Identification of hyperreactive cysteines within ryanodine receptor type 1 by mass spectrometry

The skeletal-type ryanodine receptor (RyR1) undergoes covalent adduction by nitric oxide (NO), redox-induced shifts in cation regulation, and non-covalent interactions driven by the transmembrane redox potential that enable redox sensing. Tight redox regulation of RyR1 is thought to be primarily mediated through highly reactive (hyperreactive) cysteines. Of the 100 cysteines per subunit of RyR1, approximately 25-50 are reduced, with 6-8 considered hyperreactive. Thus far, only Cys-3635, which undergoes selective adduction by NO, has been identified. In this report, RyR1-enriched junctional sarcoplasmic reticulum is labeled with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM, 1 pmol/microg of protein) in the presence of 10 mm Mg(2+), conditions previously shown to selectively label hyperreactive sulfhydryls and eliminate redox sensing. The CPM-adducted RyR1 is separated by gel electrophoresis and subjected to in-gel tryptic digestion. Isolation of CPM-adducted peptides is achieved by analytical and microbore high-performance liquid chromatography utilizing fluorescence and UV detection. Subsequent analysis using two direct and one tandem mass spectrometry methods results in peptide masses and sequence data that, compared with the known primary sequence of RyR1, enable unequivocal identification of CPM-adducted cysteines. This work is the first to directly identify seven hyperreactive cysteines: 1040, 1303, 2436, 2565, 2606, 2611, and 3635 of RyR1. In addition to Cys-3635, the nitrosylation site, six additional cysteines may contribute toward redox regulation of the RyR1 complex.     

Three-dimensional reconstruction of the recombinant type 2 ryanodine receptor and localization of its divergent region 1

Isoform 2 of the ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. In the present study, two kinds of RyR2 cDNA were constructed, one encoding the wild type mouse RyR2 (RyR2(wt)) and the other encoding modified RyR2, into which was inserted a cDNA encoding green fluorescent protein (GFP). GFP was inserted into the divergent region 1 (DR1) of RyR2, after the Asp-4365 (RyR2(D4365-GFP)). HEK293 cells expressing both RyR2(wt) and RyR2(D4365-GFP) cDNAs showed caffeine- and ryanodine-sensitive calcium release, demonstrating that both wild type and modified RyR2s form functional calcium release channels. Cells expressing the fusion protein, RyR2(D4365-GFP), were readily identified by their fluorescence due to the presence of GFP, indicating that the inserted GFP folded properly. Both expressed RyR2s were purified from cell lysates in a single step by affinity chromatography using a GST-FKBP12.6 as the affinity ligand. Cryoelectron microscopy of purified RyR2s showed structurally intact receptors, and three-dimensional reconstructions were obtained by single particle image processing. The three-dimensional reconstruction of RyR2(wt) appeared very similar to that of the native RyR2 purified from dog heart. The location of the inserted GFP, and consequently of DR1, was mapped on the three-dimensional structure of RyR2 to one of the subunit's characteristic domains, domain 3, also known as the "handle" domain. This study describes the first internal fusion of a protein into a ryanodine receptor, and it demonstrates the potential of this technology for localizing functional and structural domains on the three-dimensional structure of RyR.     

Lobe-dependent regulation of ryanodine receptor type 1 by calmodulin

Calmodulin activates the skeletal muscle Ca(2+) release channel RYR1 at nm Ca(2+) concentrations and inhibits the channel at microm Ca(2+) concentrations. Using a deletion mutant of calmodulin, we demonstrate that amino acids 2-8 are required for high affinity binding of calmodulin to RYR1 at both nm and microm Ca(2+) concentrations and are required for maximum inhibition of the channel at microm Ca(2+) concentrations. In contrast, the addition of three amino acids to the N terminus of calmodulin increased the affinity for RYR1 at both nm and microm Ca(2+) concentrations, but destroyed its functional effects on RYR1 at nm Ca(2+). Using both full-length RYR1 and synthetic peptides, we demonstrate that the calmodulin-binding site on RYR1 is likely to be noncontiguous, with the C-terminal lobe of both apocalmodulin and Ca(2+)-calmodulin binding to amino acids between positions 3614 and 3643 and the N-terminal lobe binding at sites that are not proximal in the primary sequence. Ca(2+) binding to the C-terminal lobe of calmodulin converted it from an activator to an inhibitor, but an interaction with the N-terminal lobe was required for a maximum effect on RYR1. This interaction apparently depends on the native sequence or structure of the first few amino acids at the N terminus of calmodulin.     

MCF-7 breast carcinoma cells express ryanodine receptor type 1: functional characterization and subcellular localization

Breast carcinoma-derived MCF-7 cells are frequently used in biomedical research. However, few reports exist regarding the characterization of signaling mechanisms in these cancerous cells involved in intracellular Ca²⁺ dynamics. Consequently, the aim of these experiments was to characterize the ryanodine receptor/Ca²⁺ release channel (RyR) present in MCF-7 cells. Ryanodine (100 nM), cADPR (5 μM), and caffeine (10 mM) promoted cytoplasmic Ca²⁺ mobilization; in contrast, ryanodine at inhibitory concentration (100 μM) decreased the basal Ca²⁺ level. Fluorescent probes demonstrated that RyR is located mainly in endomembranes. Some degree of co-localization with inositol trisphosphate receptor (IP₃R) was observed, whereas coincidence with thapsigargin-sensitive Ca²⁺-ATPase (SERCA) was more limited. Molecular cloning resulted in the detection exclusively of RyR isoform 1. For the first time, it is shown that MCF-7 cells express functional RyR.     

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca²⁺ release during excitation–contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose–response profiles and previously unreported effects on RyR were further tested using [³H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca²⁺. Two of these hits activated RyR1 only at micromolar Ca²⁺, highlighting them as potential enhancers of excitation–contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation–contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca²⁺ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.     

Dantrolene stabilizes domain interactions within the ryanodine receptor

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.     

Homer regulates gain of ryanodine receptor type 1 channel complex

Homer proteins form an adapter system that regulates coupling of group 1 metabotropic glutamate receptors with intracellular inositol trisphosphate receptors and is modified by neuronal activity. Here, we demonstrate that Homer proteins also physically associate with ryanodine receptors type 1 (RyR1) and regulate gating responses to Ca(2+), depolarization, and caffeine. In contrast to the prevailing notion of Homer function, Homer1c (long form) and Homer1-EVH1 (short form) evoke similar changes in RyR activity. The EVH1 domain mediates these actions of Homer and is selectively blocked by a peptide that mimics the Homer ligand. 1B5 dyspedic myotubes expressing RyR1 with a point mutation of a putative Homer-binding domain exhibit significantly reduced (approximately 33%) amplitude in their responses to K(+) depolarization compared with cells expressing wild type protein. These results reveal that in addition to its known role as an adapter protein, Homer is a direct modulator of Ca(2+) release gain. Homer is the first example of an "adapter" that also modifies signaling properties of its target protein. The present work reveals a novel mechanism by which Homer directly modulates the function of its target protein RyR1 and excitation-contraction coupling in skeletal myotubes. This form of regulation may be important in other cell types that express Homer and RyR1.     

Postulated role of interdomain interaction between regions 1 and 2 within type 1 ryanodine receptor in the pathogenesis of porcine malignant hyperthermia

We have demonstrated recently that CICR (Ca2+-induced Ca2+ release) activity of RyR1 (ryanodine receptor 1) is held to a low level in mammalian skeletal muscle ('suppression' of the channel) and that this is largely caused by the interdomain interaction within RyR1 [Murayama, Oba, Kobayashi, Ikemoto and Ogawa (2005) Am. J. Physiol. Cell Physiol. 288, C1222-C1230]. To test the hypothesis that aberration of this suppression mechanism is involved in the development of channel dysfunctions in MH (malignant hyperthermia), we investigated properties of the RyR1 channels from normal and MHS (MH-susceptible) pig skeletal muscles with an Arg615-->Cys mutation using [3H]ryanodine binding, single-channel recordings and SR (sarcoplasmic reticulum) Ca2+ release. The RyR1 channels from MHS muscle (RyR1MHS) showed enhanced CICR activity compared with those from the normal muscle (RyR1N), although there was little or no difference in the sensitivity to several ligands tested (Ca2+, Mg2+ and adenine nucleotide), nor in the FKBP12 (FK506-binding protein 12) regulation. DP4, a domain peptide matching the Leu2442-Pro2477 region of RyR1 which was reported to activate the Ca2+ channel by weakening the interdomain interaction, activated the RyR1N channel in a concentration-dependent manner, and the highest activity of the affected channel reached a level comparable with that of the RyR1MHS channel with no added peptide. The addition of DP4 to the RyR1MHS channel produced virtually no further effect on the channel activity. These results suggest that stimulation of the RyR1MHS channel caused by affected inter-domain interaction between regions 1 and 2 is an underlying mechanism for dysfunction of Ca2+ homoeostasis seen in the MH phenotype.     

Functional expression of recombinant type 1 ryanodine receptor in insect cells

We have investigated the biochemical properties of the rabbit ryanodine receptor type 1 (RyR1) from skeletal muscle functionally expressed in insect sf 21 cells infected with recombinant baculovirus. Equilibrium [3H]ryanodine binding assays applied to total membrane fractions from sf 21 cells expressing recombinant RyR1 showed a non-hyperbolic saturation curve (Hill coefficient = 2.1). The [3H]ryanodine binding was enhanced by 1 mM AMP-PCP and 10 mM caffeine, whereas 10 mM Mg(2+) and 5 microM ruthenium red reduced the specific binding. The dependence of [3H]ryanodine binding on ionic strength showed positive cooperativity (Hill coefficient = 2.2) with a plateau at 1 M KCl. The recombinant RyR1 showed a bell-shaped [3H]ryanodine binding curve when free [Ca(2+)] was increased, with an optimal concentration around 100 microM.Confocal microscopy studies using the Ca(2+) ATPase selective inhibitor, thapsigargin coupled to fluorescein and ryanodine coupled to Texas red demonstrated that the recombinant RyR1 and the Ca(2+) ATPase co-localize to the same intracellular membrane. No significant RyR1 fluorescence was observed at the plasma membrane.Fluo-4-loaded sf 21 cells expressing recombinant RyR1 responded to activating-low ryanodine concentrations (100 nM) or caffeine (10 mM) with a sharp rise in intracellular Ca2 followed by a sustained phase, in contrast, sf 21 cells expressing the human bradykinin type 2 receptor did not respond to ryanodine or caffeine.These results demonstrate the expression of recombinant RyR1 in sf 21 cells with functional properties similar to what has been previously reported for native RyR1 in mammalian tissues, however, some differences were observed in [3H]ryanodine binding assays compared to native rabbit RyR1. Hence, the baculovirus expression system provides a generous source of protein to accomplish structure-function studies and an excellent model to assess functional properties of wild type and mutant RyR1.     

Functionally accepted insertions of proteins within protein domains

Experiments were designed to explore the tolerance of protein structure and folding to very large insertions of folded protein within a structural domain. Dihydrofolate reductase and beta-lactamase have been inserted in four different positions of phosphoglycerate kinase. The resultant chimeric proteins are all overexpressed, and the host as well as the inserted partners are functional. Although not explicitly designed, functional coupling between the two fused partners was observed in some of the chimeras. These results show that the tolerance of protein structures to very large structured insertions is more general than previously expected and supports the idea that the natural sequence continuity of a structural domain is not required for the folding process. These results directly suggest a new experimental approach to screen, for example, for folded protein in randomized polypeptide sequences.     

A drug and ATP binding site in type 1 ryanodine receptor

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Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.     

Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1

The functional relevance of putative Ca(2+) binding motifs previously identified with Ca(2+) overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca(2+)-imaging techniques, [(3)H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [(3)H]ryanodine binding measurements, both Ca(2+) activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [(3)H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca(2+) concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3-5) did not affect functional responses in 1B5 myotubes except that the EC(50) for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [(3)H]ryanodine binding experiments, the Ca(2+)-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.     

Dynamic regulation of ryanodine receptor type 1 (RyR1) channel activity by Homer 1

Homer, a family of scaffolding proteins originally identified in neurons, is also expressed in skeletal muscle. Previous studies showed that splice variants of Homer 1 (H1) amplify the gain of the ryanodine receptor type 1 (RyR1) channel complex. Using [3H]ryanodine ([3H]Ry) to probe the conformational state of RyR1, the actions of long- and short-forms of H1 are examined singly and in combination. At < or =200 nM, H1 long-forms (H1b or H1c possessing coiled-coil (CC) domains) and short-forms (H1a or H1EVH1 lacking CC domains) enhance specific [3H]Ry binding to RyR1. However, at a concentration > 200 nM, either H1 form completely inhibited [3H]Ry binding. Importantly, the combinations of H1c+H1EVH1, or H1b+H1a acted in an additive manner to enhance or inhibit [3H]Ry-binding activity. H1a and H1c individually or in combination produced the same dynamic pattern in regulating purified RyR1 channels reconstituted in planar lipid bilayers. In combination, their net action on RyR1 channels depends on total concentrations of H1. These data provide a mechanism by which constitutively and transiently expressed H1 forms can tightly regulate RyR1 channel activity in response to changing levels of expression and degradation of H1 proteins.     

Addressing protein localization within the nucleus

Bridging the gap between the number of gene sequences in databases and the number of gene products that have been functionally characterized in any way is a major challenge for biology. A key characteristic of proteins, which can begin to elucidate their possible functions, is their subcellular location. A number of experimental approaches can reveal the subcellular localization of proteins in mammalian cells. However, genome databases now contain predicted sequences for a large number of potentially novel proteins that have yet to be studied in any way, let alone have their subcellular localization determined. Here we ask whether using bioinformatics tools to analyse the sequence of proteins whose subnuclear localizations have been determined can reveal characteristics or signatures that might allow us to predict localization for novel protein sequences.     

Probing luminal negative charge in the type 3 ryanodine receptor

In this study, we have investigated block of potassium (K(+)) current by neomycin, a large polycation, from the luminal face of the type 3 ryanodine receptor (RyR3). Previous studies have shown that neomycin is an open channel blocker of RyR2 that interacts with negatively charged residues in the mouth of the conduction pathway to partially occlude it. In the current study, we have used neomycin as a probe to investigate proposed negatively charged regions in the luminal pore mouth of RyR3. Luminal neomycin induces concentration- and voltage-dependent partial block to a subconductance state in RyR3. Blocking parameters calculated in this study show that neomycin has a higher affinity for RyR3 than RyR2, but block may occur at the same site within the pore mouth. The change in affinity may be due to altered negative charge density at the site of interaction.     

Phosphorylation of the cardiac ryanodine receptor by cAMP-dependent protein kinase

The phosphorylation of canine cardiac and skeletal muscle ryanodine receptors by the catalytic subunit of cAMP-dependent protein kinase has been studied. A high-molecular-weight protein (Mr 400,000) in cardiac microsomes was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. A monoclonal antibody against the cardiac ryanodine receptor immunoprecipitated this phosphoprotein. In contrast, high-molecular-weight proteins (Mr 400,000-450,000) in canine skeletal microsomes isolated from extensor carpi radialis (fast) or superficial digitalis flexor (slow) muscle fibers were not significantly phosphorylated. In agreement with these findings, the ryanodine receptor purified from cardiac microsomes was also phosphorylated by cAMP-dependent protein kinase. Phosphorylation of the cardiac ryanodine receptor in microsomal and purified preparations occurred at the ratio of about one mol per mol of ryanodine-binding site. Upon phosphorylation of the cardiac ryanodine receptor, the levels of [3H]ryanodine binding at saturating concentrations of this ligand increased by up to 30% in the presence of Ca2+ concentrations above 1 microM in both cardiac microsomes and the purified cardiac ryanodine receptor preparation. In contrast, the Ca2+ concentration dependence of [3H]ryanodine binding did not change significantly. These results suggest that phosphorylation of the ryanodine receptor by cAMP-dependent protein kinase may be an important regulatory mechanism for the calcium release channel function in the cardiac sarcoplasmic reticulum.     

A FRET-based analysis of SNPs without fluorescent probes

Fluorescence resonance energy transfer (FRET) is a simple procedure for detecting specific DNA sequences, and is therefore used in many fields. However, the cost is relatively high, because FRET-based methods usually require fluorescent probes. We have designed a cost-effective way of using FRET, and developed a novel approach for the genotyping of single nucleotide polymorphisms (SNPs) and allele frequency estimation. The key feature of this method is that it uses a DNA-binding fluorogenic molecule, SYBR Green I, as an energy donor for FRET. In this method, single base extension is performed with dideoxynucleotides labeled with an orange dye and a red dye in the presence of SYBR Green I. The dyes incorporated into the extended products accept energy from SYBR Green I and emit fluorescence. We have validated the method with ten SNPs, which were successfully discriminated by end-point measurements of orange and red fluorescence intensity in a microplate fluorescence reader. Using a mixture of homozygous samples, we also confirmed the potential of this method for estimation of allele frequency. Application of this strategy to large-scale studies will reduce the time and cost of genotyping a vast number of SNPs.     

Unaltered ryanodine receptor protein levels in ischemic cardiomvopathy

Previous studies on sarcoplasmic reticulum calcium release channel (ryanodine receptor) demonstrated that protein levels are unchanged in myocardium from hearts with end-stage failing dilated cardiomyopathy. In ischemic cardiomyopathy, ryanodine receptor mRNA levels were shown to be decreased but no data on protein levels are available. Accordingly, protein levels of ryanodine receptor, calsequestrin, and sarcoplasmic reticulum calcium-ATPase (SR-Ca²⁺-ATPase) were measured by Western blot analysis in nonfailing human myocardium (n = 7) and in end-stage failing myocardium due to ischemic cardiomyopathy (n = 14). Protein levels of calsequestrin which is the major sarcoplasmic reticulum calcium storage protein were similar in nonfailing myocardium and in myocardium from end-stage failing hearts with ischemic cardiomyopathy. Ryanodine receptor protein levels, normalized to total protein or calsequestrin were also unchanged in ischemic cardiomyopathy. In contrast, protein levels of SR-Ca²⁺-ATPase normalized to total protein or calsequestrin were decreased by 31 and 30%, respectively (p < 0.05). The data indicate that (I) sarcoplasmic reticulum calcium uptake sites are decreased relative to the release sites in ischemic cardiomyopathy, and (2) alterations of sarcoplasmic proteins are similar in ischemic and dilated cardiomyopathy.     

A variably spliced region in the type 1 ryanodine receptor may participate in an inter-domain interaction

The aim of the present study was to examine residues that are variably spliced in the juvenile and adult isoforms of the skeletal-muscle RyR1 (type 1 ryanodine receptor). The juvenile ASI(-) splice variant is less active than the adult ASI(+) variant and is overexpressed in patients with DM (myotonic dystrophy) [Kimura, Nakamori, Lueck, Pouliquin, Aoike, Fujimura, Dirksen, Takahashi, Dulhunty and Sakoda (2005) Hum. Mol. Genet. 14, 2189-2200]. In the present study, we explore the ASI region using synthetic peptides corresponding to rabbit RyR1 residues Thr3471-Gly3500 either containing [PASI(+)] or lacking [PASI(-)] the ASI residues. Both peptides increased [3H]ryanodine binding to rabbit RyR1s, increased Ca2+ release from sarcoplasmic reti-culum vesicles and increased single RyR1 channel activity. The peptide PASI(-) was more active in each case than PASI(+). [3H]Ryanodine binding to recombinant ASI(+)RyR1 or ASI(-)-RyR1 was enhanced more by PASI(-) than PASI(+), with the greatest increase seen when PASI(-) was added to ASI(-)RyR1. The activation of the RyR channels is consistent with the hypo-thesis that the peptides interrupt an inhibitory inter-domain inter-action and that PASI(-) is more effective at interrupting this interaction than PASI(+). We therefore suggest that the ASI(-) sequence interacts more tightly than the ASI(+) sequence with its binding partner, so that the ASI(-)RyR1 is more strongly inhibited (less active) than the ASI(+)RyR1. Thus the affinity of the binding partners in this inter-domain interaction may deter-mine the activities of the mature and juvenile isoforms of RyR1 and the stronger inhibition in the juvenile isoform may contribute to the myopathy in DM.