A noncontiguous, intersubunit binding site for calmodulin on the skeletal muscle Ca2+ release channel.

Both apocalmodulin (Ca(2+)-free calmodulin) and Ca(2+)-calmodulin bind to and regulate the activity of skeletal muscle Ca(2+) release channel (ryanodine receptor, RYR1). Both forms of calmodulin protect sites after amino acids 3630 and 3637 on RYR1 from trypsin cleavage. Only apocalmodulin protects sites after amino acids 1982 and 1999 from trypsin cleavage. Ca(2+)-calmodulin and apocalmodulin both bind to two different synthetic peptides representing amino acids 3614-3643 and 1975-1999 of RYR1, but Ca(2+)-calmodulin has a higher affinity than apocalmodulin for both peptides. Cysteine 3635, within the 3614-3643 sequence of RYR1, can form a disulfide bond with a cysteine on an adjacent subunit within the RYR1 tetramer. The second cysteine is now shown to be between amino acids 2000 and 2401. The close proximity of the cysteines forming the intersubunit disulfide to the two sites that bind calmodulin suggests that calmodulin is binding at a site of intersubunit contact, perhaps with one lobe bound between amino acids 3614 and 3643 on one subunit and the second lobe bound between amino acids 1975 and 1999 on an adjacent subunit. This model is consistent with the finding that Ca(2+)-calmodulin and apocalmodulin each bind to a single site per RYR1 subunit (Rodney, G. G., Williams, B. Y., Strasburg, G. M., Beckingham, K., and Hamilton, S. L. (2000) Biochemistry 39, 7807-7812).

The skeletal muscle Ca 2ϩ release channel (also known as the ryanodine receptor, RYR1) is a homotetramer containing four 565-kDa subunits; four-fifths of the molecule is cytoplasmic, and the remaining one-fifth is membrane-spanning and/or luminal to the sarcoplasmic reticulum (SR). 1 Ca 2ϩ and several endogenous proteins that bind within the large cytoplasmic domain modulate the activity of RYR1. In the muscle fiber, these endogenous modulators probably act together to regulate RYR1 channel activity and, ultimately, Ca 2ϩ release from the SR. One of these modulators, calmodulin (CaM), binds to RyR1 at both nanomolar and micromolar Ca 2ϩ concentrations (1). Apocalmodulin (apoCaM) is a weak partial agonist, whereas Ca 2ϩ -calmodulin (Ca 2ϩ CaM) is an inhibitor of RyR1 (1). CaM in both its Ca 2ϩ -bound and Ca 2ϩ -free forms binds to one site per RYR1 subunit (2).
ApoCaM and Ca 2ϩ CaM bound to RYR1 protect sites after amino acids 3630 and 3637 from tryptic digestion (3), suggesting that this sequence may form part of both the apoCaM and Ca 2ϩ CaM binding sites. Evidence that the Ca 2ϩ CaM and apoCaM binding sites are overlapping includes the following: (a) apoCaM and Ca 2ϩ CaM have the same number of binding sites (2); (b) the binding sites for both apoCaM and Ca 2ϩ CaM are destroyed by trypsin at the same rate (3); (c) the two forms of CaM protect each other's binding sites from tryptic destruction (3); (d) the protected cleavage sites within the 3614 -3643 region are the same in high and low Ca 2ϩ ; (e) the binding of [ 35 S]E1234Q (a mutant CaM that cannot bind Ca 2ϩ at any of the four Ca 2ϩ binding sites) is completely inhibited by Ca 2ϩ CaM (2); (f) no cooperativity in binding has been detected 2 ; (g) mutations W3620A or L3624A in RYR1 greatly decrease the affinity of both apoCaM and Ca 2ϩ CaM binding for RYR1 (4); (h) the 3614 -3643 peptide binds the C-lobe but not the N-lobe of both apoCaM and Ca 2ϩ CaM (5); and (i) the interaction of 3614 -3643 with CaM increases the Ca 2ϩ affinity only of the C-lobe of CaM (5). These findings support a model in which the C-lobe of both apoCaM and Ca 2ϩ CaM binds to the sequence between amino acids 3614 -3643. The C-lobe binding sites for apoCaM and Ca 2ϩ CaM are overlapping, nonidentical sites, and CaM shifts N-terminally within the site when Ca 2ϩ binds to the C-lobe (2). The binding of Ca 2ϩ to the C-lobe of CaM is responsible for its conversion from an activator to an inhibitor of RYR1 (2). Samso and Wagenknecht (6) have localized the binding site for CaM in the three-dimensional structure of RYR1. They found that the two forms of CaM bind on the sides of the cytoplasmic domain of RYR1 to overlapping locations with centers separated by 33 Å. This large translocation, especially in light of a common C-lobe binding site (2,3,5), suggests that the binding sequence is moving a relatively large distance when the channel changes its conformation in response to CaM and Ca 2ϩ and/or there is a major movement of one lobe of calmodulin when Ca 2ϩ binds to both CaM and RYR1.
Additional information about the location of the CaM binding site in the RYR1 tetramer has come from oxidation studies. An intersubunit disulfide bond can be formed by treating RYR1 with oxidizing agents (7)(8)(9), and the formation of this cross-link is blocked by bound Ca 2ϩ CaM or apoCaM (7). These data suggest that the CaM binding site may be localized to a site of intersubunit contact. Consistent with this, one of the cysteines involved in this cross-link is found within the amino acid 3614 -3643 binding site for the C-lobe of CaM (10).
These findings raise the possibility that the CaM could be interacting simultaneously with two subunits in the RYR1 tetramer. Since the C-lobe of CaM appears to bind primarily within the 3614 -3643 sequence, the site on the second subunit might represent an interaction site for the N-lobe of CaM. We have demonstrated the importance of the N-lobe in binding and regulation of RYR1 using N-terminal CaM mutants (5). The N-lobe of CaM appears to bind at an unidentified location distinct from that of the C-lobe (5). We now identify a new sequence that could be the binding site for the N-lobe of CaM on an adjacent subunit in the RYR1 tetramer.

Materials
propanesulfonic acid (MOPS), 3-[cyclyhexylamino]-1-propane sulfonic acid (CAPS), bovine brain calmodulin, and bovine serum albumin were obtained from Sigma. Amplify and [ 3 H]N-ethymaleimide were obtained from Amersham Biosciences. All antibodies except Ab 5029 were prepared in rabbit immunized with synthetic peptides coupled to keyhole limpet hemocyanin. Immunizations were performed either by our laboratory or by Pelfreeze Laboratories (Rogers, AR) using Freund's complete adjuvant, and the antibodies were purified as we have previously described (8). Ab 5029 was graciously provided by Dr. Andrew Marks (Columbia University, College of Physicians and Surgeons). E1234Q CaM was graciously provided by Dr. Kathleen Beckingham (Rice University).

Methods
SR Membrane Preparation-SR membranes were prepared as previously described (11) from rabbit hind leg and back strap white skeletal muscle and purified using sucrose gradient centrifugation.
Generation of Trypsin-digested Complexes of RYR-SR membranes (10 mg of protein/ml) in 300 mM NaCl, 50 mM MOPS (pH 7.4), 100 M CaCl 2 were treated with trypsin for 1-5 min at 37°C at a trypsin/ protein ratio of 1:1000. Digestion was halted by the addition of a 10-fold excess of soybean trypsin inhibitor. After solubilization in 2% CHAPS, proteolyzed complexes were purified over 5-20% linear sucrose gradients containing 0.4% CHAPS, 300 mM NaCl, 50 mM MOPS (pH 7.4) centrifuging at 24,000 rpm in a Beckman SW28 rotor for 18 h. Fractions were collected, and fractions containing the RYR1 proteolytic complex were identified using a Bio-Rad protein assay. Fractions from the sucrose gradient were compared by SDS-PAGE.
Protein Sequencing-Proteins were prepared for sequencing as described previously (12). N-terminal sequencing was performed in the Core Protein Facility by Dr. Richard Cook at Baylor College of Medicine.
Western Blots-Protein samples subjected to SDS-PAGE were transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA) for 16 -18 h at 25 V in 5% methanol, 10 mM CAPS (pH 11.0) at 4°C. Incubation with primary and secondary antibodies was performed as described by Wu et al. (8). The blots were developed using the Super-Signal chemoluminescence substrate (Pierce).
Two-dimensional SDS-PAGE-The purified proteolytic complex was treated with 5 mM N-ethymaleimide for 20 min at 4°C before solubilization in SDS sample buffer. 7.5% polyacrylamide gels were used for the first dimension electrophoresis. Lanes were excised from the first dimensional gel, and the gel strips were incubated with 25-50 mM DTT in the sample buffer without SDS and bromophenol blue for 1 h at room temperature (23°C). The gel strip was then loaded on top of a 12.5% polyacrylamide gel for the second dimensional electrophoresis. The gap between the gel strip and the second dimensional resolving gel was sealed with melted agarose (1% agarose, 2% SDS, 50 mM DTT), and the gels were electrophoresed for 1-2 h at 120-170 V at 4°C. The twodimensional gels were subsequently stained with Coomassie Brilliant Blue.
Electrophoretic Mobility Shift Assays-Analysis of the interaction of the peptides with CaM by nondenaturing gel electrophoresis was performed as we have previously described (15).

ApoCaM Protects New Sites from Tryptic
Cleavage-Our previous studies using CaM to protect cleavage sites on RYR1 were primarily performed at high Ca 2ϩ concentrations. Under these conditions, the major cleavage sites protected by CaM were after amino acids 3630 and 3637 (3). With longer digestion times and in Ͻ10 nM Ca 2ϩ we have now identified two additional protected sites. The fragmentation patterns obtained by digestion of RYR1 in membranes at nanomolar Ca 2ϩ concentration in the presence and absence of CaM are shown in Fig. 1  (A, lanes a and b, and B, lanes a and b). To aid in the visualization of the protected fragments, the purified proteolytic complexes were also labeled with [ 3 H]N-ethylmaleimide and visualized by fluorography (Fig. 1A, lanes c and d, and Fig. 2, lanes c and d). Protected bands that we have previously identified (3) are clearly seen in these gels, and, in addition, a new 71-kDa band is seen in the absence but not in the presence of apoCaM.
We routinely use two different one-dimensional SDS-PAGE systems to analyze the proteolytic fragments: (a) the gel system of Schä gger and von Jagow (14) for separation of smaller fragments and (b) 7.5% Laemmli gels (13) for separation of larger fragments. In the Schä gger and von Jagow gels, large fragments are difficult to separate. The separation of these high molecular weight fragments into multiple bands is more clearly achieved in the Laemmli gels (Fig. 1C). However, smaller fragments are best differentiated in the Schä gger and von Jaggow gels (Fig. 1A). Also, fragments with molecular masses less than ϳ40 kDa run with the dye front on the 7.5% Laemmli gels.
The identity of the tryptic fragments was determined by Western blotting with sequence-specific antibodies ( Fig. 3 and Table I) and with N-terminal sequencing (Table II). The tentative assignment of the C-terminal amino acid for each of the fragments is based on both the apparent size and our previously determined tryptic digestion sites (12). To assess precursor/product relationships, the intensities of the bands were analyzed by densitometry (Fig. 1C). Consistent with our previous findings, a cleavage at amino acid 3630 and/or 3637 converts fragment 1 (apparent molecular mass of 170 kDa, Nterminal amino acid 3120) to fragment 6 (ϳ128 kDa, N-terminal amino acid 3120). Fragment 6 is found in variable amounts from preparation to preparation. In reduced membranes, fragment 6 is rapidly converted to fragment 9, but this cleavage is slowed if the membranes are oxidized (see below). Fragment 2 (ϳ158 kDa, also beginning with amino acid 3120) is directly converted to fragment 9 (ϳ110 kDa, beginning with amino acid 3631). Both Ca 2ϩ CaM and apoCaM protect the sites after arginine 3630 and 3637 from trypsin cleavage (3) (Figs. 1 and 2).
A second site protected by apoCaM is also seen (Fig. 1A). When the digestion was performed at Ͻ10 nM Ca 2ϩ concentration, fragment 12 is found in samples digested in the absence but not in the presence of CaM (Fig. 1A, lane a versus lane b). N-terminal amino acid sequencing of this band identified it as beginning with amino acid 1509. To determine whether there is another site protected by apoCaM, we looked for both the precursor of this fragment at a higher molecular weight and the other part of a larger precursor fragment at a lower molecular weight. The fluorogram shows that the broad band around 110 -125 kDa appears to be even broader when the digestion was performed in the presence of apoCaM (Fig. 1A, lanes b and  d). We find a fragment with an apparent molecular mass of about 123 kDa (fragment 7) with an N-terminal sequence beginning with amino acid 1509. Furthermore, there is at least three times more of this fragment (determined by N-terminal sequencing) in the 110 -120-kDa band if apoCaM is present during the proteolysis compared with that found in the absence of CaM. Fragment 7 but not fragment 12 was recognized by an antibody to amino acid 2391 (Fig. 4). These findings suggest that apoCaM protects a site within fragment 7 from tryptic cleavage. The size of the piece removed from fragment 7 to form fragment 12 should be around 50 kDa, and, indeed, bands in the 50-kDa region were recognized by Ab 2391 (Fig. 3). We sequenced the broad 50-kDa band and found that it is composed of multiple fragments, including a sequence beginning with amino acid 1983 (fragment 14); a sequence beginning with either amino acid 2402, 2403, or 2404 (fragment 15, carboxylterminal end with a jagged cut); and smaller and variable amounts of a fragment beginning with amino acid 2000 (fragment 16). In some preparations, there were also small amounts of a fragment of the sarcoplasmic reticulum Ca 2ϩ ATPase, SERCA 1, in this band. When CaM was present during the proteolysis very little of either fragment 14 or 16 could be detected in the ϳ50-kDa band by N-terminal amino acid sequencing, suggesting that these two fragments are derived from cleavage of fragment 7. The appearance of the fragment 14 with an N-terminal sequence beginning after arginine 1982 appears to correlate most closely with the appearance of fragment 12 and the disappearance of fragment 7, suggesting that it represents the first cleavage. The cleavage at 1999 may represent a second protected site, or it may arise as a secondary cleavage after the primary cut at arginine 1982.
A Functionally Inactive ApoCaM ((Nϩ3)CaM) Also Protects the 1982 Site from Tryptic Cleavage-ApoCaM is a weak partial agonist of the channel. We found that the addition of 3 amino acids to the N terminus of CaM produces a mutant CaM (designated (Nϩ3)CaM) that binds with higher affinity than wild-type apoCaM but does not activate the channel (5). The cleavage of the fragment 7 to fragments 12 and 14 is also protected by (Nϩ3)CaM (Fig. 2), suggesting that this represents a direct protection of this site from proteolysis rather than a protection arising allosterically. CaM was added to one sample to a final concentration of 5 M, and the samples were incubated for an additional 30 min. Trypsin (10 g) was then added, and the sample was incubated for 2 min at 37°C. The proteolysis was stopped with soybean trypsin inhibitor, and the cleaved RYR1 complex was isolated as described under "Experimental Procedures." Following isolation, 100-l aliquots of the RYR1 peak were incubated with 5 l of [ 3 H]N-ethymaleimide (51.7 Ci/mmol, 1 mCi/ml) for 30 min on ice, followed by the addition of DTT to 25 mM. Samples were developed for autoradiography as described under "Experimental Procedures." A, fragments obtained with (lane b) and without (lane a) apoCaM analyzed on Schä gger gels. Samples were electrophoresed on 10% Schä gger gels and stained with Coomassie Brilliant Blue. Bio-Rad molecular weight markers are shown to the left. For fluorography, the gel was immersed in Amplify (Amersham Biosciences) for 20 min, washed quickly three times with distilled water, and dried. The dried gel was exposed to film at  trypsin does not greatly alter the tryptic digestion pattern. When we examined the effect of CaM on the proteolysis patterns at high Ca 2ϩ concentrations (Fig. 3A), we found that fragment 12 is present in the proteolytic complexes prepared both with and without CaM (Fig. 3A, lane a versus lane b), indicating that Ca 2ϩ CaM did not prevent the cleavage after arginine 1982. Ca 2ϩ CaM did protect the previously described 158-kDa band (fragment 2) to 110-kDa (fragment 9) conversion (a cleavage after amino acid 3630/3637). E1234QCaM, a Mutant CaM That Does Not Bind Ca 2ϩ , Protects the Site at Arginine 1982 from Tryptic Cleavage on the Ca 2ϩ -bound RYR1-We have demonstrated a cleavage site that is protected by apoCaM on the Ca 2ϩ -free RYR1, raising the question of whether the protection is due to the conformation of CaM or RYR1 or both. A mutant CaM that cannot bind Ca 2ϩ at any of the four Ca 2ϩ binding sites (E1234Q) was tested for the ability to protect the 1982 cleavage site on the Ca 2ϩbound RYR1. Fig. 3B shows that the conversion of fragment 7 to fragments 12 and 14 occurs in high Ca 2ϩ in the presence and absence of Ca 2ϩ CaM (Fig. 3B, lanes a and b). The presence of E1234Q, however, partially prevents this conversion (Fig. 3B,  lane c, and C). We conclude that Ca 2ϩ -free CaM can protect the 1982 cleavage site on the Ca 2ϩ -bound RYR1, indicating that the protection has more to do with the conformation of CaM than any Ca 2ϩ -driven changes in RYR1.
Other Tryptic Fragments of RYR1 Were Identified with Nterminal Sequencing and Western Blotting with Sequence-specific Antibodies-Other tryptic fragments of RYR1 were identified in these experiments and are listed in Table I. These were all consistent with the fragments that we had previously identified (12). Western blotting with four different antipeptide antibodies is shown in Fig. 4.
A Synthetic Peptide Binds Ca 2ϩ CaM-In an attempt to identify the amino acids involved in binding CaM, we made a series of synthetic peptides corresponding to the RYR1 sequence around the protected cleavage site (Table III). These peptides were screened for ability to bind to CaM at both high and low Ca 2ϩ concentrations. In this gel system, the peptide alone does not enter the gel due to its positive charge. The complex of the peptide and CaM can be seen above the free CaM band, and the extent of interaction was assessed by the disappearance of the free CaM band. Representative gels using low and high Ca 2ϩ concentrations are shown in Fig. 5, A and B, respectively. The summarized data (I/I 0 ; the ratio of intensity of the CaM band in the presence of the peptide compared with that of CaM alone) from the densitometric analysis of the CaM band at low and high Ca 2ϩ with increasing peptide/CaM ratios are shown in Fig. 5C. By nondenaturing gel electrophoreses, only one peptide, 1975-1999, binds CaM. 1975-1999 binds Ca 2ϩ CaM with an affinity comparable with the 3614 -3649 peptide (16). ApoCaM (Fig. 5) also binds to the 1975-1999 peptide but with lower apparent affinity. Other methods to detect the interaction (pull-down assays with biotinylated 1975-1999 and strepavidin beads, and tryptophan fluorescence with a tryptophan substituted for the 1978 phenylalanine) also show a weak interaction of the peptide with apoCaM (data not shown). These findings suggest that although Ca 2ϩ CaM binds to the 1975-1999 sequence, its binding does not protect the 1982/1999 cleavage sites. In contrast, apoCaM binds more weakly to this sequence but does protect the cleavage site. There are a number of possible explanations for the different abilities of Ca 2ϩ CaM and apoCaM to protect the 1982/1999 cleavage sites: (a) both apoCaM and Ca 2ϩ CaM bind to this sequence, but Ca 2ϩ CaM does not protect the site (perhaps due to its conformation or to a slightly shifted interaction site); (b) apoCaM may bind at a different site on RYR1 and protect the cleavage site either sterically or allosterically; (c) the conformational change that occurs in RYR1 as a result of the inhibitory action of Ca 2ϩ CaM may increase the exposure of the 1983/1999 cleavage site; or (d) the binding affinities to the peptides do not accurately reflect the affinities of these regions for CaM in the native protein. Whether the 1975-1999 synthetic peptide binds only the N-lobe of CaM is not yet known.
Both CaM Binding Sites Are Close to a Site of Intersubunit Cross-linking-Cysteine 3635, located within the binding site for the C-lobe of CaM, can be cross-linked to a cysteine located on an adjacent subunit within the RYR1 tetramer (10). This cross-linking occurs in both 200 M Ca 2ϩ and at less than 10 nM Ca 2ϩ concentrations (Fig. 6) and is blocked by apoCaM and Ca 2ϩ CaM (3, 7, 10). The close proximity of a neighboring subunit to the C-lobe binding site raises the question of whether the N-lobe of CaM is binding to a different subunit from the one that binds the C-lobe. To address this question, we cross-linked RYR1 with the disulfide-inducing agent diamide and treated the membranes with trypsin. After stopping the proteolysis with soybean trypsin inhibitor, the membranes were solubilized in CHAPS, and the RYR1 proteolytic complex was purified. The proteolytic patterns under both reducing and nonreducing conditions were analyzed on both Schä gger and von  c and d, autoradiograms of samples a and b. B, proteolysis was performed as described in the legend for A, except the ability of E1234Q to protect the fragmentation (lane b) was compared with that of Ca 2ϩ CaM (lane c). Samples were electrophoresed on Schä gger and Jaggow gels. Coomassie-stained gel was used. Lane a is again the digestion pattern obtained at high Ca 2ϩ in the absence of CaM. C, densitometer analysis of the 58-kDa band from three independent experiments performed as described for B. *, p Ͻ 0.01. Jaggow (Fig. 7A, lanes a and b) and Laemmli gels (Fig. 7B,  lanes a and b). A number of minor bands at high molecular weight can be seen in the unreduced sample. Differences be-  a and b in panels A and B. A Western blot of a 7.5% gel with antibodies 2391 and 2727 (Fig. 7, C and D) shows that the fragment containing the "2391 sequence" but not the "2727 sequence" may be involved in formation of higher molecular weight complexes. The data obtained in this experiment suggest that the RYR1 fragments that are most likely to be involved in the cross-linking are fragments 1, 2, and 9 and a component the 50-kDa band.
To confirm the identity of the fragments involved in crosslinking, the cross-linked proteolytic complexes were analyzed using the two-dimensional gel electrophoresis approach first described by Wang and Richards (17). In a previous study using this approach with a calpain-digested and purified RYR1 complex, we localized the second cysteine to a region between amino acids 1400 and 2100 (8). We have also found that all off-diagonal spots can be eliminated by preincubation of the membranes with CaM prior to cross-linking. 2 Our data (3,(7)(8)(9)(10)12) are consistent with a single major inter-subunit cross-linking event.
The two-dimensional gel of RYR1 that was cross-linked with diamide to produce dimers (also designated oxidized RYR1), proteolyzed with trypsin in nanomolar Ca 2ϩ , and then purified, is shown in Fig. 8A. It should be noted that there are some subtle differences in the tryptic digest pattern obtained with oxidized RYR1, including (as discussed below) larger quantities of fragment 6, and the cleavage at 1999 predominates over cleavage at 1983. Western blots of the two-dimensional gels using Ab 2391 and 2727 are shown in Fig. 8, B and C, respectively. Four major off-diagonal spots (a-d) are clearly detected and appear to represent complexes prior to reduction of a-c and b-d. Using N-terminal Edman sequencing of the two upper bands, we found that both spots a and b begin with the Nterminal amino acid sequence AVVAXFRM, indicating that  4. Identification of fragments using Western blotting. Samples prepared as described in the legends to Figs. 1 and 2 were electrophoresed on either Schä gger and von Jagow or 7.5% Laemmli gels and transferred to Immobilon. Western blotting was performed as described under "Experimental Procedures." A, on the left are the molecular weight standards, which are the same as those listed in their N-terminal amino acid is 3631 and corresponds to fragments 6 and 9, respectively. Spots c and d both begin with amino acid 2000, and both correspond to fragment 16. We did not detect the sequence beginning at 1983, presumably because both cleavages occur in these experiments using oxidized RYR1. In all of our oxidized preparations, fragment 16 was present in higher amounts than fragment 14, suggesting that oxidized RYR1 protein is more readily digested at this site than reduced RYR1. This is consistent with our findings with calpain fragments, where calpain cleaved a new site around amino acid ϳ2100 in the oxidized RYR1 (8).
From the size of these fragments and our previous identification of tryptic fragments, we propose that the fragments containing cysteine 3635 (spot a, corresponding to fragment 6, and spot b, corresponding to fragment 9) are cross-linked by diamide treatment to a cysteine located in the fragment 16, found in both spots c and d. DISCUSSION How CaM can function as both an activator and an inhibitor of RYR1 is unknown. In addition, there appears to be a very large translocation of CaM within the three-dimensional structure of RYR1 when Ca 2ϩ binds to both CaM and RYR1. Clues to the molecular mechanisms contributing to these phenomena can be obtained from an analysis of the binding sites for apoCaM and Ca 2ϩ CaM on RYR1.
A binding site for CaM on the native RYR1 was identified using the ability of bound CaM to protect sites on RYR1 from tryptic cleavage (10). A synthetic peptide composed of amino  5. Nondenaturing gels. Nondenaturing gels were performed as previously described (17). Calmodulin in 50 mM MOPS (pH 7.4), 1 mM EGTA with or without 1.  4). Each sample was divided into two equal aliquots, and diamide was added to one of each of these to a final concentration of 100 M. Following an incubation of 10 min on ice, the reaction was stopped by the addition of N-ethylmaleimide to 5 mM. Samples (20 g) were electrophoresed on 7.5% Laemmli gels and stained with Coomassie Brilliant Blue. A, 7.5% SDS-PAGE. a, low Ca 2ϩ , no diamide; b, low Ca 2ϩ , diamide; c, high Ca 2ϩ , no diamide; d, high Ca 2ϩ , diamide. The arrows indicate the 565-kDa band (lower arrow) and a dimer of the 565-kDa band (upper arrow), which in a 7.5% gels is found close to the top of the gel. B, following staining and destaining, the intensity of the 565-kDa band was analyzed by densitometry. *, p Ͻ 0.05. acids 3614 -3643 was then shown to bind both apoCaM and Ca 2ϩ CaM, and cysteine 3635 was identified as one of the amino acids involved in the intersubunit disulfide bond (2,10). ApoCaM requires additional amino acids between 3634 and 3643, whereas Ca 2ϩ CaM needs only amino acids 3614 -3634. We recently found, however, that only the C-lobe of CaM binds to this sequence (5). This raised questions about the binding site for the N-lobe.
Deletion of amino acids 2-8 of calmodulin greatly decreases the affinity of calmodulin for RYR1 at both nanomolar and micromolar Ca 2ϩ concentrations (5). These amino acids are also required for maximal inhibition of the channel at micromolar Ca 2ϩ concentrations. In contrast, the addition of 3 amino acids to the N terminus of calmodulin increases its affinity for RYR1 at both nanomolar and micromolar Ca 2ϩ concentrations but destroys its functional effects on RYR1 in nanomolar Ca 2ϩ (5). These studies suggest that the N-lobe of CaM is binding to RYR1, but its binding site is likely to be noncontiguous with the C-lobe site. In the current study, we have attempted to identify the location of the binding site for the N-lobe of CaM.
To identify the N-lobe binding site, we used trypsin digestion of the membrane-bound RYR1. Tryptic fragments were identified by N-terminal sequencing and Western blotting. Fig. 9 illustrates the tryptic digest patterns detected in the presence (yellow lines) and absence of apoCaM (red lines). In low Ca 2ϩ , we found that, in addition to the protected sites after arginines 3630 and 3637, there are sites after amino acid 1982 (and, possibly 1999) protected by apoCaM. Since apoCaM is an activator of RYR1, the protection could be arising from a burying of the site due to the conformational change in RYR1. However, an inactive CaM with 3 additional N-terminal amino acids, (Nϩ3)CaM also protects the 1982/1999 sites, suggesting that this site is not protected as the result of a change in the conformation of the binding site arising from the ability of apoCaM to activate RYR1. (Nϩ3)CaM is a high affinity antagonist of the Ca 2ϩ -free RYR1.
The sites after amino acids 1982 and 1999 are not protected by Ca 2ϩ CaM, although the 1975-1999 sequence binds Ca 2ϩ CaM. It is possible that Ca 2ϩ binding to CaM bound to this sequence produces a conformational change in CaM itself or a movement of CaM within the binding site that uncovers this site. Another possible explanation of the lack of protection of this site by Ca 2ϩ CaM is that there is an increased exposure of the site arising from the Ca 2ϩ CaM-driven inhibition of RYR1 activity. This possibility is currently being investigated. The lack of protection by Ca 2ϩ CaM does not, however, appear to arise from a conformational change driven solely by Ca 2ϩ binding to RYR1, since a Ca 2ϩ binding site mutant of CaM (E1234Q) can protect the 1983/1999 cleavage sites. E1234Q is a partial agonist of RYR1 at all Ca 2ϩ concentrations (2) and provides some protection of the 1982 and 1999 cleavage sites on the Ca 2ϩ -bound RYR1, suggesting that the apoCaM binding site is similar in the Ca 2ϩ -bound and Ca 2ϩ -free RYR1.
The site identified in the protection and peptide studies (amino acids 1975-1999) is clearly not close to the C-lobe binding site in the primary sequence of RYR1, but is it on the same subunit? Cysteine 3635 is one of the cysteines involved in the formation of an intersubunit disulfide bond within the RYR1 tetramer (10), raising the possibility that the N-lobe binding site is also on a second subunit. One approach to assessing this is to identify the location of the second cysteine and determine whether it is close to the site protected by apoCaM. Consistent with the idea that the N-lobe binding site is on a second subunit, we previously demonstrated that the second cysteine was located somewhere between amino acids 1400 and 2100 (8), a sequence that contains the apoCaM-protected tryptic site. To narrow the location of the cross-linking cysteine, we used diamide as a disulfide-inducing cross-linker and tryptic digestion to generate fragments. We found that fragments containing cysteine 3635 could be cross-linked to a 50-kDa fragment that had an N-terminal sequence corresponding to amino acids 2000 and from its size was predicted to extend to amino acid 2401. These studies place the cysteine involved in the intersub-unit cross-link with cysteine 3635 in close vicinity of the apoCaM-protected cleavage site at amino acid 1982, suggesting that the N-lobe of CaM is binding to the subunit that is adjacent to the binding site for the C-lobe.
In summary, we propose that CaM binds to RYR1 with its C-lobe bound between amino acids 3614 and 3643 and its N-lobe bound to the adjacent subunit between amino acids 1975 and 1999, and Ca 2ϩ binding to CaM allows its movement within these two sequences to inhibit channel activity.