanomolar range (5,6). MCa presents a strong sequence homology (82% amino acid sequence identity) with imperatoxin A (IpTxA), a toxin active on RyR1 and isolated from another scorpion venom (7)(8)(9)(10). Besides the fact that MCa and IpTxA represent two of the most high affinity effectors of RyR1, they also share some amino acid sequence homology with a specific domain (domain A) of the II-III loop of Ca v ␣ 1.1 , the subunit that carries the voltage sensor of DHPR (Fig. 1). Although the exact role of the domain A in the EC-coupling process is highly debated (11)(12)(13), we have recently shown by using plasmon resonance measurements that it is the single II-III loop sequence interacting with RyR1 (14). Nevertheless, this exceptional form of homology between a channel sequence and a toxin could be indicative that domain A possesses some kind of yet unresolved function in RyR1 regulation. Therefore, studying the MCa effects on RyR1 may produce several interesting hints on how to proceed further on investigating the role of domain A in EC coupling.
In this work, we synthesized several MCa analogues in which amino acid residues, also present in homologous position in domain A, were substituted by alanine residues. We then analyzed the effect of MCa and the analogues on [ 3 H]ryanodine * This work was supported by INSERM, CEA, and UJF. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a CIFRE fellowship (ANRT and Cellpep). binding, Ca 2ϩ -dependent activation of RyR1, channel activity, and Ca 2ϩ release from SR vesicles. We identified an amino acid residue critical for the interaction of MCa with RyR1 and for the induction of a long-lasting subconductance state. The data also demonstrate a clear relationship in the ability of MCa to potentiate [ 3 H]ryanodine binding, induce a subconductance state, and produce Ca 2ϩ release from heavy SR vesicles. To prove the physiological relevance of RyR1 as the main target of MCa, we tested the effect of the peptide on internal Ca 2ϩ release in intact myotubes. We show that the addition of 100 nM MCa in Ca 2ϩ -free extracellular medium induces an increase in cytosolic Ca 2ϩ concentration and a complete inhibition of CMCinduced Ca 2ϩ release.

EXPERIMENTAL PROCEDURES
Chemical Synthesis-N-␣-Fmoc-L-amino acids, 4-hydroxymethylphenyloxy resin, and reagents used for peptide synthesis were obtained from PerkinElmer Life Sciences. The MCa and analogues were obtained by the solid-phase peptide synthesis (15) using an automated peptide synthesizer (Model 433A, Applied Biosystems Inc.). Analogues were obtained by point mutation (Ala instead of one amino acid in the sequence of maurocalcine native-like) and named Ala/Lys 8 , Ala/Lys 19 , Ala/Lys 20 , Ala/Lys 22 , Ala/Arg 23 , Ala/Arg 24 , and Ala/Thr 26 . Peptide chains were assembled stepwise on 0.25 meq of hydroxymethylphenyloxy resin (1% cross-linked; 0.89 meq of amino group/g) using 1 mmol of N-␣-Fmoc amino acid derivatives. The side chain-protecting groups were as follows: trityl for Cys and Asn; tert-butyl for Ser, Thr, Glu, and Asp; pentamethylchroman for Arg; and tert-butyloxycarbonyl for Lys. N-␣-Amino groups were deprotected by treatment with 18 and 20% (v/v) piperidine/N-methylpyrrolidone for 3 and 8 min, respectively. The Fmoc-amino acid derivatives were coupled (20 min) as their hydroxybenzotriazole active esters in N-methylpyrrolidone (4-fold excess). After peptide chain assembly, the peptide resin (approximately 1.8 g) was treated between 2 and 3 h at room temperature in constant shaking with a mixture of trifluoroacetic acid/H 2 O/thioanisol/ethanedithiol (88: 5/5/2, v/v) in the presence of crystalline phenol (2.25 g). The peptide mixture was then filtered, and the filtrate was precipitated by adding cold t-butylmethyl ether. The crude peptide was pelleted by centrifugation (3.000 ϫ g for 10 min), and the supernatant was discarded. The reduced peptide was then dissolved in 200 mM Tris-HCl buffer, pH 8.3, at a final concentration of 2.5 mM and stirred under air to allow oxidation/folding (between 50 and 72 h, room temperature). The target products, MCa and analogues, were purified to homogeneity, first by reversed-phase high pressure liquid chromatography (PerkinElmer Life Sciences, C 18 Aquapore ODS, 20 m, 250 ϫ 10 mm) by means of a 60-min linear gradient of 0.08% (v/v) trifluoroacetic acid ϭ 0 -30% acetonitrile in 0.1% (v/v) trifluoroacetic acid/H 2 O at a flow rate of 6 ml/min ( ϭ 230 nm). A second step of purification of MCa and analogues was achieved by ion-exchange chromatography on a carboxymethyl cellulose matrix using 10 mM (buffer A) and 500 mM (buffer B) sodium phosphate buffers, pH 9.0 (60-min linear gradient from 0 to 60% buffer B at a flow rate of 1 ml/min). The homogeneity and identity of MCa or analogues were assessed by the following: (i) analytical C 18 reversed-phase high pressure liquid chromatography (Merck, C 18 Li-Chrospher, 5 m, 4 ϫ 200 mm) using a 60-min linear gradient of 0.08% (v/v) trifluoroacetic acid/0 -60% acetonitrile in 0.1% (v/v) trifluoroacetic acid/H 2 O at a flow rate of 1 ml/min; (ii) amino acid analysis after acidolysis (6 N HCl/2% (w/v) phenol, 20 h, 118°C, N 2 atmosphere); and (iii) mass determination by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Heavy SR Vesicles Preparation-Heavy SR vesicles were prepared following a modified method of Kim et al. (16) as described in Marty et al. (17). Protein concentration was measured by the Biuret method.
[ 3 H]Ryanodine Binding Assay-Heavy SR vesicles (1 mg/ml) were incubated at 37°C for 2.5 h in an assay buffer composed of 5 nM [ 3 H]ryanodine (unless otherwise stated), 150 mM NaCl, 2 mM EGTA, variable concentrations of CaCl 2 to adjust for pCa, and 20 mM HEPES, pH 7.4. MCa or peptide mutants were added to the assay buffer just prior to the addition of heavy SR vesicles. [ 3 H]Ryanodine bound to heavy SR vesicles was measured by filtration through Whatmann GF/B glass filters followed by three washes with 5 ml of ice-cold washing buffer composed of 150 mM NaCl, 20 mM HEPES, pH 7.4. Filters were then soaked overnight in 10 ml of scintillation mixture (Cybscint, ICN), and bound radioactivity was determined by scintillation spectrometry. Nonspecific binding was measured in the presence of 20 M cold ryanodine. Each experiment was performed in triplicate and repeated at least twice. All of the data are presented as the mean Ϯ S.E.
Ca 2ϩ Release Measurements-Ca 2ϩ release from heavy SR vesicles was measured using the Ca 2ϩ -sensitive dye, antipyrylazo III. The absorbance was monitored at 710 nm by a diode array spectrophotometer (MOS-200 Optical System, Biologic, Claix, France). Heavy SR vesicles (50 g) were actively loaded with Ca 2ϩ at 37°C in a 2-ml buffer containing 100 mM KCl, 7.5 mM sodium pyrophosphate, 20 mM potassium MOPS, pH 7.0, supplemented with 250 M antipyrylazo III, 1 mM ATP/MgCl 2 , 5 mM phosphocreatine, and 12 g/ml creatine phosphokinase (18). Ca 2ϩ loading was started by sequential additions of 50 and 20 M of CaCl 2 . In these loading conditions, no calcium-induced calcium release interferes with the observations. At the end of each experiment, Ca 2ϩ remaining in the vesicles was determined by the addition of Ca 2ϩ ionophore A23187 (4 M) and the absorbance signal calibrated by two consecutive additions of 20 M CaCl 2 .
RyR1 Ca 2ϩ Channel Reconstitution and Single-channel Recording Analysis-Measurements of channel activity were carried out using purified RyR1 incorporated into planar lipid bilayers. RyR1 was purified from rabbit SR vesicles as previously described (19). The bilayers were formed using phosphatidylethanolamine, phosphatidylserine, and L-phosphatidylcholine in a ratio of 5:4:1 dissolved in n-decane up to the final lipid concentration of 20 mg/ml (20). Bilayers were formed across a 200-or 250-m diameter aperture of a Delrin cap using a symmetrical buffer solution (250 mM KCl, 100 M EGTA, 150 M CaCl 2 , 20 mM PIPES, pH 7.2). The chamber into the small aliquot of purified RyR1 was added and defined as the cis (cytoplasmic) side, whereas the other chamber labeled as the trans (luminal) side was kept on ground potential. To ensure the orientation of the incorporated RyR1, we tested the effect of free Ca 2ϩ concentration on both side. After successful incorporation of the RyR1 channel, free calcium concentration in the cis chamber was adjusted to 238 nM by the addition of EGTA. Electrical signals were filtered at 1 kHz through an 8-pol low-pass Bessel filter and digitized at 3 kHz using Axopatch 200 and pCLAMP 6.03 (Axon Instruments, Union City, CA).
Total recording time in each experiment was 10 -20 min for any experimental condition tested. After changing conditions, at least 5 min were allowed for equilibration, which appeared to be enough to reach the new equilibrium of the parameters. Single channel measurements were carried out at 20 -22°C. The free Ca 2ϩ concentration was calculated using the computer program and affinity constants published by Fabiato (21). Open probabilities were calculated using the common 50% criteria with a medial dead zone of 5%. Current amplitude distribution was analyzed using Origin (Microcal Software, Northampton, MA).
Values of the open probability are expressed as the means Ϯ S.E.
Ca 2ϩ Imaging from Culture Myotubes-Changes in cytosolic calcium levels were monitored using the calcium-dependent fluorescent dye Fluo-4 (Molecular Probes). The myotubes were loaded for 1 h at room temperature in the presence of 5 M Fluo-4-AM. Uptake of the dye was facilitated by the addition of 0.02% pluronic acid (Sigma). After loading, myotubes were washed for 1 h to allow ester hydrolysis of the dye. Fluorescence changes were measured by confocal laser-scanning microscopy using a LEICA TCS-SP2 operating system in the xyt mode. Fluo4 was excited at a wavelength of 488 nm, and the fluorescence was collected from 500 to 570 nm. Images were collected every 1.6 s for 2-4 min and then analyzed frame by frame with the data analysis software provided by Leica. Fluorescence curves are expressed as a function of time as ⌬F/F, where F represents the base-line fluorescence and ⌬F represents the fluorescence variations. Fig. 1 presents the primary structure of the different MCa analogues that we synthesized. The structural determination of MCa shows that the stretch of basic residues (from Lys 20 to Arg 24 ) forms a single basic-rich surface (22). We will define the residues forming this basic surface as residues belonging to the "basic class." The opposite surface of the toxin contains four acidic residues (Asp 2 , Glu 12 , Asp 15 , and Glu 29 ) and one basic residue (Lys 8 ). We will define this acidic surface as the "acidic face." This electrostatic charge distribution creates a marked anisotropy in which the role in MCa pharmacology is unknown. We choose to substitute one by one all of the MCa amino acid residues common between MCa and the domain A of the II-III loop of Ca v ␣ 1.1 subunit. Most of these residues belong to the basic class with the exception of Thr 26 and therefore were replaced by neutral alanine residues. We also synthesized the mutant [Ala 8 ]MCa because Lys 8 is not conserved in the II-III loop but is located on the toxin face opposite to the basic surface (23). All of the peptides were folded/oxidized in a 200 mM Tris-HCl buffer, pH 8.3, by a 72-h exposure to air and purified to homogeneity by preparative C 18 reversed-phase high pressure liquid chromatography. Both analysis of elution profiles and circular dichroism spectra indicate that all of the peptides have proper secondary structures (data not shown).

Effects of MCa on [ 3 H]Ryanodine
Binding to RyR1-We recently described that MCa stimulates the binding of [ 3 H]ryanodine onto heavy SR vesicles that contain RyR1 (6).  (Fig. 2D). In the presence of MCa (saturating 100 nM), [ 3 H]ryanodine binding on SR vesicles still shows a classical bell-shaped dependence to external Ca 2ϩ . Interestingly, we observed that the [ 3 H]ryanodine binding inhibition by high Ca 2ϩ concentration (pCa 3) was slightly decreased, whereas the [ 3 H]ryanodine binding stimulation by low Ca 2ϩ concentration (pCa 6 and 6.5) was increased. Indeed, while in the absence of MCa, ryanodine binding was almost completely inhibited (Ͼ99%) at pCa 3. In the presence of MCa, 39% of the maximum [ 3 H]ryanodine binding was still observed at pCa 3. Similarly, at pCa 6.5, ryanodine binding is at 70% of its maximum in the presence of MCa, whereas it is only at 4.8% of its maximum in the absence of MCa.   Fig. 3B shows that the effect of MCa is not altered by the presence of up to 1 M [Ala 24 ]MCa, demonstrating that this analogue is not able to bind onto RyR1. We also checked whether or not the absence of the effect of [Ala 24 ]MCa on [ 3 H]ryanodine binding is related to experimental pCa conditions. Indeed, no [ 3 H]ryanodine binding stimulation was observed at pCa values ranging between 2 and 7 (Fig. 3C).

Characterization of the Effects of MCa Mutants on [ 3 H]Ryanodine Binding to
Characterization of the Effects of MCa and MCa Mutants on Ca 2ϩ Release from SR Vesicles-To investigate the effect of MCa on Ca 2ϩ release from heavy SR vesicles, we first actively loaded the vesicles by two consecutive additions of Ca 2ϩ (50 M and 20 M) in the presence of ATP-Mg 2ϩ , pyrophosphate, and ATP-regenerating system. After Ca 2ϩ loading reaches equilibrium, the addition of 1 M MCa to the external medium induces Ca 2ϩ release as shown in Fig. 4A. Similar results were obtained with 100 nM MCa (data not shown), but 1 M MCa was chosen to allow a comparison with the effects of the lower affinity MCa analogues. External Ca 2ϩ level reaches a plateau that corresponds to a new equilibrium between Ca 2ϩ release and Ca 2ϩ uptake rates as evidenced by the additional change produced by 4 M A23187 calcium ionophore. The release of Ca 2ϩ induced by 1 M MCa was prevented by preincubating the SR vesicles with 10 M ruthenium red, demonstrating that MCa-induced calcium release occurs through RyR (Fig. 4B). Similarly, the application of 1 M MCa prevents the release of Ca 2ϩ induced by the addition of 500 M CMC, and conversely, the application of 500 M CMC prevents an additional release of Ca 2ϩ by 1 M MCa (Fig. 4C). These data again demonstrate that MCa re-  (Fig. 4E). The present observation made for [Ala 24 ]MCa is in complete agreement with its lack of effect on [ 3 H]ryanodine binding (Fig. 3B) (Fig. 5A). This subconductance state represents 60% of the full conductance state, and the channel spends 54.8 Ϯ 6.2% of its time in this subconductance level. A much less frequent smaller LLSS (48% of the full conductance) was described in earlier reports (5,6), but the experimental conditions were slightly different (purified RyR1 versus junctional SR vesicles and K ϩ current versus Cs ϩ current in previous reports (5,6)). We next examined whether the LLSS induced by MCa could be correlated to Ca 2ϩ release from heavy SR vesicles and stimulation of [ 3 H]ryanodine binding. Therefore, we tested the effect of [Ala 24 ]MCa on the conductance level of RyR1 in lipid bilayers (Fig. 5B). Indeed, this mutant peptide has no effect on [ 3 H]ryanodine binding (Fig. 3A) nor on the Ca 2ϩ release from SR vesicles (Fig. 4D). As expected, we show that the application of teristic LLSS. With this analogue, the LLSS are characterized by a conductance corresponding to 54% of the full-conductance state. Under this condition, the channel spends on average 28 Ϯ 8.6% of its time in this state (data not shown). The lesser probability for RyR1 to develop in the LLSS in the presence of [Ala 20 ]MCa compared with MCa may be related to a lesser ability to stimulate [ 3 H]ryanodine binding (Fig. 3A). Other analogues including [Ala 23 ]MCa also produced LLSS (data not shown).
Intracellular Ca 2ϩ Release in Intact Myotubes Induced by MCa-We next analyzed the effect of MCa on cytosolic Ca 2ϩ variations by changes in Fluo-4 fluorescence levels in cultured myotubes. Extracellular application of 100 nM MCa on a group of representative myotubes produces a fast increase in intracellular Ca 2ϩ level (Fig. 6A). The time course of the change in fluorescence level (⌬F/F) shows that Ca 2ϩ increase occurs within 3 s followed by a rapid decrease back to the basal level (Fig. 6B). MCa produces a similar elevation in cytosolic Ca 2ϩ when extracellular medium is supplemented with 50 M La 3ϩ and deprived of Ca 2ϩ by the addition of 1 mM EGTA (Fig. 6B). This observation demonstrates that MCa-induced Ca 2ϩ mobilization occurs from internal sources. An analysis of the average change in fluorescence level confirms that there is no significant difference in cytosolic Ca 2ϩ elevation in the absence or presence of extracellular Ca 2ϩ (Fig. 6C). In similar experimental conditions, external application of 250 M CMC also induces a rapid and transient Ca 2ϩ elevation (Fig. 6D) (24). A prior incubation of the myotube with 100 nM MCa almost totally prevents CMC-induced peak Ca 2ϩ elevation (Fig. 6D). This observation was confirmed by an average quantification of fluorescence variation (mean ⌬F/F ϭ 79 Ϯ 67 (n ϭ 53) for CMC alone versus 4 Ϯ 7 (n ϭ 91) for CMC after MCa) (Fig. 6E). We also analyzed the cell variability in response to CMC alone or CMC after MCa application (Fig. 6F). In the presence of CMC, most myotubes (85.6%) produce important changes in fluorescence (⌬F/F Ͼ 50%), whereas after incubation with MCa, an opposite profile of responses is observed with 47% of the cells showing Ͻ10% change in fluorescence (⌬F/F ϭ 0 -10%). These results demonstrate that the CMC-sensitive Ca 2ϩ stores are also MCa-sensitive. Because CMC is known to act on intracellular Ca 2ϩ stores by the bias of RyR (2), this is another indication that MCa acts on RyR-dependent Ca 2ϩ stores. To confirm that the effect of MCa on Ca 2ϩ elevation is the result of an interaction with RyR, we also tested the effect of the mutant [Ala 24 ]MCa. We show that it is unable (i) to produce a Ca 2ϩ release from myotubes (Fig. 7A) and (ii) to modify CMC effect (Fig. 7B). DISCUSSION In skeletal muscles, RyR1 appears to represent the main target of MCa. We have previously shown that this toxin stimulates [ 3 H]ryanodine binding, stabilizes RyR1 into a subconductance state, and produces Ca 2ϩ release from heavy SR vesicles. In this work, we further investigated the biochemical and functional properties of MCa. Using synthetic mutated analogues of MCa, we defined some critical amino acid residues of MCa that are required for its effects on RyR1. We also show for the first time that MCa is able to induce intracellular Ca 2ϩ release in intact myotubes. This effect is conserved in the absence of external Ca 2ϩ , demonstrating that the increase in cytoplasmic Ca 2ϩ concentration is due to the release of Ca 2ϩ from internal stores. In these conditions, Ca 2ϩ release induced by CMC, a RyR1 agonist, is completely inhibited by the preapplication of MCa. Conversely, the application of the inactive [Ala 24 ]MCa analogue does not induce any Ca 2ϩ release from internal stores nor does it inhibit CMC-induced Ca 2ϩ release. These data demonstrate that Ca 2ϩ release induced by MCa occurs through RyR1 activation. The cellular effect of MCa is of the greatest importance because it demonstrates that the toxic effect of the toxin can be explained by its action on RyR1 despite the intracellular location of this target. This result also reveals that MCa has the ability to cross the plasma membrane of skeletal muscle cells, a property that is shared by some other basic peptides (25). Cell penetration is expected to occur rapidly as Ca 2ϩ elevation can be observed a few seconds after external application of MCa.
We have identified Arg 24 as a crucial amino acid residue for the effects of MCa. Indeed, the replacement of Arg 24 (26). There are interesting functional similarities between ryanodine and MCa. Both appear able to induce subconductance states, and MCa favors [ 3 H]ryanodine binding. These data strongly suggest the existence of a positive synergy between the binding sites of the two molecules on RyR1. This synergy is further evidenced by the fact that MCa increases by a factor of 6.1-fold the affinity of [ 3 H]ryanodine for RyR1. In addition, in the presence of MCa, we observed a shift to lower Ca 2ϩ concentrations of the Ca 2ϩ stimulatory effect on [ 3 H]ryanodine binding and a shift to higher Ca 2ϩ concentrations of the Ca 2ϩ inhibitory effect on [ 3 H]ryanodine binding. These data are in favor of a model in which the binding of either one of these two molecules, ryanodine or MCa, produces a chain of conformational events, leading to the appearance of subconductance states. All of the active MCa analogues that we tested stimulated [ 3 H]ryanodine binding with higher EC 50 values than MCa itself. We also measured a reduced ability to stimulate [ 3 H]ryanodine binding. Although the substituted amino acid residues of MCa do not appear decisive for the interaction with RyR1, they seem to contribute to some extent to the binding site recognition. In addition, they also appear to participate to the conformational events that lead to the modification of the low affinity ryanodine binding sites. We would expect that more drastic substitutions within MCa lead to modifications in the level of RyR1 subconductance states and/or in the time spent in these subconductance states. Indeed, it has been observed that IpTxA, which shares 82% sequence identity with MCa, induces a subconductance state of RyR significantly different from the one triggered by MCa (25% of the full-conductance state for IpTxA versus 58% for MCa) (9,10). Moreover, in contrast to what is observed with IpTxA, LLSS induced by MCa and its analogues are polarity-independent. Similarly, the domain A of the ␣ 1 subunit of DHPR that shares sequence similarities with both MCa and IpTxA also induces a subconductance state different from those observed with the toxin (65 and 86% of the full-conductance state for domain A) (6). Based on sequence identity, it is probable that MCa and IpTxA bind more than one common site(s) or regions on the RyR1 tetramer. Interestingly, the binding of two structurally related molecules on the same channel can produce highly different conductance states of RyR1. Therefore, it is tempting to postulate that once MCa or IpTxA is bound on RyR1, structural modification of these ligands could produce important changes in the conductance properties of RyR1. This concept of RyR1 conductance modulation could be applied to domain A to provide an explanation for its role in the regulation of RyR1 function. Recently, it was proposed that the threedimensional structural surface of MCa and IpTxA mimics that of domain A (23). It would be interesting to investigate whether conformational changes in domain A during membrane depolarization are of nature to modify RyR1 channel conductance.