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J. Biol. Chem., Vol. 277, Issue 2, 993-1001, January 11, 2002

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Ca²⁺-dependent Dual Functions of Peptide C

THE PEPTIDE CORRESPONDING TO THE Glu⁷²⁴-Pro⁷⁶⁰ REGION (THE SO-CALLED DETERMINANT OF EXCITATION-CONTRACTION COUPLING) OF THE DIHYDROPYRIDINE RECEPTOR 1 SUBUNIT II-III LOOP^*

Takeshi Yamamoto, John Rodriguez^§, and Noriaki Ikemoto^¶

From  Boston Biomedical Research Institute, Watertown, Massachusetts 02472, ^§ Harvard University, Cambridge, Massachusetts 02138, and ^¶ Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, June 22, 2001, and in revised form, October 15, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Both in vivo and in vitro studies suggest that the Glu⁷²⁴-Pro⁷⁶⁰ (peptide C) region of the dihydropyridine receptor 1 II-III loop is important for excitation-contraction coupling, although its actual function has not yet been elucidated. According to our recent studies, peptide C inhibits Ca²⁺ release induced by T-tubule depolarization or peptide A. Here we report that peptide C has Ca²⁺-dependent dual functions on the skeletal muscle ryanodine receptor. Thus, at above-threshold [Ca²⁺]s (0.1 µM) peptide C blocked peptide A-induced activation of the ryanodine receptor (ryanodine binding and Ca²⁺ release); peptide C also blocked T-tubule depolarization-induced Ca²⁺ release. However, at sub-threshold [Ca²⁺]s (<0.1 µM), peptide C enhanced ryanodine binding and induced Ca²⁺ release. If peptide A was present, together with peptide C, both peptides produced additive activation effects. Neither peptide A nor peptide C produced any appreciable effect on the cardiac muscle ryanodine receptor at both high (1.0 µM) and low (0.01 µM) Ca²⁺ concentrations. These results suggest the possibility that the in vivo counterpart of peptide C retains both activating and blocking functions of the skeletal muscle-type excitation-contraction coupling.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The functional coupling between the voltage-sensing DHP¹ receptor and the RyR Ca²⁺ channel in skeletal muscle-type E-C coupling seems to be mediated by a physical interaction between these receptors by mediation of one of the cytoplasmic loops of the DHP receptor 1 subunit, the so-called II-III loop (1, 2). However, the important question regarding which region or regions of the II-III loop are involved in the voltage-dependent interaction with the RyR has not yet been settled. According to the studies with myotubes expressing chimeric II-III loop, the region encompassing the residues Phe⁷²⁵-Pro⁷⁴² of the II-III loop (3) or its extended region encompassing Leu⁷²⁰-Leu⁷⁶⁴ region (4) plays a critical role in both orthograde and retrograde communications between the DHP receptor and the RyR.

On the other hand, studies with shorter synthetic peptides corresponding to various regions of the II-III loop suggested that the important functions of E-C coupling are localized in the two different regions of the loop. Namely, the peptides corresponding to the overlapping regions encompassing Glu⁶⁶⁶-Glu⁷²⁶ (5) and Thr⁶⁷¹-Leu⁶⁹⁰ (peptide A; see Refs. 6-10) produced a strong activation of the RyR. This suggested that the putative activator of E-C coupling resides in the peptide A region, although whether the activating function is localized in the Arg⁶⁸¹-Leu⁶⁹⁰ (peptide A-10; see Ref. 11) region or in the Thr⁶⁷¹-Glu⁶⁸⁰ region (12) has not yet been settled. Another peptide corresponding to the Glu⁷²⁴-Pro⁷⁶⁰ region of the II-III loop, peptide C, inhibited peptide A-mediated activation of the RyR (6, 13, 14) and also produced a small inhibition of depolarization-induced tension development in the skinned muscle fiber (15). Thus, the information derived from the peptide probe studies suggested that these two regions of the II-III loop are involved in E-C coupling (13).

However, according to the recent report of Proenza et al. (16), scrambling of the amino acid sequence in the peptide A-10 region, which produced a severe loss of the activating function of peptide A-10 in our in vitro experiments (11), produced no detectable changes in E-C coupling in the dysgenic myotubes. Similarly, chimeric construct, in which the Leu⁷²⁰-Leu⁷⁶⁴ region is the skeletal muscle-type sequence, but the rest is identical to the housefly II-III loop with the sequence quite dissimilar to the skeletal muscle sequence, produced essentially the same E-C coupling activity as the wild-type skeletal muscle system (17). Furthermore, according to the more recent report of Ahern et al. (18) deletion of the Thr⁶⁷¹-Leu⁶⁹⁰ peptide A region from the 1 subunit produced virtually no effect on Ca²⁺ conductance, charge movement, and Ca²⁺ transients. These reports raised the possibility that the only Leu⁷²⁰-Leu⁷⁶⁴ region, containing the Phe⁷²⁵-Pro⁷⁴² region (the so-called determinant of skeletal-type E-C coupling), may be sufficient for the in vivo E-C coupling without the requirement of the peptide A region.

Although the chimera approach suggested that the Leu⁷²⁰-Leu⁷⁶⁴ region (containing the determinant region) is important for in vivo E-C coupling as described above, the actual functions of this region have not yet been elucidated. What kind of function or functions does this region retain? Can we find any of the functions required for E-C coupling, such as activation, re-priming, and physical linking of the II-III loop with the RyR? As described previously (6, 13-15), peptide C that corresponds to essentially the same region as the Leu⁷²⁰-Leu⁷⁶⁴ region suppresses activation of the RyR by peptide A and T-tubule depolarization, suggesting that this region may be required for at least the blocking or re-priming process of E-C coupling. According to the recent report by Stange et al. (12), the peptide corresponding to the Leu⁷²⁰-Gln⁷⁶⁵ (a slightly extended version of peptide C) activated the RyR Ca²⁺ channel, suggesting that this region may contain the potential activating function, as well. The main aim of the present study is to gain further insight into the functions to be assigned to the peptide C region of the II-III loop. The peptide probe has permitted us to perform functional and site-localization assays in a highly quantitative manner. Using this advantage we re-investigated the effects of peptide C, as well as peptide A, on the ryanodine binding and Ca²⁺ release activities at both higher ([Ca²⁺]s >0.1 µM; above-threshold or contracting) and lower ([Ca²⁺]s <0.1 µM; sub-threshold or relaxing) concentrations of Ca²⁺. An interesting new finding in the present study is that peptide C enhanced ryanodine binding and induced SR Ca²⁺ release at sub-threshold [Ca²⁺] for muscle contraction, but at above-threshold [Ca²⁺] it produced an antagonistic effect against the channel activation by peptide A and T-tubule depolarization. On the other hand, peptide A significantly enhanced ryanodine binding and induced SR Ca²⁺ release in both ranges of Ca²⁺ (0.01-1.0 µM). In the present study, we also carried out the peptide-mediated site-directed fluorescent labeling using peptide C as a site-directing carrier at various [Ca²⁺]s ranging in both sub- and above-threshold concentrations. The results of the labeling experiment suggest that there are two classes of peptide C binding; one, Ca²⁺-independent and the other, Ca²⁺-dependent. Based upon these findings, we propose that the Ca²⁺-independent binding of peptide C produces activation of the RyR, whereas the Ca²⁺-dependent binding produces the antagonistic effect.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation-- Triad-enriched microsomal fraction of skeletal muscle was prepared from the rabbit back paraspinous and hind leg skeletal muscle by a method of differential centrifugation as described previously (19). Microsomes from the final centrifugation were homogenized in a sample solution containing 0.3 M sucrose, 0.15 M potassium gluconate, proteolytic enzyme inhibitors (0.1 mM phenylmethanesulfonyl fluoride, 1 µg/ml leupeptin, 2.0 µg/ml soybean trypsin inhibitor), 20 mM MES, pH 6.8, to a final concentration of 20-30 mg/ml, frozen immediately in liquid N₂, and stored at 78 °C.

Cardiac muscle microsomes were prepared as follows: rabbit ventricular cardiac muscle was homogenized in 4 volumes of 0.3 M sucrose, 20 mM MOPS, pH 7.2, 0.1 mM phenylmethanesulfonyl fluoride, 5 µg/ml leupeptin, 2.0 µg/ml soybean trypsin inhibitor, and 50 µM BAPTA in a Waring blender at high speed. During the homogenization the pH value was adjusted to 7.2. The homogenate was then centrifuged at 6,000 × g for 10 min. The pellet was discarded, and the supernatant was filtered through Whatman filter paper. The filtrate was centrifuged at 10,000 × g for 50 min. The pellet was discarded, and supernatant was filtered through Whatman filter paper. The filtrate was centrifuged at 17,000 × g for 50 min. The resultant pellet was resuspended in the above solution to a final concentration of 20-30 mg/ml, frozen immediately in liquid N₂, and stored at 78 °C.

Peptides Used and Peptide Synthesis-- We used two II-III loop peptides corresponding to the Thr⁶⁷¹-Leu⁶⁹⁰ (peptide A) and Glu⁷²⁴-Pro⁷⁶⁰ (peptide C) regions of the II-III loop of the DHP receptor 1 subunit of the rabbit skeletal muscle (6). 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.

Reagents Used-- [³H]Ryanodine was purchased from PerkinElmer Life Sciences. Recombinant calpain II was purchased from Calbiochem. SAED was obtained from Pierce.

[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; PerkinElmer Life Sciences), 0.15 M KCl, 1 mM BAPTA, various concentrations of CaCl₂ (to create various levels of well defined Ca²⁺ concentration), and 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 three times with 5 ml (in each washing step) 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 µM non-radioactive ryanodine. The assays were carried out in duplicate, and each datum point was obtained by averaging the duplicates.

Site-specific MCA Labeling of the RyR by Mediation of Peptide C-- Site-specific fluorescent labeling of the peptide C binding site of the RyR moiety of the triad was performed using the cleavable heterobifunctional cross-linking reagent, SAED (20) in the following way. First, peptide-SAED conjugates were formed by incubating 0.5 mM peptide C with 0.5 mM SAED in 20 mM HEPES, pH 7.5, for 60 min at 22 °C in the dark. The reaction was quenched by 20 mM lysine. Free SAED was removed using Sephadex G15 gel filtration. The peptide-SAED conjugate (~5 µM) was mixed with 2 mg/ml triad protein in the sample solution (see "Preparation") containing 1 mM BAPTA and various concentrations of CaCl₂ (to create various levels of well defined Ca²⁺ concentration) and incubated for 5 min in the dark. Then the reaction mixture was photolyzed with UV light in a Pyrex tube at 4 °C for 2 min. -Mercaptoethanol was added (100 mM final concentration) to cleave the disulfide bond of SAED. After incubation on ice for 1 h, the mixture was centrifuged at 100,000 × g for 15 min, and the sedimented vesicles were re-suspended in the sample solution to a final protein concentration of ~ 20 mg/ml.

RyR Digestion by Calpain II-- Fluorescence-labeled microsomes (1 mg/ml) were mixed with recombinant calpain II at the ratio of 6 units of calpain to 1 mg of SR protein in a buffer containing 150 mM NaCl and 20 mM MOPS, pH 7.2. Digestion was started with the addition of 3 mM CaCl₂. After the digestion for 6 min at 22 °C, the reaction was stopped by addition of 3 mM BAPTA.

Fluorescence Gel Analysis-- Fluorescence gel was illuminated with a 360-nm UV lamp through a UG-1 filter (Schott), and the images were obtained with a digital camera (Olympus C-2020) using a 440-nm interference filter with 40 nm of bandwidth. The fluorescence intensity was analyzed with NIH image software.

Assays of Depolarization-induced Ca²⁺ Release-- To induce Ca²⁺ release by T-tubule depolarization, we employed the K⁺ to Na⁺ replacement protocol, which was originally devised in the skinned fiber system by Lamb and Stephenson (21) and was adopted to our triad system (22). The T-tubule moiety (1.0 mg/ml) of the triad was first polarized by incubating in the base solution (150 mM potassium gluconate, 15 mM NaCl, 20 mM imidazole, pH 6.8) containing 5.0 mM MgATP, 100-150 µM CaCl₂, and an ATP-regenerating system (2.5 mM phosphoenolpyruvic acid and 10 units/ml pyruvate kinase) for 10 min. Then the T-tubule moiety was depolarized by mixing in a stopped-flow apparatus (Bio-Logic SFM-4) 30 µl of the solution (Solution A) containing the polarized triads with 120 µl of depolarization solution (150 mM sodium gluconate, 15 mM NaCl, 20 mM imidazole, pH 6.8; Solution B).

The time course of Ca²⁺ release was monitored in a stopped-flow apparatus (Bio-Logic SFM-4 with a MOS-200 optical system; excitation at 440 nm, emission at 510 nm using an interference filter with 40 nm of bandwidth) using fluo-3 as a Ca²⁺ indicator as described previously (13, 22, 23). Six to ten traces (each representing 1,000 data points) of the fluo-3 signal were averaged for each experiment.

Assays of Ca²⁺ Release Induced by the II-III Loop Peptides-- To induce Ca²⁺ release triggered by several voltage-independent agonists of the RyR as a control, the microsomes (0.4 mg/ml) were incubated in a solution containing 0.15 M potassium gluconate, 1 mM MgATP, 40-50 µM CaCl₂, 20 mM MES, pH 6.8, for 5 min for active Ca²⁺ loading. Then 1 volume of the above solution was mixed with 1 volume of a release solution containing 0.15 M potassium gluconate, 5.0 µM fluo-3, 20 mM MES, pH 6.8, and various concentrations of peptide C or peptide A or both. The time course of SR Ca²⁺ release was monitored in a stopped-flow apparatus (Bio-Logic SFM-4) using fluo-3 as a Ca²⁺ indicator as described previously (24). Six to ten traces (each representing 1,000 data points) of the fluo-3 signal were averaged for each stopped-flow measurement, and several measurements were repeated for each experiment. In the experiment shown in Fig. 3A and the upper part of Fig. 4, 30 µM BAPTA was added to Solution B. This concentration of BAPTA, together with an endogenous calcium, weakly buffered the [Ca²⁺] of the reaction mixture at 0.035 µM, as determined by calibration.

Curve Analysis and Statistics-- Most Ca²⁺ release time courses could be fitted by the equation: y = A {1  exp ( k × t)}, where A is the maximum amount of Ca²⁺ release, k is the rate constant of Ca²⁺ release, and t is the reaction time; A*k gives the initial rate of Ca²⁺ release, because (dy/dt)_t = 0 = A*k. For the calculation of the maximal rate of Ca²⁺ release in an early phase in the experiments shown in Fig. 3, A and B, in which some traces could not be fitted by an exponential equation, the release curve was fitted first by y = at⁵ + bt⁴ + ct³ + dt² + et + f and then the rate was calculated from the maximal value of the dy/dt curve. Paired t test was employed to determine the statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In our previous studies (6, 11, 13, 14), we reported that peptide A enhances ryanodine binding and induces a rapid Ca²⁺ release, whereas peptide C antagonizes the activation of ryanodine binding and Ca²⁺ release by peptide A. However, most of our previous studies were carried out at [Ca²⁺]s higher than 0.2 µM; many experiments were carried out at or higher than 1.0 µM. Because the in vivo studies of E-C coupling described in the Introduction (3, 4, 16) have been carried out probably in a rather low range of the [Ca²⁺], we decided to investigate the effects of peptide A and peptide C in a broad range of [Ca²⁺] (0.01-1.0 µM) with particular focus to the lower range of [Ca²⁺].

Peptide A Activates the RyR in Both High and Low Ca²⁺ Concentration Ranges in an RyR1-specific Manner-- Fig. 1, A and B shows the effects of 50 µM peptide A (a maximally activating concentration) on the ryanodine binding activity of the RyR of skeletal muscle and cardiac muscle microsome preparations, respectively, at different Ca²⁺ concentrations in the assay solution. Incidentally, the Ca²⁺ of our assay solution is equivalent to the cytoplasmic Ca²⁺ in the case of the whole-cell system. In both cases of skeletal muscle (Fig. 1A) and cardiac muscle (Fig. 1B), the amount of ryanodine bound (in the absence of added peptides) decreased considerably upon decreasing the [Ca²⁺] (Fig. 1, A and B, upper panel) as is well known in the literature (25, 26). Presumably, this reflects the fact that the population of the Ca-bound RyR molecules (Ca-RyR), which is the species that binds ryanodine, decreases with the decrease in the [Ca²⁺]. In case of the skeletal muscle microsome preparation (Fig. 1A), 50 µM peptide A produced a significant (about 3-fold) enhancement of the ryanodine binding activity at all Ca²⁺ concentrations examined. Interestingly, the relative magnitude of activation of the RyR by peptide A {(ryanodine bound in the presence of added peptide A)/(ryanodine bound in the absence of added peptide) in %} is essentially the same over the broad range of [Ca²⁺] from 0.01 to 1.0 µM, as shown in the lower part of Fig. 1A. In contrast to the above, the same concentration (50 µM) of peptide A produced virtually no effect on the ryanodine binding activity of the cardiac muscle microsome preparation at all Ca²⁺ concentrations examined as shown in Fig. 1B. These results indicate that peptide A activates the RyR1 (skeletal muscle isoform of the RyR) but not the RyR2 (cardiac muscle isoform of the RyR).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   A, the [Ca2+] dependence of [3H]ryanodine binding to the rabbit skeletal muscle microsomes (skeletal muscle isoform of the RyR; RyR1) in the absence and in the presence of added peptide A (50 µM). Upper figure, the amounts of ryanodine bound (pmol/mg) at various concentrations of Ca2+ (0.01-1.0 µM). Lower figure, relative enhancement of ryanodine binding by 50 µM peptide A, % control. Note that the magnitude of enhancement of ryanodine binding by peptide A, if expressed as the % enhancement of the control, is essentially identical (about 340% control) at all Ca2+ concentrations examined (lower figure). Each datum point represents the mean + S.E. of six or more experiments carried out in duplicate. B, the [Ca2+] dependence of [3H]ryanodine binding to the rabbit cardiac muscle microsomes (cardiac muscle isoform of the RyR; RyR2) in the absence and in the presence of added peptide A (50 µM). Upper figure, the amounts of ryanodine bound (pmol/mg) at various concentrations of Ca2+ (0.01-1.0 µM). Lower figure, relative enhancement of ryanodine binding by 50 µM peptide A, % control. Each datum point represents the mean ± S.E. of five experiments carried out in duplicate.

Peptide C Shows Ca²⁺-dependent Dual Functions-- As previously described (6, 13), at a higher [Ca²⁺] (e.g. at 1.0 µM), peptide C produced no appreciable effect on the RyR, but if present together with peptide A, peptide C inhibited peptide A-induced activation of the RyR (i.e. peptide A-dependent enhancement of ryanodine binding, production of the active conformational state of the RyR and induction of SR Ca²⁺ release) in a concentration-dependent manner (6, 13). In the present study we carried out similar experiments in a broader range of the Ca²⁺ concentrations by using the skeletal muscle microsome preparation (Fig. 2A) and the cardiac muscle microsome preparation (Fig. 2B). In the experiments shown in Fig. 2A, we determined, at each of different [Ca²⁺]s (0.01, 0.03, 0.1, 0.3, and 1.0 µM), the peptide C concentration dependence of the ryanodine binding activity of the RyR1 in the absence (left column) or in the presence of a maximally activating concentration (50 µM) peptide A (right column). To facilitate the comparison of the concentration-dependent activity patterns obtained at different [Ca²⁺]s, all data are expressed in the changes in the amount of ryanodine bound produced by the added peptide C at each [Ca²⁺] investigated. Peptide C alone (namely in the absence of added peptide A; see the left column of Fig. 2A) produced no appreciable effect at [Ca²⁺] 0.1 µM, in agreement with our previous results. Interestingly, however, peptide C produced a small but statistically significant enhancement of ryanodine binding at 0.01 µM Ca²⁺. Thus, it appears that peptide C retains the ability to activate the RyR without requiring the Ca²⁺ (unlike peptide A; see above). When the data obtained in the presence of added peptide A are analyzed as a function of the [Ca²⁺] (Fig. 2A, right column), it reveals very interesting [Ca²⁺]-dependent dual (activation and inhibition) functions of peptide C. In agreement with our previous studies, at higher [Ca²⁺]s (0.3 and 1.0 µM), peptide C blocked peptide A activation in a concentration-dependent manner. At 0.1 µM Ca²⁺ or at even lower [Ca²⁺]s (0.03 and 0.01 µM), peptide C produced an appreciable activation, in addition to peptide A activation (the maximally activating concentration of peptide C being 50-100 µM). Thus, it appears that at [Ca²⁺]s lower than 0.1 µM (i.e. sub-threshold Ca²⁺ concentrations for muscle contraction), either peptide A or peptide C works as an activator of the RyR, and if both work together they seem to produce additive activation effects.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   A, [Ca2+]-dependent dual effects of peptide C on the ryanodine binding activity of the skeletal muscle microsomes (RyR1); at lower Ca2+ concentrations, peptide C activates the RyR (enhancement of ryanodine binding) both in the absence and the presence of peptide A; at higher Ca2+ concentrations, peptide C inhibits the RyR (reduction of ryanodine binding) in the presence of peptide A. Ryanodine binding assays were carried out in the presence of various BAPTA/calcium buffers, as described under "Experimental Procedures." Each datum point represents the difference between the amount of [3H]ryanodine bound in the presence of peptide C and that of the control (left column, 0 peptide C; right column, 0 peptide C and 50 µM peptide A) in pmol/mg protein. Each datum point represents the mean ± S.E. of six to eight experiments carried out in duplicate. *, p < 0.05; §, p < 0.01. B, lack of effect of peptide C on the ryanodine binding activity of the cardiac muscle microsomes (RyR2); at both low (0.01 µM) and high (1.0 µM) Ca2+ concentrations, peptide C has no effect on the RyR2 in the absence and in the presence of peptide A. Ryanodine binding assays were carried out in the presence of BAPTA/calcium buffers. Each datum point represents the difference between the amount of [3H]ryanodine bound in the presence of peptide C and that of the control (left column, 0 peptide C; right column, 0 peptide C and 50 µM peptide A) in pmol/mg protein. Each datum point represents the mean ± S.E. of five experiments carried out in duplicate.

Fig. 2B shows the results of the same type of ryanodine binding experiments as shown in Fig. 2A except that the cardiac muscle microsome preparation was used in this case. Because the most prominent blocking and activation patterns of the RyR1 by peptide C were observed at the Ca²⁺ concentrations of 1.0 and 0.01 µM, respectively, we investigated the effects of peptide C on the RyR2 at these two Ca²⁺ concentrations. As seen in Fig. 2B, peptide C produced virtually no effects on the ryanodine binding activity of the RyR2 at both high and low Ca²⁺ concentrations (left column). Fig. 2B also shows that even in the presence of 50 µM peptide A peptide C has no effect on the RyR2 at both Ca²⁺ concentrations (right column). Thus, it is concluded that the Ca²⁺-dependent dual functions of peptide C described here represent the events specific to the skeletal muscle isoform of the RyR.

We previously reported that a chimeric peptide C, whose Gln⁷²⁶-Gly⁷⁴³ segment is a cardiac-type sequence, and the rest is a skeletal muscle-type sequence, showed much less blocking effect on peptide A activation at 1.0 µM Ca²⁺ (13). We found that the chimeric peptide C had virtually no activation effect at 0.03 and 0.01 µM Ca²⁺ (data not shown). This indicates that the skeletal-type sequence in the Gln⁷²⁶-Gly⁷⁴³ segment (approximately corresponding to the determinant region) is important for both activating (at low Ca²⁺) and blocking (at high Ca²⁺) functions of peptide C.

The left panel of Fig. 3A shows the time course of Ca²⁺ release from the skeletal muscle microsomes induced by various concentrations of peptide C alone at 0.035 µM Ca²⁺ at weakly buffered conditions.² Importantly, peptide C induced Ca²⁺ release in a concentration-dependent manner. In agreement with the ryanodine binding data (see Fig. 1A and Fig. 2A), 100 µM peptide C produced about the same amount of Ca²⁺ release as produced by 10 µM peptide A. The addition of various concentrations of peptide C, together with 10 µM peptide A, produced additional Ca²⁺ release again in a concentration-dependent manner (right panel of Fig. 3A). Fig. 3B shows the time courses of Ca²⁺ release induced by various concentrations of peptide C alone (left panel) and various concentrations of peptide C, together with 10 µM peptide A (right panel), at 0.7 µM Ca²⁺. Addition of increasing concentrations of peptide C alone produced no appreciable effect, in agreement with the fact that peptide C alone was without effect on ryanodine binding at [Ca²⁺]s in the range of 0.1 to 1.0 µM. Addition of increasing concentrations of peptide C in the presence of 10 µM peptide A inhibited peptide A-induced Ca²⁺ release in a [peptide C]-dependent manner, in good agreement with the ryanodine binding data obtained either at 0.3 or 1.0 µM (see Fig. 2A and our previous studies (6, 13)). Table I depicts the maximal rate of Ca²⁺ release in an early phase of the release reaction and the statistic variations.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Ca2+-dependent dual effects of peptide C on Ca2+ release. A, at a lower Ca2+ (0.035 µM), peptide C induces Ca2+ release in a concentration-dependent manner; together with 10 µM peptide A, peptide C produces additive activation effects. B, at a higher Ca2+, peptide C alone produces virtually no Ca2+ release, but peptide C inhibits peptide A-induced Ca2+ release in a concentration-dependent manner. Ca2+ release was induced by mixing the actively calcium-loaded SR with various concentrations of peptide C alone, or together with 10 µM peptide A, in a stopped-flow apparatus, and the time course of Ca2+ release was monitored with fluo-3 ("Experimental Procedures"). Each stopped-flow trace of the Ca2+ release time courses represents the result of signal averaging of five to seven experiments; each experiment consisting of six to ten repeats of the same stopped-flow reaction. Statistic variation (S.E.) among these experiments is indicated at every 1-s (A) or 0.5-s (B) interval in each Ca2+ release time course. For each Ca2+ release curve shown, the release time course trace with added peptides was subtracted by the control trace with no added peptide.

As seen from the above data, the mode of action of peptide C is quite distinguishable from that of peptide A in that the former shows activating and inhibiting functions at relaxing and contracting concentrations of Ca²⁺, respectively, whereas the latter has only activating function regardless of Ca²⁺. In an attempt to gain some clues for the explanation of such dual functions of peptide C, we carried out the peptide-mediated site-specific MCA incorporation at various concentrations of Ca²⁺ (corresponding to the concentrations used in the ryanodine binding experiments of Fig. 1A and Fig. 2A). The amount of MCA that has been incorporated by mediation of peptide A or peptide C is presumably proportional to the amount of the peptide bound to the RyR. This permits us to assess the Ca²⁺ dependence of the peptide binding to the RyR from the fluorescence intensity of the protein-incorporated MCA, as shown in Fig. 4. In this experiment, the fluorescence probe MCA was incorporated into the RyR1 moiety of the triad by mediation of peptide A (left) or by peptide C (right) in a site-specific manner at different concentrations of Ca²⁺ as indicated, and the MCA-labeled RyR was subjected to digestion with calpain II (see "Experimental Procedures"). As demonstrated in Ref. 27, calpain II cleaved the RyR into two fragments, a 150-kDa N-terminal fragment and a 400-kDa C-terminal fragment (see Coomassie Blue-stained gels; calpain, before calpain digestion and +calpain, after calpain digestion). Fig. 4 (top row) shows the fluorescence staining gel pattern of the SR preparation after calpain digestion. As seen, the site of peptide A-mediated MCA incorporation is localized in the 400-kDa C-terminal calpain fragment, whereas the site of peptide C-mediated MCA incorporation is in the 150-kDa N-terminal calpain fragment. These results suggest that peptide A and peptide C bound to the 400-kDa C-terminal region and the 150-kDa N-terminal region of the RyR, respectively, at all Ca²⁺ concentrations investigated. Fig. 4 (bottom row) shows the relative fluorescence intensity of the MCA-labeled band as a function of the Ca²⁺ concentration at which MCA incorporation (or peptide binding) took place. Interestingly, there is a clear difference in the Ca²⁺-dependent MCA labeling pattern between peptide A and peptide C. Namely, the [Ca²⁺] dependence of the incorporation is much sharper in the case of peptide A-mediated incorporation compared with the case of peptide C-mediated incorporation. It is noted that the [Ca²⁺] dependence curve of peptide A-mediated incorporation shows essentially the same pattern as that of peptide A activation of the ryanodine binding activity shown in Fig. 1A. Because the calcium-bound form of the RyR (Ca-RyR) seems to be responsible for appreciable ryanodine binding (see above), the fact that the [Ca²⁺] dependence curves of ryanodine binding and MCA incorporation are essentially identical to each other suggests that the binding of peptide A takes place to the Ca-RyR but not to the Ca-unbound RyR. In contrast, there was a significant amount of MCA incorporation at 0.01 µM Ca²⁺ in the case when peptide C was used as a site-directing carrier. Thus, its [Ca²⁺] dependence curve shows two clearly resolvable components (Ca²⁺-independent and Ca²⁺-dependent components). This suggests that unlike peptide A, peptide C can bind to both Ca-unbound RyR and Ca-RyR. It is tempting to speculate that peptide C binding to the Ca-unbound RyR is responsible for the activation by this peptide, whereas binding to the Ca-RyR causes the antagonistic effect on the channel activation.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   The fluorescence intensity of the MCA-labeled calpain fragments of the RyR (the 400-kDa band in the case of peptide A-mediated incorporation, and the 150-kDa band in the case of peptide C mediation) varies with the Ca2+ concentration at which the fluorescence labeling was performed. Upper panel, Coomassie Blue-stained gel picture, calpain, microsomes before calpain digestion; +calpain, microsomes after calpain digestion (see "Experimental Procedures"). Upper panel, fluorescence gel picture, fluorescence labeling was performed at various Ca2+ concentrations adjusted by strong BAPTA-Ca buffers, followed by digestion with calpain II ("Experimental Procedures"). Lower panel, the fluorescence intensity of the incorporated MCA as a function of the Ca2+ concentration. The incorporated fluorescence was determined by densitometric scan of the gel and expressed as % maximal incorporation (taking the value at 1.0 µM Ca2+ as 100%). The dotted line in the peptide C curve shows the peptide A curve as the reference. Note that the peptide A curve shows the essentially same Ca2+ dependence profile as that of enhancement of ryanodine binding by the peptide. However, the peptide C curve is less Ca2+-dependent, suggesting that peptide C-mediated MCA incorporation (or peptide C binding) consists of Ca2+-independent and Ca2+-dependent components. The whole set of this experiment was repeated four times for the reproducible results. Error bar represents mean ± S.E. of four experiments.

Peptide C Inhibits T-tubule Depolarization-induced Ca²⁺ Release-- Peptide C antagonizes the activation by peptide A at the above-threshold Ca²⁺ as described above. Then the important question to address is whether peptide C also blocks depolarization-induced Ca²⁺ release. In the experiment shown in the bottom row of Fig. 5, triads were first incubated with 100 µM peptide C during the priming/Ca²⁺ loading process and then various types of Ca²⁺ release were induced at about 0.3 µM Ca²⁺ by depolarizing the T-tubule or by adding caffeine or polylysine. The corresponding control time courses of Ca²⁺ release without pre-incubation with peptide C are shown in the upper row of Fig. 5. Table II depicts various kinetic parameters and statistic variations and significance of the data shown in Fig. 5. As seen in Fig. 5 and Table II, 100 µM peptide C inhibited depolarization-induced Ca²⁺ release almost completely without producing any appreciable effect on Ca²⁺ release induced by caffeine or polylysine. Thus, it seems that the inhibitory effect of peptide C at the above-threshold concentrations of Ca²⁺ is exerted specifically upon depolarization- or peptide A-induced Ca²⁺ release. It would be nice to carry out the same type of experiment at the sub-threshold concentrations of Ca²⁺, but we did not perform such an experiment for the following reason. Because peptide C induces Ca²⁺ release at low Ca²⁺ concentrations, partial depletion of the SR calcium by peptide C will become an inhibitory factor of the subsequent Ca²⁺ release, making the interpretation of the results ambiguous.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Pre-incubation of triads with peptide C blocked depolarization-induced Ca2+ release but produced no effect on the other types of Ca2+ release (induced by caffeine or polylysine). Triads were incubated with 100 µM peptide C during the priming/Ca2+ loading process, and Ca2+ release was induced as described under "Experimental Procedures." Each stopped-flow trace of the Ca2+ release time courses represents the result of signal averaging of three to four experiments; each experiment consisting of six to ten repeats of the same stopped-flow reaction. Statistic variation (S.E.) among these experiments is indicated at every 0.5-s interval in each Ca2+ release time course.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

There is a considerable amount of inconsistency concerning the proposed locations of critical domains of the DHP receptor 1 II-III loop deduced from the in vivo and in vitro studies. As described in the Introduction, major controversy exists in the assignment of the II-III loop domain critical for the regulation of E-C coupling. The in vitro finding that peptide A produces conspicuous activation effects on the RyR, whereas peptide C antagonizes the activating function of peptide A (6, 13-15), led to the view that the corresponding two regions of the loop (peptide A, Thr⁶⁷¹-Leu⁶⁹⁰ and peptide C, Glu⁷²⁴-Pro⁷⁶⁰) are involved in the voltage-dependent activation and re-priming of the RyR Ca²⁺ channel, respectively. The recent report that not only peptide A but also p720-765 (the peptide essentially identical to peptide C) activated the RyR1 Ca²⁺ channel suggested that these two domains may be involved in the activation of the skeletal muscle Ca²⁺ release channel (12). On the other hand, the in vivo finding with myotubes expressing chimeric cardiac DHP receptor suggested that the region corresponding to peptide C alone may be sufficient to perform skeletal-type E-C coupling. Thus, the replacement of the Phe⁷²⁵-Pro⁷⁴² region, or its extended Leu⁷²⁰-Leu⁷⁶⁴ region, of the II-III loop from the cardiac muscle-type to the skeletal muscle-type sequence rescued the skeletal-type E-C coupling (3, 4), suggesting that this region may retain the capability to mediate the key processes of E-C coupling (both orthograde and retrograde communications between the DHP receptor and the RyR). Furthermore, according to the recent reports, scrambling of the critical sequence of the peptide A region produced no appreciable effect on the in vivo E-C coupling (16) and that replacement of a major portion of the II-III loop other than the Leu⁷²⁰-Leu⁷⁶⁴ region with the sequence quite dissimilar to the skeletal muscle II-III loop produced little or no effect on E-C coupling (17). Furthermore, deletion of the peptide A region of the II-III loop had little effect on E-C coupling in vivo (18).

Despite the above-mentioned controversy regarding peptide A, there seems to be a broader agreement in that the peptide C region of the II-III loop may play some physiological roles in E-C coupling. However, its actual functions have not yet been elucidated. The main goal of the present study is to gain more information about this question. The Phe⁷²⁵-Pro⁷⁴² region, the so-called determinant of skeletal muscle-type E-C coupling (3), corresponds to the N-terminal half of our peptide C, and the critical Leu⁷²⁰-Leu⁷⁶⁴ region represents a slightly extended version of peptide C (Glu⁷²⁴-Pro⁷⁶⁰). According to our recent studies of ryanodine binding and Ca²⁺ release (13), peptide C alone produced virtually no appreciable effect on the RyR, but if added together with peptide A it blocked peptide A activation in a concentration-dependent manner. In the present study, we decided to re-investigate the effects of peptide C under extended conditions to examine other functions that peptide C may possibly have. In agreement with our previous studies, at Ca²⁺ concentrations higher than 0.1 µM, peptide C alone produced no appreciable effect, but if present together with peptide A, peptide C blocked the activation by peptide A. The most interesting new finding in the present study is that at Ca²⁺ concentrations lower than 0.1 µM, peptide C in fact enhanced ryanodine binding and induced Ca²⁺ release, indicating that peptide C retains the capability of being the activator of E-C coupling. This is consistent with the recent report that p720-765 (a peptide corresponding to the Leu⁷²⁰-Gln⁷⁶⁵ region of the II-III loop) increased Po (12), although the [Ca²⁺] dependence of the activation effect was not investigated.

The [Ca²⁺]-dependent transition from the activation mode (at lower Ca²⁺) to the inhibition mode of peptide C (at higher Ca²⁺) took place in the vicinity of 0.1 µM Ca²⁺. Because this transition point is essentially identical to the threshold Ca²⁺ for relaxation/contraction of muscle (0.1-0.2 µM; see Ref. 28), the Ca²⁺-dependent activation/blocking functions of peptide C described is quite intriguing. Peptide C does not seem to retain any Ca²⁺ binding site with appreciable affinity. Therefore, the Ca²⁺-dependent switch from the activation mode to the inhibition mode must be controlled by the Ca-unbound and the Ca-bound states of the RyR. The peptide C-mediated site-directed MCA labeling study presented here provided some clues to the possible mechanism underlying the Ca²⁺-dependent dual functions of peptide C. As shown in the present study of the [Ca²⁺] dependence of peptide C-mediated MCA incorporation (the measure of peptide C binding), peptide C appears to bind to both Ca-unbound RyR and Ca-bound RyR. This finding has provided a useful clue to account for the data concerning the Ca²⁺-dependent dual functions of peptide C. Because the activation of peptide C takes place at a very low Ca²⁺ such as 0.01 µM, at which a majority of the RyR population must be in the form of Ca-unbound RyR, we can assume that the peptide C binding to the Ca-unbound RyR seems to be responsible for the activation. Consequently, peptide C binding to the Ca-bound RyR seems to be responsible for its antagonistic function against the channel activation. This antagonism is exerted when the RyR channel is activated by peptide A or T-tubule depolarization but not when the channel is activated by caffeine or polylysine as shown in the present study. This opens a new possibility that peptide C may bind to different sites in a Ca²⁺-dependent manner. As seen in the experiment shown in Fig. 4, the peptide C-mediated MCA labeling, i.e. peptide C binding, occurs exclusively to the 150-kDa N-terminal calpain fragment regardless of Ca²⁺ concentrations. However, it may well be that there is a Ca²⁺-dependent shift of the peptide C binding site within the 150-kDa N-terminal region, as in the case of calmodulin binding, in which the precise location of the calmodulin binding site on the RyR shows a slight shift in a Ca²⁺-dependent manner (29).

One of the interesting findings in the present study is that neither peptide A nor peptide C produces any appreciable effects on the cardiac isoform of the RyR. Because the skeletal muscle-type II-III loop-mediated E-C coupling mechanism does not seem to be operating in the cardiac system (30, 31), this suggests that both peptide A and peptide C retain at least some of the in vivo features of E-C coupling. The present finding that peptide C blocked T-tubule depolarization (and peptide A-induced release, as well) without affecting Ca²⁺ release induced by pharmacological agents may also suggest the usefulness of peptide C as a physiologically relevant probe. Although some of the functions of peptide A and peptide C described here may not necessarily represent the functions of their in vivo counterparts, we tentatively propose the following alternative hypotheses. (a) At relaxing concentrations of the cytoplasmic Ca²⁺, where the in vivo E-C coupling experiments are carried out, both peptide A and peptide C regions of the II-III loop work as the activator of E-C coupling. Deleterious modifications made in the peptide A region (cf. recent in vivo E-C coupling experiments described above) would then be compensated by the activating function residing in the peptide C region, leaving the E-C coupling function virtually intact. (b) The peptide A region plays a little role in E-C coupling in vivo, and the peptide C region alone is sufficient for both activation (at low Ca²⁺) and blocking/priming (at high Ca²⁺) of E-C coupling. However, we encounter one problem in either of these hypotheses. That is, as seen in both ryanodine binding and Ca²⁺ release assays the activating function of peptide C was significantly lower than that of peptide A. It might well be that the activating function intrinsically present in peptide C is intensified under in vivo conditions.

	ACKNOWLEDGEMENTS

We thank Dr. Graham D. Lamb for comments on this work and Dr. Renne C. Lu, Dr. Paul Leavis, Gina Pagani, and Elizabeth Gowell for help in the synthesis and purification of the peptides.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant AR 16922.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Boston Biomedical Research Inst., 64 Grove St., Watertown, MA 02472. Tel.: 617-658-7774; Fax: 617-972-1761; E-mail: ikemoto@bbri.org.

Published, JBC Papers in Press, October 26, 2001, DOI 10.1074/jbc.M105837200

² Unlike the ryanodine binding assay, which permitted one to use strong BAPTA-Ca buffers, in the Ca²⁺ release assay with fluo-3 we could use only a weak BAPTA-Ca buffer, but we calibrated the free Ca²⁺ concentration at the steady state of Ca²⁺ uptake (viz. at time 0 of the Ca²⁺ release reaction) as described under "Experimental Procedures."

	ABBREVIATIONS

The abbreviations used are: DHP, dihydropyridine; BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; E-C, excitation-contraction; MCA, methyl coumarin acetamido; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; RyR, ryanodine receptor; SAED, sulfosuccinimidyl 2-[7-azido-4-methyl-coumarin-3-acetamido] ethyl-1,3'-dithiopropionate; SR, sarcoplasmic reticulum.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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