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J. Biol. Chem., Vol. 277, Issue 2, 984-992, January 11, 2002

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T-tubule Depolarization-induced Local Events in the Ryanodine Receptor, as Monitored with the Fluorescent Conformational Probe Incorporated by Mediation of Peptide A^*

Takeshi Yamamoto and Noriaki Ikemoto^§¶

From the  Boston Biomedical Research Institute, Watertown, Massachusetts 02472 and ^§ Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, March 15, 2001, and in revised form, October 15, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

There is a considerable controversy about the postulated role of the Thr⁶⁷¹-Leu⁶⁹⁰ (peptide A) region of the dihydropyridine (DHP) receptor 1 II-III loop. Here we report that peptide A introduced the fluorescence probe methyl coumarin acetamido (MCA) in a well defined region of the ryanodine receptor (RyR), A-site, in a specific manner. Depolarization of the T-tubule moiety of the triad induced a rapid increase of the fluorescence intensity of the MCA attached to the A-site. Other RyR agonists, which activate the RyR without mediation of the DHP receptor (e.g. caffeine, polylysine, and peptide A), induced Ca²⁺ release without producing such an MCA fluorescence increase. Both magnitudes of the fluorescence change and Ca²⁺ release increased with the increase in the degree of T-tubule depolarization. MCA fluorescence increase at the A-site and subsequent sarcoplasmic reticulum Ca²⁺ release were blocked by blocking of the DHP receptor-to-RyR communication. These results may be accounted for by two alternative models as follows. (a) Upon T-tubule depolarization a portion of the DHP receptor comes close to the RyR, forming a hydrophobic interface (within such an interface the A-site is located), or (b) T-tubule depolarization may produce a local conformational change in the A-site-containing region of the RyR that is not necessarily within the DHP receptor/RyR junction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The concept that one of the cytoplasmic loops of the DHP¹ receptor 1 subunit (II-III loop) plays a critical role in skeletal muscle-type E-C coupling emerged from an earlier finding of Tanabe et al. (1, 2) that replacement of the II-III loop of the cardiac DHP receptor with the skeletal muscle-type sequence conferred the skeletal muscle-type E-C coupling activity in dysgenic myotubes expressing chimeric DHP receptors. According to further studies with chimeras (3), replacement of the Phe⁷²⁵-Pro⁷⁴² region of the II-III loop from the cardiac to the skeletal sequence conferred the skeletal type E-C coupling, leading to the proposal that the critical functions required for the skeletal-type E-C coupling are localized in this region (the so-called "determinant"). It was also shown that the skeletal Leu⁷²⁰-Leu⁷⁶⁴ region (which is approximately identical to our peptide C described below and that contains the determinant region) rescued both skeletal-type orthograde and retrograde communications between the DHP receptor and the RyR (4). In support of the concept that the II-III loop plays the critical role in E-C coupling, a recombinant peptide corresponding to the II-III loop in fact activated the RyR Ca²⁺ channel in the in vitro system (5).

Further studies with shorter peptides 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 residues Glu⁶⁶⁶-Glu⁷²⁶ (6) and Thr⁶⁷¹-Leu⁶⁹⁰ (peptide A, Refs. 7-11) produced a strong activation of the RyR. This suggests that the putative activator of E-C coupling may reside in the peptide A region, although whether the critical activating function is localized in the Arg⁶⁸¹-Leu⁶⁹⁰ region (peptide A-10, Ref. 12) or in the Thr⁶⁷¹-Glu⁶⁸⁰ region (13) has not yet been settled. Interestingly, peptide C corresponding to the Glu⁷²⁴-Pro⁷⁶⁰ region of the II-III loop (note that this is essentially identical to the Leu⁷²⁰-Leu⁷⁶⁴ sequence containing the determinant region described above) inhibited peptide A-mediated activation of the RyR (7, 14) and also produced a moderate inhibition of depolarization-induced tension development in the skinned muscle fiber (15). These results led us to our recent proposal that the voltage-dependent activation and blocking (re-priming) processes of skeletal-type E-C coupling are mediated by alternative binding of the peptide A and the peptide C regions of the II-III loop to the RyR, respectively (14, 16). According to the more recent studies, peptide C (7, 14) and its slightly extended version corresponding to the Leu⁷²⁰-Gln⁷⁶⁵region (13) activate the RyR under certain conditions, suggesting that peptide C can perform either activating or inhibitory function depending upon the conditions.

The above view that the peptide A region plays an important role in the activation process of E-C coupling has been questioned by several investigators. According to Proenza et al. (17), a moderate degree of scrambling of the amino acid sequence in the peptide A-10 region (see above) produced no detectable changes in E-C coupling in the dysgenic myotubes, although the same scrambling produced a severe loss of the activating function of peptide A-10 in case of the in vitro experiments (12). Furthermore, according to Wilkens et al. (18) replacement of the Leu⁷²⁰-Leu⁷⁶⁴ region of the housefly II-III loop, which has the sequence structure highly dissimilar to the skeletal muscle 1_S II-III loop, with the skeletal muscle sequence-restored skeletal muscle-type E-C coupling. Furthermore, according to the more recent report of Ahern et al. (19) deletion of the Thr⁶⁷¹-Leu⁶⁹⁰ peptide A region from the 1 subunit produced virtually no effect on Ca²⁺ conductance, charge movement, and Ca²⁺ transients. Thus, the in vivo evidence accumulated so far is inconsistent with the view that the peptide A region may play an active role in the in vivo E-C coupling.

To test the physiological significance of the information obtained from the in vitro studies with peptide A, we addressed in this study two key questions as follows. (a) Do these II-III loop peptides, peptide A and peptide C, bind to the RyR in a site-specific manner? (b) Can the fluorescence probe that is attached to the peptide A-binding site or to the peptide C-binding site report the local events that are relevant to the physiological coupling between the DHP receptor and the RyR? As shown in our recent publications (12, 14, 20, 21), the fluorescence conformational probe, MCA, can be incorporated into the designated site on the RyR in a site-specific manner using an appropriate RyR-specific ligand (e.g. the channel blocker, neomycin (14, 20), and an agonist of the RyR, polylysine (21)) as a site-directing carrier. Here we report that the site-directed fluorescence labeling technique using peptide A as a site-directing carrier permitted us to introduce the MCA probe into the 160-kDa segment at the C-terminal side of the amino acid residue 1400 of the RyR, indicating that peptide A binds to this region of the RyR in a specific manner. Furthermore, depolarization of the T-tubule moiety of the triad, but not any of chemical/pharmacological agonists of the RyR, produced a rapid increase in the fluorescence intensity of the MCA attached to the peptide A-binding site. The magnitude of the depolarization-induced Ca²⁺ release was approximately proportional to that of the MCA fluorescence change as determined at various degrees of T-tubule depolarization. Inhibition of T-tubule depolarization and T-tubule-to-RyR signal transmission resulted in the inhibition of both MCA fluorescence change and Ca²⁺ release. Various agonists of the RyR other than T-tubule depolarization, such as caffeine, polylysine, and peptide A, induced Ca²⁺ release but did not produce any appreciable change in the MCA fluorescence. These results suggest that depolarization in the T-tubule produces dramatic changes either in the DHP receptor/RyR interface or in the cytoplasmic domain of the RyR or both. It is tempting to speculate that the site of peptide A-mediated MCA labeling is localized in the DHP receptor/RyR-interacting interface, but an alternative possibility cannot be excluded that the MCA labeling site is localized in the non-junctional cytoplasmic region of the RyR (cf. Fig. 7 models).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Preparation-- Triad-enriched microsomal fractions were prepared from the rabbit back paraspinous and hind leg skeletal muscles by a method of differential centrifugation as described previously (22). 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.

Peptides Used and Peptide Synthesis-- We used two peptides, peptide A and peptide C, corresponding to the Thr⁶⁷¹-Leu⁶⁹⁰ and Glu⁷²⁴-Pro⁷⁶⁰ regions of the II-III loop of the DHP receptor 1 subunit of the rabbit skeletal muscle, respectively (7). The 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-- Anti-RyR polyclonal antibody was kindly provided by Dr. Kevin P. Campbell. Anti-residue 416, anti-residue 1417, anti-residue 2727 antibodies were kindly provided by Dr. Susan L. Hamilton. Anti-residue 5029 antibody was kindly provided by Dr. Andrew R. Marks. [³H]Ryanodine was purchased from PerkinElmer Life Sciences. Recombinant calpain II was purchased from Calbiochem. Sulfosuccinimidyl 3-((2-(7-azido-4-methylcoumarin-3-acetamido)ethyl)dithio) propionate (SAED) was from Pierce.

Site-specific MCA Labeling of the Peptide A- and Peptide C-binding Site of the RyR-- Site-specific fluorescent labeling of the peptide A- and peptide C-binding sites of the RyR moiety of the triad was performed using the cleavable hetero-bifunctional cross-linking reagent SAED (21) in the following way. First, peptide-SAED conjugates were formed by incubating 0.5 mM peptide with 0.5 mM SAED in 20 mM HEPES, pH 7.5, for 60 min at 22 °C in the dark. Both peptide A-SAED and peptide C-SAED conjugates retained essentially the same activities as those of the unmodified peptides. The reaction was quenched by 20 mM lysine. Free SAED was removed using Sephadex G15 gel filtration. The peptide-SAED conjugate (5 µM final concentration) was mixed with 2 mg/ml triad protein in the sample solution (see "Preparation" under "Experimental Procedures") containing 1 mM BAPTA/calcium buffer (1.0 µM free Ca²⁺) in the dark, and the mixture was incubated at 4 °C for about 5 min to ensure the access of the peptide-SAED conjugate to the peptide-binding sites. The incubation time of 5 min seemed to be sufficient to introduce the peptide-SAED conjugated to its target site located in the junctional triad, as judged from the fact that this incubation time was sufficient to produce a maximal MCA labeling and a maximal MCA fluorescence response upon T-tubule depolarization. Then, the mixture was photolysed 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. Gels containing electrophoretically separated protein bands were illuminated with a 360-nm UV lamp through the UG-1 filter (Schott), and the fluorescence images were obtained with a digital camera (Olympus C-2020) using a 440-nm interference filter with 40-nm bandwidth.

Proteolytic Cleavage of the RyR Polypeptide Chain-- For digestion with calpain II, fluorescently 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 solution containing 150 mM NaCl and 20 mM MOPS, pH 7.2. Digestion was started by adding 3 mM CaCl₂. After the digestion for 6 min at 22 °C, the reaction was stopped by adding 3 mM BAPTA.

For digestion with trypsin, fluorescently labeled microsomes (1 mg/ml) were mixed with L-1-tosylamido-2-phenylethyl chloromethyl ketone trypsin at the protein/trypsin ratios of 4000:1, 2000:1, 1000:1, 500:1 in a solution containing 150 mM NaCl and 20 mM MOPS (pH7.2) at 22 °C. After digestion for 6 min, the digestion was terminated by adding a 10-fold excess of soybean trypsin inhibitor.

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 (23) and was adopted to our triad system (24). 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 (solution B) having various ionic compositions to produce various degrees of depolarization (see Table I).

The time course of Ca²⁺ release was monitored in a stopped flow apparatus (Bio-Logic SFM-4 with MOS-200 optical system; excitation at 440 nm, emission at 510 nm using an interference filter with 40-nm bandwidth) using fluo-3 as a Ca²⁺ indicator as described previously (14, 20, 25). Six to 10 traces (each representing 1,000 data points) of the fluo-3 signal were averaged for each experiment.

Spectrometric Monitoring of Depolarization-induced Conformational Change in the Physiologic E-C Coupling Sites of the RyR-- The fluorescence change of the MCA that had been incorporated to the peptide A- or peptide C-binding site of the RyR in a site-specific fashion (see above) was induced by various degrees of T-tubule depolarization. The time course of the MCA fluorescence change was monitored with the stopped-flow fluorometer (Bio-Logic SFM-4 with MOS-200 optical system: excitation at 368 nm, emission at 455 nm using an interference filter with 70 nm bandwidth) as described previously (14, 19). Ten to 15 traces (each representing 1,000 data points) of the MCA signal were averaged for each experiment.

Assays of Ca²⁺ Release Induced by the Voltage-independent Agonists-- 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 one volume of the above solution was mixed with one volume of a release solution containing 0.15 M potassium gluconate, 5.0 µM fluo-3, 20 mM MES, pH 6.8, and various agonists (peptide A, polylysine, and caffeine). The time course of SR Ca²⁺ release was monitored in a stopped flow apparatus using fluo-3 as a Ca²⁺ indicator as described previously (25). Six to 10 traces (each representing 1,000 data points) of the fluo-3 signal were averaged for each stopped-flow measurement. Several such measurements (n = 3-5) were repeated for each experiment shown in the figure.

Control Assays of the Effect of the Voltage-independent Agonists on the MCA Fluorescence-- The time courses of fluorescence change of the protein-bound MCA upon mixing with various RyR agonists were monitored with the stopped-flow fluorometer as described previously (25). Ten to 15 traces (each representing 1,000 data points) of the MCA signal were averaged for each experiment.

Calculation of Kinetic Parameters and Statistics-- The time courses of MCA fluorescence change and Ca²⁺ release were fitted by the equation: y = A (1-exp(kt)), where A is the maximum amount of Ca²⁺ release, k is the rate constant of Ca²⁺ release, t is reaction time, and Ak is the initial rate of Ca²⁺ release since (dy/dt)_t=0 = A*k. Unpaired t test was employed to determine the statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Site-specific Fluorescence Labeling of the II-III Loop Peptide Binding Regions of the RyR-- In our recent studies (12, 14, 20, 24), we incorporated the conformation-sensitive fluorescent probe MCA into the trans-membrane channel domain using the Ca²⁺ channel blocker neomycin as a site-directing carrier and monitored conformational changes in the channel domain induced by various types of RyR agonists. In the present study, we introduced the MCA probe into the putative II-III loop binding region of the RyR using peptide A and peptide C as site-directing carriers. For this purpose, the triad-enriched SR fraction was incubated with the SAED-peptide A or the SAED-peptide C conjugate (azido-MCA-S-S-peptide A or azido-MCA-S-S-peptide C) followed by photo-cross linking of the conjugate with the triad via the azido group. Then peptide A or peptide C (site-directing carrier) was removed from the cross-linking site by cleaving the S-S bond of SAED, leaving the MCA that had been covalently attached to the cross-linked site (details are provided under "Experimental Procedures").

Fig. 1A depicts Coomassie Blue staining, Western blot, and MCA fluorescence-labeling patterns of the electrophoretically separated protein bands of the triad-enriched SR preparation that has been subjected to the site-directed MCA labeling by mediation of peptide A or peptide C. Fig. 1A also contains the set of staining patterns of the sample that was labeled with MCA first and then digested with calpain II. The corresponding pictures of the undigested sample (calpain) and digested sample (+calpain) are arranged side by side to facilitate the comparison. As seen in lane 9 (peptide A) and lane 11 (peptide C), the MCA fluorescence is associated almost exclusively with the 550-kDa band in both cases when the MCA labeling has taken place by mediation of peptide A and peptide C. The MCA-labeled 550-kDa band was identified as the RyR protein by immuno-staining with the RyR-specific polyclonal antibody (lane 3). The result indicates that both peptide A and peptide C have bound specifically to the RyR moiety out of numerous proteins present in the triad preparation. As shown in Fig. 1B, photolysis of the mixture of the triads and the peptide-SAED conjugate in the presence of an excess amount of unmodified peptide (cold chase experiment) resulted in a considerably reduced amount of MCA incorporation (for the quantities and statistics, see the legend to Fig. 1B). This suggests that the peptide-SAED conjugate bound to the same site as the unmodified peptide bound and that the sites of the peptide-mediated MCA labeling basically represent the sites of the peptide binding.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   A, site-specific labeling of the RyR moiety with the fluorescent conformational probe MCA by mediation of peptide A or peptide C. Note that photo-affinity cross-linking of the triad with the conjugate of SAED with peptide A or peptide C permitted specific MCA fluorescence labeling almost exclusively at the RyR out of the many proteins present in the triad preparation. Digestion of the RyR with calpain II produced two fragments with ~150 and 400 kDa. The 400-kDa fragment corresponds to the segment at the C-terminal side of the calpain cleavage site as evidenced by its reactivity with anti-residue 5029 antibody (Ab 5029), whereas the 150-kDa fragment corresponds with the segment at the N-terminal side as shown by its reactivity with anti-residue 416 antibody (Ab 416). The MCA incorporated by mediation of peptide A is almost exclusively in the 400-kDa fragment, whereas the MCA incorporated by mediation of peptide C is almost exclusively in the 150-kDa fragment. The whole set of the experiment shown in this figure was repeated at least five times for the reproducible results. B, cold chase experiments showing the competition of the un-conjugated (cold chase) peptide A (left panel) and peptide C (right panel) with the peptide-SAED conjugates. Sample 1 (lanes 1 and 1'), photo-affinity MCA labeling was performed by mediation of 5 µM peptide A-SAED conjugate. Sample 2 (lanes 2 and 2'), photo-affinity MCA labeling was performed by mediation of 5 µM peptide A-SAED conjugate in the presence of 500 µM peptide A. Sample 3 (lanes 3 and 3'), photo-affinity MCA labeling was performed by mediation of 10 µM peptide C-SAED conjugate. Sample 4 (lanes 4 and 4'), photo-affinity MCA labeling was performed by mediation of 10 µM peptide C-SAED conjugate in the presence of 500 µM peptide C. Lanes 1, 2, 3, and 4, Coomassie Blue-stained gels. Lanes 1', 2', 3', and 4', fluorescence gels. The density of the peptide A-mediated MCA fluorescence labeling in the presence of 500 µM peptide A was 23.1 ± 3.5% that of the control (without cold chase) (n = 4). The density of the peptide C-mediated MCA fluorescence labeling in the presence of 500 µM peptide C was 31.8 ± 5.2% that of the control (without cold chase) (n = 4).

To localize the sites of MCA labeling (i.e. the sites of binding of peptide A and peptide C) in the primary structure of the RyR, the MCA-labeled RyR was cleaved at the residue 1400 (26) with calpain II, which produced ~150 and ~400 kDa fragments (Fig. 1A, lane 2). The 150- and 400-kDa fragments must have originated from the N- and C-terminal segments of the RyR, respectively, because the former was stained with anti-residue 416 antibody (Fig. 1A, lane 6), but the latter was stained with anti-residue 5029 antibody (Fig. 1A, lane 8). Interestingly, the MCA fluorescence was found almost exclusively in the 400-kDa calpain fragment if the MCA labeling was mediated by peptide A (Fig. 1A, lane 10). If the MCA labeling was mediated by peptide C, however, the MCA fluorescence was found almost exclusively in the 150-kDa fragment (Fig. 1A, lane 12).

Tryptic digestion cleaved the RyR more extensively, producing shorter fragments (Fig. 2). In this figure, MCA-labeling patterns were compared with Western blot patterns obtained with various site-specific anti-RyR monoclonal antibodies. In case of peptide A-mediated labeling (upper panel), after relatively extensive digestion, the major intensity of MCA fluorescence was localized in a 160-kDa tryptic sub-fragment. Because this sub-fragment was stained with anti-residue 1417 antibody (Ab 1417) but not with anti-residue 2727 antibody (Ab 2727), the peptide A-mediated MCA labeling site, i.e. peptide A-binding site, seemed to be localized in the region of the RyR encompassing residues 1400-2726, as illustrated at the bottom of Fig. 2. There is an additional lower molecular mass (145 kDa) tryptic sub-fragment labeled with MCA that matches approximately with the band intensely stained with anti-residue 416 antibody (Ab 416). We assume that the 145-kDa fluorescent sub-fragment has derived not from the N-terminal 150-kDa calpain fragment but from the 160-kDa (peptide A binding region) tryptic sub-fragment described above for the following reasons. First, the molecular mass of the Ab 416-stained band (150 kDa) was always higher than that of the 145-kDa MCA-labeled sub-fragment. Second, not even a trace of MCA labeling was detected in the 150-kDa N-terminal region of the RyR, as described in Fig. 1A. The lower panel of Fig. 2 shows the result of similar site-localization experiments with peptide C-mediated MCA labeling. In this case, the MCA-labeling site was localized in the 150-kDa sub-fragment that was stained with anti-residue 416 antibody (Ab 416) after a partial tryptic digestion. After more extensive digestion, the shortest fluorescent sub-fragment was a 100-kDa subfragment that was stained intensely with Ab 416, indicating that peptide C binds the 100-kDa region of the N-terminal segment as indicated in the diagram at the bottom of Fig. 2. The above results suggest that both II-III loop peptides, peptide A and peptide C, bind exclusively to the RyR moiety out of many proteins present in the triad preparation. However, peptide A and peptide C introduced the fluorescence MCA probe to the clearly distinguishable regions of the RyR. Thus, it is concluded that peptide A and peptide C bind to the RyR in protein-specific and domain-specific manners. Although it may well be that the peptide binding domain actually consists of a multiple number of binding sites distributed in the different places of the primary structure, to facilitate discussion these distinguished regions will be called A-site (peptide A-binding site) and C-site (peptide C-binding site), respectively.

View larger version (55K): [in this window] [in a new window]   Fig. 2.   Peptide mapping of the MCA-labeling sites (peptide A- and peptide C-binding sites). To produce shorter peptide fragments to further localize the labeling sites, the RyR that had been labeled with MCA by mediation of either peptide A, or peptide C was digested with trypsin at various trypsin/SR protein ratios (no digestion (1st lane), 4000:1 (2nd lane), 2000:1 (3rd lane), 1000:1 (4th lane), 500:1 (5th lane)) at 22 °C. Electrophoretically separated bands on the 6% SDS gel were transferred to Immobilon-P membrane. The blotted sample was reacted with various primary antibodies (Ab 416, Ab 1417, Ab 2727, and Ab 5029) overnight at 22 °C followed by the treatment with alkaline phosphatase-conjugated second antibodies for 3 h and stained with diaminobenzidine. Correlation of the digestion pattern with the fluorescence-labeling pattern permitted us to localize the MCA labeling sites in shorter peptides. In the case of peptide A-mediated MCA incorporation (upper panel), the shortest recognizable peptide showing the intense MCA fluorescence was a 160-kDa sub-fragment that reacted with Ab 1417 antibody but not with Ab 2727. Thus, the peptide A-mediated MCA incorporation site (i.e. peptide A-binding site) must be within the region encompassing residue 1400 (calpain cleavage site) and residue 2726 (see the diagram shown at the bottom). In the case of peptide C-mediated incorporation (lower panel), the shortest recognizable fluorescent band was a 100-kDa sub-fragment, which reacted with Ab 416 but not with Ab 1417. This indicates that the peptide C-binding site is in the 100-kDa segment located at the N-terminal side of the peptide A binding region, as shown in the diagram. The whole set of experiments shown in this figure was repeated four times for reproducible results.

The MCA Probe Attached to the A-site, Not That Attached to the C Site, Reports T-tubule Depolarization-induced Local Events in the Coupled Triad-- The MCA-labeled triad preparation by mediation of either peptide A or peptide C showed almost intact activity of depolarization-induced Ca²⁺ release as the unlabeled preparation, although there was a significant decrease in the rate of depolarization-induced Ca²⁺ release; at G9, 60.01 ± 11.9 nmol/mg/s (n = 4) in the MCA-labeled triads (see Table II) versus 435.8 ± 73.9 nmol/mg/s (n = 5) in the case of the unlabeled triads (cf. Ref. 14). Therefore, the labeled triads can serve as a useful in vitro model of E-C coupling. We examined whether any of these activators produce appreciable changes in the fluorescence intensity of the MCA that had been incorporated into either the A or C site on the RyR in a protein-specific manner. We found that depolarization of the T-tubule moiety of the triad induced a rapid increase in the fluorescence intensity of the MCA bound to the A-site but no appreciable change in the MCA bound to the C-site.

Fig. 3 depicts the time courses of the increase in the fluorescence intensity of the MCA bound to the A-site (left panel) and Ca²⁺ release (right panel) induced by various degrees of T-tubule depolarization after the K⁺-to-Na⁺ replacement protocol shown in Table I ("Experimental Procedures"). In the control mixing with no K⁺-to-Na⁺ replacement, viz. with no depolarization (G1), there was no appreciable change in the MCA fluorescence and no induced Ca²⁺ release. Upon increasing the degree of T-tubule depolarization (the degree of depolarization is expressed as the ratio of (Na⁺ concentration after mixing)/(the Na⁺ concentration before mixing), the magnitude of the MCA fluorescence change increased. Concomitantly, the magnitude of induced Ca²⁺ release increased in proportion to the increased magnitude of the MCA fluorescence change. The MCA fluorescence change was much faster than that of Ca²⁺ release at all degrees of T-tubule depolarization tested so far, as seen from Fig. 3 and Table II. Table II depicts the values of various kinetic parameters (A, the amplitude; k, the rate constant; Ak, the initial rate) and the statistic variations of these values calculated from the data shown in Fig. 3. This indicates that the MCA fluorescence change represents a local event occurring in the A-site before Ca²⁺ release, suggesting that the former is a causative mechanism for the latter.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   The time courses of depolarization-induced changes in the fluorescence intensity of the MCA probe attached to the peptide A-binding site and the subsequent Ca2+ release at various magnitudes of depolarization. The T-tubule moiety of the triad was depolarized to various degrees (G1, G3, G6, G9) by the K+-to-Na+ replacement protocol (cf. Table I) at the Ca2+ concentration of 0.3 µM (the Ca2+ concentration immediately after the stopped-flow mixing), and the changes in the fluorescence intensity of the MCA incorporated to the peptide A-binding site and induced Ca2+ release were monitored as described under "Experimental Procedures." The magnitude of MCA fluorescence change increases with the magnitude of depolarization in a G-dependent manner. In parallel to this, the rate and the size of Ca2+ release also increased. The MCA fluorescence change was much faster than that of Ca2+ release at each G, indicating that the observed MCA fluorescence change (conformational change) represents the event that precedes Ca2+ release. Each MCA fluorescence trace presented here was obtained by signal-averaging a total of 50-70 traces originated from five experiments. Each Ca2+ release trace presented here was obtained by signal-averaging a total of 24-40 traces originated from four experiments. The values of kinetic parameters and the statistic variations calculated from the data of this figure are shown in Table II. Note that the time scale of MCA fluorescence change is 10 times faster than that of Ca2+ release.

Fig. 4 depicts the time courses of MCA fluorescence change at the A-site (left column) and those of SR Ca²⁺ release (right column) when the RyR was stimulated by several DHP receptor-independent agonists (peptide A, polylysine, and caffeine). Interestingly, none of these agonists produced any appreciable change in the MCA fluorescence at the A-site within the time scale of the stopped-flow measurements (0.5 s as shown). Table III depicts the values of various kinetic parameters (A, the amplitude; k, the rate constant; Ak, the initial rate) and the statistic variations of these values calculated from the data shown in Fig. 4. It seemed rather puzzling that even peptide A did not produce MCA fluorescence change. Therefore, we investigated the possibility that peptide A might be accessible to the A-site in the coupled triad at a rather slow rate and might produce a slow MCA fluorescence increase by carrying out hand-mixing experiments. However, it was found that even at a 5-min time scale 50 µM peptide A produced no statistically significant MCA fluorescence change (the F/F_{o/t=5 min} = 0.35 ± 0.12%, n = 7). Thus, the rapid MCA fluorescence increase at the A-site observed here does not represent a local conformational change induced by peptide A. Instead, the results suggest that T-tubule depolarization induces the characteristic events in the particular region of the coupled triad (see "Discussion," Fig. 7 models), and within such region the A-site and the attached MCA happened to be located.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Various agonists that activate the RyR by direct stimulation produce Ca2+ release without producing the fluorescence change of the MCA attached to the peptide A-binding site. The Ca2+-loaded triad vesicles were mixed with various voltage-independent agonists such as 3 mM caffeine, 100 nM polylysine, and 50 µM peptide A. These agonists induce a large Ca2+ release but produce no appreciable change in the MCA fluorescence, indicating that the MCA labeling site (i.e. peptide A-binding site) is in the domain outside of the agonist binding-channel activation pathways. The values of kinetic parameters and the statistic variations calculated from the data of this figure are shown in Table III.

The experiment shown in Fig. 5 further supports the above notion. In this experiment, the triads were incubated in the priming solution containing nimodipine (antagonist of the DHP receptor at 1 µM, a sufficient concentration to uncouple the DHP receptor-to-RyR communication (27)) and monensin/valinomycin mixture (an agent to prevent the formation of the trans-T-tubule membrane Na⁺/K⁺ gradient) and then mixed with the depolarization solution (G9). As seen, both of these agents almost completely abolished both MCA fluorescence change and subsequent Ca²⁺ release (the values of various kinetic parameters and the statistic variations are shown in Table II). This indicates that the observed MCA fluorescence change (in the A-site domain) is under the control of the DHP receptor. It should be noted that neither of these agents produced any appreciable effects on Ca²⁺ release induced by the voltage-independent agonists such as peptide A, polylysine, and caffeine.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   There was no MCA fluorescence change if the depolarization procedure took place in the presence of nimodipine or in the presence of ionophores (monensin and valinomycin). 1 µM nimodipine (upper panel) or the mixture of 10 µM valinomycin and 10 µM monensin (lower panel) prevented the MCA fluorescence change from occurring even when a maximal degree of ionic replacement (G9) was applied. Consequently, the subsequent Ca2+ release was almost completely blocked. The values of kinetic parameters and the statistic variations calculated from the data of this figure are shown in the bottom part of Table II.

In the experiment shown in Fig. 6 and Table IV, we carried out the same type of depolarization-induced Ca²⁺ release experiment as above using the triad preparation in which MCA was labeled to the C-site. As seen, T-tubule depolarization induced Ca²⁺ release but produced no appreciable change in the fluorescence intensity of the MCA that had been attached to the C-site. The values of kinetic parameters and the statistic variations are shown in the table attached to the legend to Fig. 6. Overall, the above results suggest that the postulated depolarization-induced characteristic events are localized in the region where the peptide A-mediated MCA labeling has taken place.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   T-tubule depolarization produced no appreciable change in the fluorescence intensity of the MCA incorporated to the peptide C-binding site of the RyR. The site-specific MCA incorporation into the RyR moiety of the triad was performed by mediation of peptide C as a site-directing carrier, and T-tubule depolarization (G9) was performed as described under "Experimental Procedures." The values of kinetic parameters and the statistic variations calculated from the data of this figure are shown in Table IV.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we addressed the two key questions as to (a) whether peptide A and peptide C bind to the RyR in a protein- and site-specific manner and (b) whether the fluorescent probe that is attached to the peptide-binding sites can report the local events that are relevant to the physiological coupling between the DHP receptor and the RyR. The site-directed fluorescence probe-labeling technique we used in this study (original description, Ref. 21) involves the use of a hetero-bifunctional cleavable photo-affinity cross-linking reagent, SAED, with such a structure, (azido group)-(fluorescent adduct MCA)-S-S-(succinimidyl). Upon forming the conjugate of a selected ligand via the reaction of its reactive amino group with succinimidyl of SAED, the ligand delivers the conjugate to its binding site in a site-directed fashion serving as a site-directing carrier. Photo-cross-linking of the conjugate via the azido group of SAED followed by removal of the ligand moiety by cleaving the S-S bond under reducing conditions permits site-specific covalent labeling of the ligand-binding site with MCA. In our recent studies, we used neomycin (a blocker of the RyR Ca²⁺ channel, which is known to bind to the trans-membrane channel domain, Ref. 28) as the site-directing carrier to introduce MCA to the channel domain and investigated conformational changes occurring in the channel domain upon activation of the channel by various types of agonists (14, 20, 25). In the case when the site of the ligand binding has not yet been characterized as in the present case with the II-III loop peptides, the site-directed labeling technique provides a powerful tool to identify and characterize the site of peptide binding. Furthermore, if the probe is introduced successfully in the specific site or the specific region as in the present case, the protein-bound probe can serve as a reporter of the local events occurring during E-C coupling. Thus, the application of this technique in the present study has permitted us to investigate both key questions a and b outlined above.

With regard to the specificity of peptide binding, the fact that specific fluorescence labeling of the RyR could be achieved by using peptide A or peptide C as a site-directing carrier clearly indicates that both peptide A and peptide C are the RyR-specific ligands. The present results also suggest that peptide A and peptide C bind to the specific domains; hence, their binding is not only protein-specific but also domain-specific. Thus, the chief fluorescence labeling of the peptide A-binding site (A-site) occurred in the 160-kDa region located at the C-terminal side of the primary calpain II cleavage site at residue 1400 (26), whereas the chief fluorescence labeling of the peptide C-binding site (C-site) occurred in the 100-kDa segment located at the opposite side (i.e. N-terminal side) of the primary calpain cleavage site. We propose that in the quaternary structure the A-site and the C-site are in a close apposition to each other for several reasons. First, according to our preliminary study (29), the fluorescence energy transfer could be detected between the donor and acceptor placed at the A- and C-sites, respectively. Second, several different regions of the RyR have been identified as the II-III loop binding domains in the literature. Using deletion strategy, Yamazawa et al. (30) identified the residue-1303-1406 (D2) region as a critical region. The chimera approach by Nakai et al. (31) suggest that the critical region is in a rather long 1635-2636 stretch. On the other hand, the II-III loop affinity column assay by Leong and MacLennan (32) suggests a short 1076-1112 segment. These findings are consistent with the view that the putative a II-III loop-binding core is constructed by multiple segments that are scattered in a relatively broad range of the primary structure (29). Third, we pay a particular attention to an interesting analogy of our present results to the structure of the so-called inositol 1,4,5-trisphosphate (IP₃) binding core located in the N-terminal region of the IP₃ receptor, where the basic residues critical for IP₃ binding are positioned at both sides of the site that is highly susceptive to proteolytic cleavage (33).

The most important aspect of this study is the finding that depolarization of the T-tubule moiety of the triad preparation produced a rapid increase of the fluorescence intensity of the MCA attached to the A-site. The chemical depolarization protocol with various degrees of K⁺-to-Na⁺ replacement permits generation of various degrees of depolarization in the T-tubule moiety of the triad as described previously (23, 25). As shown here, upon increasing the magnitude of T-tubule depolarization, the magnitude of the MCA fluorescence change increased, and the magnitude of the induced Ca²⁺ release increased in a proportionate fashion. The MCA fluorescence change was much faster than Ca²⁺ release, suggesting that the local conformational change in the peptide A-binding domain reported by the attached MCA probe represents a causative and prerequisite mechanism for the Ca²⁺ channel activation. The observed MCA fluorescence signal and the induced Ca²⁺ release are in fact mediated by the voltage change in the T-tubule moiety and by the DHP receptor voltage sensor, as evidenced by the facts that dissipation of the Na⁺/K⁺ gradient across the T-tubule membrane by the monensin/valinomycin mixture and the antagonist of the DHP receptor nimodipine inhibited both MCA fluorescence change and subsequent Ca²⁺ release. In further support of this notion, the MCA signal was produced in a Ca²⁺-independent fashion (data not shown) like depolarization-induced contraction and Ca²⁺ release (21, 34, 35) but unlike other chemical and pharmacological agonists of the RyR, most of which have a stringent Ca²⁺ requirement.

In evaluation of the physiological significance of the above data, critically important is the fact that the MCA fluorescence exchange at the A-site can be seen in response solely to the one type of activation signal, i.e. T-tubule depolarization. Other voltage-independent agonists such as peptide A, polylysine, and caffeine induced Ca²⁺ release but without producing the MCA fluorescence change. This is in a sharp contrast to the results of the experiments with the triad preparation in which MCA was attached to the trans-membrane channel domain by mediation of neomycin, where all types of agonists we tested produced the MCA fluorescence change (see Table V). Presumably, the conformational change that was reported by the MCA at the channel domain represents the gating behavior of the channel common to various types of agonists of the RyR. Furthermore, T-tubule depolarization produced the fluorescence change only when the MCA probe was placed at the A-site but did not if the MCA was at the C-site.

View this table: [in this window] [in a new window]   Table V Comparison of the types of responses of the MCA attached to the peptide A binding site and the MCA attached to the neomycin binding site located at the trans-membrane channel domain +, appreciable fluorescence change; , no appreciable change.

In attempts to account for the present results, we postulate two alternative models as shown in Fig. 7, models a and b. Model a assumes that upon T-tubule depolarization a portion of the DHP receptor comes very close to the RyR, forming a highly hydrophobic DHP receptor/RyR interface. If the A-site is located within such an interface region, the fluorescence intensity of the attached MCA will show a rapid increase upon forming such a hydrophobic DHP receptor/RyR interface. Although peptide A is capable of delivering the MCA probe to the A-site and the binding of peptide A to this site induces SR Ca²⁺ release, it is incapable of inducing the DHP receptor/RyR contact, which is a specific event produced by the DHP receptor voltage-sensing. Although we are inclined to model a, we cannot exclude an alternative model shown in model b. Namely, T-tubule depolarization produces characteristic conformational change (e.g. internalization of the attached MCA probe) in some region of the RyR, which is not necessarily in the DHP receptor/RyR interacting region. Because the blocking of the DHP receptor-to-RyR communication results in the inhibition of the T-tubule depolarization-induced characteristic events regardless of the location of the A-site (either the DHP receptor/RyR interface or in the cytoplasmic domain of the RyR), it is difficult to decide either model by kinetic experiments alone. We should probably await for the information about the exact locations of the A-site and of the DHP receptor-interacting region within the three-dimensional image of the RyR. At any rate, the present finding that the T-tubule depolarization-induced characteristic events take place in the region where the A-site is located suggests that peptide A can serve as a useful tool at least for introducing the conformational probe to the physiologically important domain.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Hypothetical models illustrating that depolarization in the T-tubule moiety of the triad induces local events that cause a rapid increase in the hydrophobicity of the environment in the vicinity of the MCA attached to the RyR. We tentatively propose that such an event would take place in the DHP receptor/RyR interface as shown in model a. However, it may well be that such an event takes place in the cytoplasmic domain of the RyR as shown in model b.

	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. We also thank Drs. Susan L. Hamilton, Andrew R. Marks, and Kevin P. Campbell for their generous supply of anti-RyR antibodies.

	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.M102347200

	ABBREVIATIONS

The abbreviations used are: DHP, dihydropyridine; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid; E-C coupling, excitation-contraction coupling; 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; Ab, antibody.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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