Binding of La3+ to calmodulin and its effects on the interaction between calmodulin and calmodulin binding peptide, polistes mastoparan

Binding of La(3+) to calmodulin (CaM) and its effects on the complexes of CaM and CaM-binding peptide, polistes mastoparan (Mas), were investigated by nuclear magnetic resonance (NMR) spectroscopy, fluorescence and circular dichroism spectroscopy, and by the fluorescence stopped-flow method. The four binding sites of La(3+) on CaM were identified as the same as the binding sites of Ca(2+) on CaM through NMR titration of La(3+) to uniformly (15)N-labeled CaM. La(3+) showed a slightly higher affinity to the binding sites on the N-terminal domain of CaM than that to the C-terminal. Large differences between the (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of Ca(4)CaM and La(4)CaM suggest conformational differences between the two complexes. Fluorescence and CD spectra also exhibited structural differences. In the presence of Ca(2+) and La(3+), a hybrid complex, Ca(2)La(2)CaM, was formed, and the binding of La(3+) to the N-terminal domain of CaM seemed preferable over binding to the C-terminal domain. Through fluorescence titration, it was shown that La(4)CaM and Ca(2)La(2)CaM had similar affinities to Mas as Ca(4)CaM. Fluorescence stopped-flow experiments showed that the dissociation rate of La(3+) from the C-terminal domain of CaM was higher than that from the N-terminal. However, in the presence of Mas, the dissociation rate of La(3+) decreased and the dissociation processes from both global domains were indistinguishable. In addition, compared with the case of Ca(4)CaM-Mas, the slower dissociations of Mas from La(4)CaM-Mas and Ca(2)La(2)CaM-Mas complexes indicate that in the presence of La(3+), the CaM-Mas complex became kinetically inert. A possible role of La(3+) in the Ca(2+)-CaM-dependent pathway is discussed.     

The effects of Ca2+ and Cd2+ on the secondary and tertiary structure of bovine testis calmodulin. A circular-dichroism study.

Near-u.v. and far-u.v. c.d. spectra of bovine testis calmodulin and its tryptic fragments (TR1C, N-terminal half, residues 1-77, and TR2C, C-terminal half, residues 78-148) were recorded in metal-ion-free buffer and in the presence of saturating concentrations of Ca2+ or Cd2+ under a range of different solvent conditions. The results show the following: if there is any interaction between the N-terminal and C-terminal halves of calmodulin, it has not apparent effect on the secondary or tertiary structure of either half; the conformational changes induced by Ca2+ or Cd2+ are substantially greater in TR2C than they are in TR1C; the presence of Ca2+ or Cd2+ confers considerable stability with respect to urea-induced denaturation, both for the whole molecule and for either of the tryptic fragments; a thermally induced transition occurs in whole calmodulin at temperatures substantially below the temperature of major thermal unfolding, both in the presence and in the absence of added metal ion; the effects of Cd2+ are identical with those of Ca2+ under all conditions studied.     

Arachidonic acid induces both Na+ and Ca2+ entry resulting in apoptosis

Marked accumulation of arachidonic acid (AA) and intracellular Ca2+ and Na+ overloads are seen during brain ischemia. In this study, we show that, in neurons, AA induces cytosolic Na+ ([Na+](cyt)) and Ca2+ ([Ca2+](cyt)) overload via a non-selective cation conductance (NSCC), resulting in mitochondrial [Na+](m) and [Ca2+](m) overload. Another two types of free fatty acids, including oleic acid and eicosapentaenoic acid, induced a smaller increase in the [Ca2+](i) and [Na+](i). RU360, a selective inhibitor of the mitochondrial Ca2+ uniporter, inhibited the AA-induced [Ca2+](m) and [Na+](m) overload, but not the [Ca2+](cyt) and [Na+](cyt) overload. The [Na+](m) overload was also markedly inhibited by either Ca2+-free medium or CGP3715, a selective inhibitor of the mitochondrial Na+(cyt)-Ca2+(m) exchanger. Moreover, RU360, Ca2+-free medium, Na+-free medium, or cyclosporin A (CsA) largely prevented AA-induced opening of the mitochondrial permeability transition pore, cytochrome c release, and caspase 3-dependent neuronal apoptosis. Importantly, Na+-ionophore/Ca2+-free medium, which induced [Na+](m) overload, but not [Ca2+](m) overload, also caused cyclosporin A-sensitive mitochondrial permeability transition pore opening, resulting in caspase 3-dependent apoptosis, indicating that [Na+](m) overload per se induced apoptosis. Our results therefore suggest that AA-induced [Na+](m) overload, acting via activation of the NSCC, is an important upstream signal in the mitochondrial-mediated apoptotic pathway. The NSCC may therefore act as a potential neuronal death pore which is activated by AA accumulation under pathological conditions.     

A note on Ca2+ binding to calmodulin

Binding of Ca2+ to calmodulin has been simulated on the basis of a model that assumes two classes, two sites in each class, of Ca2+ binding sites. With properly chosen values of binding constants for the two classes of sites, and with the assumption that certain degree of positive cooperativity exists between the two sites in each class, the overall binding isotherm can be generated so that it appears to be a single-transition, non-cooperative binding curve of four equivalent sites. Thus this model offers a resolution for some of the discrepancies among Ca2+ binding studies of calmodulin.     

NMR-driven secondary and tertiary structure model of Ca2+-loaded calexcitin

Calexcitin (CE) is a Ca2+-binding protein which is expressed in neuronal cells and is a member of the sarcoplasmic Ca2+-binding protein subfamily. The peptide backbone of Ca2+-CE has been assigned by NMR and it shows that CE is composed of nine alpha-helices-forming four EF-hands and an additional helix near the C-terminus. A structural model of CE suggests the presence of a putative recessed hydrophobic pocket that may be involved in Ca2+-mediated protein-ligand interactions. This feature is unique to CE and is absent in other SCPs, such as those from Branchiostoma and Nereis, and from calerythrin.     

Evidence that a Ca2+ chelator and a calmodulin blocker interfere with the structure of inter-Sertoli junctions

Ca2+ dependence of tight junction structure has been well documented in cultured epithelial tissues, and regulatory mechanisms have been identified. To analyse the possible control exerted on inter-Sertoli junctions, we exposed guinea-pig seminiferous tubules to the presence of a Ca2+ chelator (EGTA) and to a calmodulin blocker (Trifluoperazine, TFP) in vitro, for times ranging from 30 to 120 min. We observed the morphology of junctional complexes and the basal cytoplasmic regions in sections and replicas. Sertoli cell response to Ca2+ depletion involved several events: retraction of cells toward the base of the tubule and a consequent stretching of the points of fusion, augmented density of the cytoplasm, and destabilization of the array of intramembrane particles. Exposure of tubules to TFP resulted in disruption of the interactions between actin filaments and membrane junctional specialization, as well as a disorganization of other cytoskeletal elements. Thus, in vitro, junction integrity appears to be related to Ca2+ level, and Ca2+ depletion apparently interferes with Ca2+ distribution inside the cell and on microfilaments involved in junction regulation. Our results do not provide direct evidence for any particular mechanism of action of TFP, but a multiple effect is evident. TFP, which affects Ca2+ regulation and membrane fluidity, probably acts indirectly on junction-associated filaments. Both the experimental conditions tested suggest a Ca2+-mediated regulatory role of microfilaments of this complex junction.     

Ca2+ and Mg2+-binding and a putative calmodulin type Ca2+-binding site in Synechococcus Photosystem II

Thylakoids and Photosystem II particles prepared from the cyanobacterium Synechococcus PCC 7942 washed with a HEPES/glycerol buffer exhibited low rates of light-induced oxygen evolution. Addition of either Ca²⁺ or Mg²⁺ to both thylakoids and Photosystem II particles increased oxygen evolution independently, maximal rates being obtained by addition of both ions. If either preparation was washed with NaCl, light induced O₂ evolution was completely inhibited, but re-activated in the same manner by Ca²⁺ and Mg²⁺ but to a lower level. In the presence of Mg²⁺, the reactivation of O₂ evolution by Ca²⁺ allowed sigmoid kinetics, implying co-operative binding. The results are interpreted as indicating that not only Ca²⁺, but also Mg²⁺, is essential for light-induced oxygen evolution in thylakoids and Photosystem II particles from Synechococcus PC 7942. The significance of the reactivation kinetics is discussed. Reactivation by Ca²⁺ was inhibited by antibodies to mammalian calmodulin, indicating that the binding site in Photosystem II may be analogous to that of this protein.Abbreviation HEPES n-2-Hydroxyethylpiperazine--2-ethane sulphonic acid     

Ca2+ binding and conformational change in two series of point mutations to the individual Ca(2+)-binding sites of calmodulin.

Two series of site-directed mutations to the individual Ca(2+)-binding sites of Drosophila melanogaster calmodulin have been generated and studied. In each mutant, a conserved glutamic acid residue at position 12 in all of the Ca(2+)-binding loops has been mutated in one site. In one series the residue is changed to glutamine; in the second series the change is to lysine. The Ca(2+)-binding properties of these mutants and the wild-type protein under pseudo-physiological conditions are presented. In addition, Ca(2+)-induced changes to the environment of the single tyrosine residue (Tyr-138) have been studied for some of the mutants. Ca2+ binding to the wild-type protein is best modeled as two pairs of sites with a higher affinity pair that shows strong cooperativity. For all but one of these eight mutant proteins, only three Ca(2+)-binding events can be detected. In three of the amino-terminal mutants, the three residual sites are (i) a pair of relatively high affinity sites and (ii) a weakened low affinity site. For all four carboxyl-terminal mutations, the residual sites are three relatively low affinity sites. In general, mutations to sites 2 and 4 prove more deleterious than mutations to sites 1 and 3. The Ca(2+)-induced conformational changes in the vicinity of Tyr-138 are relatively undisturbed by mutations of site 1. However, the changes to Tyr-138 in the carboxyl-terminal site mutants indicate that upon disruption of the cooperative binding at the high affinity sites, conformational change in the carboxyl terminus occurs in two phases. It appears that binding of Ca2+ to either carboxyl-terminal site can elicit the first phase of the response but the second phase is almost abolished when site 4 is the mutated site. The final conformations of site 3 and 4 mutants are thus significantly different.     

Communication between two globular domains of calmodulin in the presence of mastoparan or caldesmon fragment. Ca2+ binding and 1H NMR

Ca2+ binding to calmodulin was measured in the presence of mastoparan or caldesmon fragment. Mastoparan and caldesmon fragment were used as model compounds of enzymes and cytoskeleton proteins, respectively, working as the target of calmodulin. Although the Ca2+ bindings of the two globular domains of calmodulin occur independently in the absence of the target peptide (or proteins), mastoparan and caldesmon fragment increased the affinity of Ca2+ and, at the same time, produced the positive cooperative Ca2+ bindings between the two domains. The result of Ca2+ binding was compared with 1H NMR spectra of calmodulin in the presence of equimolar concentration of mastoparan. It is known that a conformation change of the C-terminal half-region (C-domain) occurs by the Ca2+ binding to C-domain. A further change in conformation of C-domain was demonstrated by the Ca2+ binding to the N-terminal half-region (N-domain) in the presence of mastoparan. It indicates that the two domains of calmodulin get into communication with each other in the associated state with the target, and we concluded that the Ca2+ binding to the N-domain is responsive to the development of calmodulin function.     

Volume changes upon addition of Ca2+ to calmodulin: Ca2+-calmodulin conformational states

Measurement of the volume change by a rapid density method upon sequential addition of calcium ion to calmodulin showed relatively large, nonuniform increases for the first 4 moles Ca2+ per mole calmodulin. Substantially larger volume increases (approximately 15 ml/mol protein) were observed upon addition of the second and fourth moles Ca2+ relative to the first and third moles added per mole calmodulin. A total volume increase of approximately 170 ml/mol protein attended the addition of 4 moles Ca2+, as expected for multidentate carboxylate coordination to metal ion. Marginal changes in volume were observed upon further additions, the data showing a remarkably sharp transition after [Ca2+]/[calmodulin] = 4. The results are consistent with an ordered binding of Ca2+ in which pair-wise additions produce similar volume changes; the volume change behavior, however, does not indicate an absence of distinct conformational states for a Ca2+(1)-calmodulin and a Ca2+(3)-calmodulin complex as has been proposed on the basis of 1H-NMR evidences.     

Mechanism of local and global Ca2+ sensing by calmodulin in complex with a Ca2+ channel

Calmodulin (CaM) in complex with Ca(2+) channels constitutes a prototype for Ca(2+) sensors that are intimately colocalized with Ca(2+) sources. The C-lobe of CaM senses local, large Ca(2+) oscillations due to Ca(2+) influx from the host channel, and the N-lobe senses global, albeit diminutive Ca(2+) changes arising from distant sources. Though biologically essential, the mechanism underlying global Ca(2+) sensing has remained unknown. Here, we advance a theory of how global selectivity arises, and we experimentally validate this proposal with methodologies enabling millisecond control of Ca(2+) oscillations seen by the CaM/channel complex. We find that global selectivity arises from rapid Ca(2+) release from CaM combined with greater affinity of the channel for Ca(2+)-free versus Ca(2+)-bound CaM. The emergence of complex decoding properties from the juxtaposition of common elements, and the techniques developed herein, promise generalization to numerous molecules residing near Ca(2+) sources.     

The calmodulin binding domain of the plasma membrane Ca2+ pump interacts both with calmodulin and with another part of the pump

Synthetic peptides corresponding to the calmodulin-binding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-21 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (James, P., Maeda, M., Fisher, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 2905-2910). Peptides C28W, C20W, and C15W bound to calmodulin with an apparent 1:1 stoichiometry in the presence of Ca2+ and inhibited the activation of the Ca2+ pump by calmodulin, while C14 was ineffective. Substituting tyrosine (C28Y) or alanine (C28A) for the tryptophan residue lowered the affinity for calmodulin. The estimated Kd values for the calmodulin-peptide complexes were 0.1 nM for C28W, 5-15 nM for C20W, C28Y, and C28A, and 700-1700 nM for C15W. The Ca2+ pump in inside-out erythrocyte membrane vesicles was activated by proteolytic removal of the endogenous calmodulin-binding domain. Addition of C20W or C28W then inhibited calmodulin-independent Ca2+ transport, while a calmodulin-binding peptide from another enzyme had no effect. The inhibition of the pump by C20W was purely competitive with Ca2+, while C28W decreased the Vmax and increased the K1/2 for Ca2+, restoring the pump activity nearly to its low basal level. The results suggest that a calmodulin-binding peptide from any enzyme has two kinds of specificity: it shares with peptides from other enzymes the ability to bind to calmodulin, but only it has the specificity to interact with its own (proteolytically activated) enzyme.     

Identification and primary structure of a calmodulin binding domain of the Ca2+ pump of human erythrocytes

Exposure of the purified Ca2+ pump of human erythrocytes to chymotrypsin led to the rapid loss of calmodulin activation. A fragment of about 12 kDa was removed from the ATPase in 1-2 min. Blotting experiments with 125I-labeled calmodulin showed that this fragment contains the calmodulin binding region. The remainder of the ATPase molecule was degraded to a number of fragments ranging from 3 to 120 kDa; none of them bound calmodulin. To isolate the calmodulin binding domain, calmodulin which had been coupled to the Denny-Jaffe reagent (a cleavable radioactive photoaffinity cross-linker) was allowed to bind to the Ca2+ pump. After illumination to couple the cross-linker to the pump, the cleavable bond was split and the calmodulin removed, leaving the pump radioactively labeled. This pump was digested with chymotrypsin, and the products were separated by gel permeation chromatography. The only radioactive peak (migrating at about 12 kDa) was further purified on reverse-phase high pressure liquid chromatography (HPLC). Amino acid analysis showed the fragment to have a minimal molecular mass of 12.4 kDa and to contain a single methionine. After attempts to sequence the peptide directly failed. CNBr digestion was carried out on the labeled ATPase, producing both soluble and insoluble labeled material. After reverse-phase HPLC purification of the soluble material, a single radioactive peak was collected. Its sequence was (Formula: see text). A portion of this peak was passed through a microcalmodulin column; it bound in the presence of Ca2+ and was eluted by EDTA, and by a mixture of EDTA and urea. Staphylococcal V8 protease digestion of the eluted peak produced the same sequence as shown above, but starting at Leu-2 and ending at Glu-32. Structural analysis of this peptide showed that it shares features with the calmodulin binding domains of other enzymes which are regulated by calmodulin.     

Role of Ca2+ activation and bilobal structure of calmodulin in nuclear and nucleolar localization

Beta2-ARs (beta2-adrenoceptors) become desensitized rapidly upon recruitment of cytosolic beta-arrestin. PDE4D5 (family 4 cAMP-specific phosphodiesterase, subfamily D, isoform 5) can be recruited in complex with beta-arrestin, whereupon it regulates PKA (cAMP-dependent protein kinase) phosphorylation of the beta2-AR. In the present study, we have used novel technology, employing a library of overlapping peptides (25-mers) immobilized on cellulose membranes that scan the entire sequence of beta-arrestin 2, to define the interaction sites on beta-arrestin 2 for binding of PDE4D5 and the cognate long isoform, PDE4D3. We have identified a binding site in the beta-arrestin 2 N-domain for the common PDE4D catalytic unit and two regions in the beta-arrestin 2 C-domain that confer specificity for PDE4D5 binding. Alanine-scanning peptide array analysis of the N-domain binding region identified severely reduced interaction with PDE4D5 upon R26A substitution, and reduced interaction upon either K18A or T20A substitution. Similar analysis of the beta-arrestin 2 C-domain identified Arg286 and Asp291, together with the Leu215-His220 region, as being important for binding PDE4D5, but not PDE4D3. Transfection with wild-type beta-arrestin 2 profoundly decreased isoprenaline-stimulated PKA phosphorylation of the beta2-AR in MEFs (mouse embryo fibroblasts) lacking both beta-arrestin 1 and beta-arrestin 2. This effect was negated using either the R26A or the R286A mutant form of beta-arrestin 2 or a mutant with substitution of an alanine cassette for Leu215-His220, which showed little or no PDE4D5 binding, but was still recruited to the beta2-AR upon isoprenaline challenge. These data show that the interaction of PDE4D5 with both the N- and C-domains of beta-arrestin 2 are essential for beta2-AR regulation.     

Probable role of amphiphilicity in the binding of mastoparan to calmodulin

Two-dimensional helical wheel diagrams and calculations of mean hydrophobic moments show mastoparan, mastoparan X, and Polistes mastoparan to have all the properties expected for amphiphilic helices. Circular dichroic properties are consistent with a random form for these peptides in dilute aqueous solution, but greater than 50% helix is apparent when the peptides are dissolved in 70% trifluoroethanol/water mixtures (v/v) or when the peptides are bound to calmodulin. Changes in the fluorescence spectra, anisotropy, and accessibility of tryptophan whose indole side chain is on the apolar surface of the amphiphilic helix imply a significant role for the apolar surface in the binding of the mastoparans and another amphiphilic peptide, melittin, to calmodulin. These data provide a useful model for designing high-affinity synthetic peptide inhibitors of calmodulin.     

Binding of Ca 2+ to normal and dicoumarol-induced prothrombin

The Ca²⁺ binding properties of normal bovine prothrombin have been studied and compared with those of an abnormal bovine prothrombin induced by dicoumarol. The normal prothrombin binds up to 10–12 Ca²⁺ per mole of protein. The three first Ca²⁺ were bound to sites which exhibited positive cooperativity. A Ca²⁺ dependent conformational change was demonstrated during the binding of the first three Ca²⁺. In contrast with normal prothrombin, the dicoumarol-induced prothrombin had only one high affinity binding site. No ligand-induced conformational change was detected in this prothrombin.     

1H-NMR studies of calmodulin. The nature of the Ca2+-dependent conformational change

The replication of M13 single-stranded DNA by the 9S DNA polymerase alpha from calf thymus has been studied in vitro. Priming conditions, the nature of the replication products and conditions for optimal elongation have been investigated. Oligonucleotides comprising only four nucleotides can serve as primers. Both ribo and deoxy oligonucleotides can be elongated. Priming by the short oligonucleotides occurs at multiple sites on the M13 genome. If replication is primed at single sites with a specific pentadecamer or with RNA in the origin of replication, specific pausing sites are observed. These pausing sites can partly be correlated with secondary structures in the template DNA. Addition of Escherichia coli single-stranded DNA binding protein leads to a weakening of pausing sites and to the synthesis of longer products. The 9S enzyme is able to proceed through most of the pausing sites resulting in the synthesis of product molecules as long as 6600 nucleotides. The 9S DNA polymerase alpha contains a potent DNA primase activity which enables it to initiate replication on a single-stranded template in the presence of the four NTPs . However, priming is also possible in the presence of ATP alone. The priming sites are not randomly distributed over the M13 DNA.     

A molecular dynamics study of the effect of Ca2+ removal on calmodulin structure

Calmodulin is a small (148 residues), ubiquitous, highly-conserved Ca(2+) binding protein serving as a modulator of many calcium-dependent processes. In this study, we followed, by means of molecular dynamics, the structural stability of the protein when one of its four bound Ca(2+) ions is removed, and compared it to a simulation of the fully Ca(2+) bound protein. We found that the removal of a single Ca(2+) ion from the N-lobe of the protein, which has a lower affinity for the ion, is sufficient to initiate a considerable structural rearrangement. Although the overall structure of the fully 4 Ca(2+) bound protein remained intact in the extended conformation, the Ca(2+)-removed protein changed its conformation into a compact state. The observation that the 3 Ca(2+) loaded protein assumes a compacted solution state is in accord with experimental observation that the NSCP protein, which binds only three Ca(2+) ions, is natively in a compact state. Examination of the folding dynamics reveals a cooperation between the C-lobe, N-lobe, and the interdomain helix that enable the conformation change. The forces driving this conformational change are discussed.     

Ca2+ Binding to Site I of the Cardiac Ca2+ Pump Is Sufficient to Dissociate Phospholamban

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca²⁺-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca²⁺. Recent cross-linking studies have suggested that PLB binding and Ca²⁺ binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca²⁺-ATPase, preventing formation of E1, the conformation that binds two Ca²⁺ (at sites I and II) with high affinity and is required for ATP hydrolysis. Here we determined whether Ca²⁺ binding to site I, site II, or both sites is sufficient to dissociate PLB from the Ca²⁺ pump. Seven SERCA2a mutants with amino acid substitutions at Ca²⁺-binding site I (E770Q, T798A, and E907Q), site II (E309Q and N795A), or both sites (D799N and E309Q/E770Q) were made, and the effects of Ca²⁺ on N30C-PLB cross-linking to Lys³²⁸ of SERCA2a were measured. In agreement with earlier reports with the skeletal muscle Ca²⁺-ATPase, none of the SERCA2a mutants (except E907Q) hydrolyzed ATP in the presence of Ca²⁺; however, all were phosphorylatable by P_i to form E2P. Ca²⁺ inhibition of E2P formation was observed only in SERCA2a mutants retaining site I. In cross-linking assays, strong cross-linking between N30C-PLB and each Ca²⁺-ATPase mutant was observed in the absence of Ca²⁺. Importantly, however, micromolar Ca²⁺ inhibited PLB cross-linking only to mutants retaining a functional Ca²⁺-binding site I. The dynamic equilibrium between Ca²⁺ pumps and N30C-PLB was retained by all mutants, demonstrating normal regulation of cross-linking by ATP, thapsigargin, and anti-PLB antibody. From these results we conclude that site I is the key Ca²⁺-binding site regulating the physical association between PLB and SERCA2a.     

Apocalmodulin and Ca2+ calmodulin bind to the same region on the skeletal muscle Ca2+ release channel.

The skeletal muscle Ca2+ release channel (RYR1) is regulated by calmodulin in both its Ca2+-free (apocalmodulin) and Ca2+-bound (Ca2+ calmodulin) states. Apocalmodulin is an activator of the channel, and Ca2+ calmodulin is an inhibitor of the channel. Both apocalmodulin and Ca2+ calmodulin binding sites on RYR1 are destroyed by a mild tryptic digestion of the sarcoplasmic reticulum membranes, but calmodulin (either form), bound to RYR1 prior to tryptic digestion, protects both the apocalmodulin and Ca2+ calmodulin sites from tryptic destruction. The protected sites are after arginines 3630 and 3637 on RYR1. These studies suggest that both Ca2+ calmodulin and apocalmodulin bind to the same or overlapping regions on RYR1 and block access of trypsin to sites at amino acids 3630 and 3637. This sequence is part of a predicted Ca2+ CaM binding site of amino acids 3614-3642 [Takeshima, H., et al. (1989) Nature 339, 439-445].