Tacrolimus (FK506) modulates calcium release and contractility of intestinal smooth muscle

Several proteins have been identified that associate with calcium release channels and potentially regulate their function. Using tacrolimus as a pharmacological tool, we investigated whether the immunophilin FKBP12 modulates ryanodine receptor channels in intestinal smooth muscle. Results with PCR demonstrated the presence of type-3 ryanodine receptor and FKBP12 in this tissue. Tacrolimus caused an irreversible increase of the intracellular calcium concentration, which was abolished by pretreatment with caffeine. The calcium channel blocker verapamil did not affect the response to tacrolimus. Tacrolimus decreased the calcium concentration in the sarcoplasmic reticulum. Caffeine, but not inositol 1,4,5-trisphosphate or heparin, abolished this effect. Finally, tacrolimus significantly and irreversibly decreased the tension generated by intestinal muscle strips. These data support our hypothesis that the immunophilin FKBP12 modulates ryanodine receptor function in smooth muscle. Interactions between such regulatory proteins and calcium release channels may play an important role in excitation-contraction coupling and other intracellular signaling processes.     

Properties of a new calcium-permeable single channel from tracheal microsomes

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.     

Deletion of amino acids 1641-2437 from the foot region of skeletal muscle ryanodine receptor alters the conduction properties of the Ca release channel.

The ryanodine receptor (RyR) of skeletal muscle contains two functional domains: a carboxyl-terminal hydrophobic domain that forms the putative conduction pore of the calcium release channel, and a large cytoplasmic domain that corresponds to the "foot structure." To understand the contribution of the foot structure to the function of the calcium release channel, we studied a RyR deletion mutant, delta(1641-2437)-RyR, in which a region that is rich in glutamate and aspartate residues (a.a. 1641-2437) was removed. The wild-type and delta(1641-2437)-RyR proteins were expressed in a Chinese hamster ovary (CHO) cell line, and functions of single calcium release channels were measured in the lipid bilayer membrane. The wild-type RyR forms functional calcium release channels with a linear current-voltage relationship similar to that of the native channel identified in the sarcoplasmic reticulum membrane of skeletal muscle, whereas the channels formed by delta(1641-2437)-RyR exhibit significant inward rectification, i.e., currents moving from cytoplasm into SR lumen were approximately 20% less than that in the opposite direction. As in to the wt-RyR channel, opening of the delta(1641-2437)-RyR channel has a bell-shaped dependence on the cytoplasmic calcium, but the calcium-dependent activation and inactivation processes of the delta(1641-2437)-RyR channel are shifted to higher calcium concentrations. Our data show that deletion of a.a. 1641-2437 from the foot region of the skeletal muscle RyR results in changes in both ion conduction and calcium-dependent regulation of the calcium release channel.     

Transient loss of voltage control of Ca2+ release in the presence of maurocalcine in skeletal muscle

In skeletal muscle, sarcoplasmic reticulum (SR) calcium release is controlled by the plasma membrane voltage through interactions between the voltage-sensing dihydropyridine receptor (DHPr) and the ryanodine receptor (RYr) calcium release channel. Maurocalcine (MCa), a scorpion toxin peptide presenting some homology with a segment of a cytoplasmic loop of the DHPr, has been previously shown to strongly affect the activity of the isolated RYr. We injected MCa into mouse skeletal muscle fibers and measured intracellular calcium under voltage-clamp conditions. Voltage-activated calcium transients exhibited similar properties in control and in MCa-injected fibers during the depolarizing pulses, and the voltage dependence of calcium release was similar under the two conditions. However, MCa was responsible for a pronounced sustained phase of Ca(2+) elevation that proceeded for seconds following membrane repolarization, with no concurrent alteration of the membrane current. The magnitude of the underlying uncontrolled extra phase of Ca(2+) release correlated well with the peak calcium release during the pulse. Results suggest that MCa binds to RYr that open on membrane depolarization and that this interaction specifically alters the process of repolarization-induced closure of the channels.     

Characteristics of irreversible ATP activation suggest that native skeletal ryanodine receptors can be phosphorylated via an endogenous CaMKII.

Phosphorylation of skeletal muscle ryanodine receptor (RyR) calcium release channels by endogenous kinases incorporated into lipid bilayers with native sarcoplasmic reticulum vesicles was investigated during exposure to 2 mM cytoplasmic ATP. Activation of RyRs after 1-min exposure to ATP was reversible upon ATP washout. In contrast, activation after 5 to 8 min was largely irreversible: the small fall in activity with washout was significantly less than that after brief ATP exposure. The irreversible activation was reduced by acid phosphatase and was not seen after exposure to nonhydrolyzable ATP analogs. The data suggested that the channel complex was phosphorylated after addition of ATP and that phosphorylation reduced the RyR's sensitivity to ATP, adenosine, and Ca(2+). The endogenous kinase was likely to be a calcium calmodulin kinase II (CaMKII) because the CaMKII inhibitor KN-93 and an inhibitory peptide for CaMKII prevented the phosphorylation-induced irreversible activation. In contrast, phosphorylation effects remained unchanged with inhibitory peptides for protein kinase C and A. The presence of CaMKIIbeta in the SR vesicles was confirmed by immunoblotting. The results suggest that CaMKII is anchored to skeletal muscle RyRs and that phosphorylation by this kinase alters the enhancement of channel activity by ATP and Ca(2+).     

Calsequestrin Is an Inhibitor of Skeletal Muscle Ryanodine Receptor Calcium Release Channels

We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca²⁺] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (∼35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.     

Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1-50 mM) and ryanodine (100 microM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all-or-none rises in intracellular calcium. Inositol 1,4,5-trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine-elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.     

Regulation of Ca2+ release from internal stores in cardiac and skeletal muscles

It is widely accepted that Ca2+ is released from the sarcoplasmic reticulum by a specialized type of calcium channel, i.e., ryanodine receptor, by the process of Ca2+-induced Ca2+ release. This process is triggered mainly by dihydropyridine receptors, i.e., L-type (long lasting) calcium channels, directly or indirectly interacting with ryanodine receptor. In addition, multiple endogenous and exogenous compounds were found to modulate the activity of both types of calcium channels, ryanodine and dihydropyridine receptors. These compounds, by changing the Ca2+ transport activity of these channels, are able to influence intracellular Ca2+ homeostasis. As a result not only the overall Ca2+ concentration becomes affected but also spatial distribution of this ion in the cell. In cardiac and skeletal muscles the release of Ca2+ from internal stores is triggered by the same transport proteins, although by their specific isoforms. Concomitantly, heart and skeletal muscle specific regulatory mechanisms are different.     

Reconstitution of purified cardiac muscle calcium release channel (ryanodine receptor) in planar bilayers

The purified ryanodine receptor of heart sarcoplasmic reticulum (SR) has been reconstituted into planar phospholipid bilayers and found to form Ca2+-specific channels. The channels are strongly activated by Ca2+ (10 nM) in the presence of ATP (1 mM) and ryanodine, and inactivated by Mg2+ (3 mM) or ruthenium red (30 microM). These characteristics are diagnostic of calcium release from heart SR. The cardiac ryanodine receptor, which has previously been identified as the foot structure, is now identified as the calcium release channel. A similar identity of the calcium release channel has recently been reported for skeletal muscle. The characteristics of the calcium release channel from skeletal muscle and heart are similar in that they: 1) consist of an oligomer of a single high molecular weight polypeptide (Mr 360,000 for skeletal muscle and 340,000 for heart); 2) exist morphologically as the foot structure; 3) are activated (ATP, Ca2+, ryanodine) and inhibited (ruthenium red and Mg2+) by a number of the same ligands. Important differences include: 1) Ca2+ activation at lower concentration of Ca2+ for the heart; 2) more dramatic stabilization by ryanodine of the open state for the skeletal muscle channel; and 3) different relative permeabilities (PCa/PK).     

SH oxidation stimulates calcium release channels (ryanodine receptors) from excitable cells

The effects of redox reagents on the activity of the intracellular calcium release channels (ryanodine receptors) of skeletal and cardiac muscle, or brain cortex neurons, was examined. In lipid bilayer experiments, oxidizing agents (2,2'-dithiodipyridine or thimerosal) modified the calcium dependence of all single channels studied. After controlled oxidation channels became active at sub microM calcium concentrations and were not inhibited by increasing the calcium concentration to 0.5 mM. Subsequent reduction reversed these effects. Channels purified from amphibian skeletal muscle exhibited the same behavior, indicating that the SH groups responsible for modifying the calcium dependence belong to the channel protein. Parallel experiments that measured calcium release through these channels in sarcoplasmic reticulum vesicles showed that following oxidation, the channels were no longer inhibited by sub mM concentrations of Mg2+. It is proposed that channel redox state controls the high affinity sites responsible for calcium activation as well as the low affinity sites involved in Mg2+ inhibition of channel activity. The possible physiological and pathological implications of these results are discussed.     

Effects of local anesthetics on single channel behavior of skeletal muscle calcium release channel

The effects of the two local anesthetics tetracaine and procaine and a quaternary amine derivative of lidocaine, QX314, on sarcoplasmic reticulum (SR) Ca2+ release have been examined by incorporating the purified rabbit skeletal muscle Ca2+ release channel complex into planar lipid bilayers. Recordings of potassium ion currents through single channels showed that Ca(2+)- and ATP-gated channel activity was reduced by the addition of the tertiary amines tetracaine and procaine to the cis (cytoplasmic side of SR membrane) or trans (SR lumenal) side of the bilayer. Channel open probability was lowered twofold at tetracaine and procaine concentrations of approximately 150 microM and 4 mM, respectively. Hill coefficients of 2.0 and greater indicated that the two drugs inhibited channel activity by binding to two or more cooperatively interacting sites. Unitary conductance of the K(+)- conducting channel was not changed by 1 mM tetracaine in the cis and trans chambers. In contrast, cis millimolar concentrations of the quaternary amine QX314 induced a fast blocking effect at positive holding potentials without an apparent change in channel open probability. A voltage-dependent block was observed at high concentrations (millimolar) of tetracaine, procaine, and QX314 in the presence of 2 microM ryanodine which induced the formation of a long open subconductance. Vesicle-45Ca2+ ion flux measurements also indicated an inhibition of the SR Ca2+ release channel by tetracaine and procaine. These results indicate that local anesthetics bind to two or more cooperatively interacting high-affinity regulatory sites of the Ca2+ release channel in or close to the SR membrane. Voltage-dependent blockade of the channel by QX314 in the absence of ryanodine, and by QX314, procaine and tetracaine in the presence of ryanodine, indicated one low-affinity site within the conduction pathway of the channel. Our results further suggest that tetracaine and procaine may primarily inhibit excitation-contraction coupling in skeletal muscle by binding to the high-affinity, regulatory sites of the SR Ca2+ release channel.     

Physical coupling between ryanodine receptor-calcium release channels

Ryanodine receptor-calcium release channels play a pivotal role in the calcium signaling that mediates muscle excitation-contraction coupling. Their membrane organization into regular patterns, functional gating studies and theoretical analysis of receptor clustering have led to models that invoke allosteric interaction between individual channel oligomers as a critical mechanism for control of calcium release. Here we show that in reconstituted "checkerboard-like" lattices that mimic in situ membrane channel arrays, each oligomer is interlocked physically with four adjacent oligomers via a specific domain-domain interaction. Direct physical coupling between ryanodine receptors provides structural evidence for an inter-oligomer allosteric mechanism in channel regulation. Therefore, in addition to established cytosolic and luminal regulation of function, these observations indicate that channel-channel communication through physical coupling provides a novel mode of regulation of intracellular calcium release channels.     

Single-channel properties of the recombinant skeletal muscle Ca2+ release channel (ryanodine receptor).

We report transient expression of a full-length cDNA encoding the Ca2+ release channel of rabbit skeletal muscle sarcoplasmic reticulum (ryanodine receptor) in HEK-293 cells. The single-channel properties of the 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate-solubilized and sucrose gradient-purified recombinant Ca2+ release channels were investigated by using single-channel recordings in planar lipid bilayers. The recombinant Ca2+ release channel exhibited a K+ conductance of 780 pS when symmetrical 250 mM KCl was used as the conducting ion and a Ca2+ conductance of 116 pS in 50 mM luminal Ca2+. Opening events of the recombinant channels were brief, with an open time constant of approximately 0.22 ms. The recombinant Ca2+ release channel was more permeable to Ca2+ than to K+, with a pCa2+/pK+ ratio of 6.8. The response of the recombinant Ca2+ release channel to various concentrations of Ca2+ was biphasic, with the channel being activated by micromolar Ca2+ and inhibited by millimolar Ca2+. The recombinant channels were activated by ATP and caffeine, inhibited by Mg2+ and ruthenium red, and modified by ryanodine. Most recombinant channels were asymmetrically blocked, conducting current unidirectionally from the luminal to the cytoplasmic side of the channel. These data demonstrate that the properties of recombinant Ca2+ release channel expressed in HEK-293 cells are very similar, if not identical, to those of the native channel.     

unc-68 encodes a ryanodine receptor involved in regulating C. elegans body-wall muscle contraction

Striated muscle contraction is elicited by the release of stored calcium ions through ryanodine receptor channels in the sarcoplasmic reticulum. ryr-1 is a C. elegans ryanodine receptor homologue that is expressed in body-wall muscle cells used for locomotion. Using genetic methods, we show that ryr-1 is the previously identified locus unc-68. First, transposon-induced deletions within ryr-1 are alleles of unc-68. Second, transformation of unc-68 mutants with ryr-1 genomic DNA results in rescue of the Unc phenotype. unc-68 mutants move poorly, exhibiting an incomplete flaccid paralysis, yet have normal muscle ultrastructure. The mutants are insensitive to the paralytic effects of ryanodine, and lack detectable ryanodine-binding activity. The Unc-68 phenotype suggests that ryanodine receptors are not essential for excitation- contraction coupling in nematodes, but act to amplify a (calcium) signal that is sufficient for contraction.     

Functional calcium release channel formed by the carboxyl-terminal portion of ryanodine receptor.

The ryanodine receptor (RyR) is one of the key proteins involved in excitation-contraction (E-C) coupling in skeletal muscle, where it functions as a Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. RyR consists of a single polypeptide of approximately 560 kDa normally arranged in a homotetrameric structure, which contains a carboxyl (C)-terminal transmembrane domain and a large amino (N)-terminal cytoplasmic domain. To test whether the carboxyl-terminal portion of RyR is sufficient to form a Ca2+ release channel, we expressed the full-length (RyR-wt) and C-terminal (RyR-C, approximately 130 kDa) RyR proteins in a Chinese hamster ovary (CHO) cell line, and measured their Ca2+ release channel functions in planar lipid bilayer membranes. The single-channel properties of RyR-wt were found to be similar to those of RyR from skeletal muscle SR. The RyR-C protein forms a cation-selective channel that shares some of the channel properties with RyR-wt, including activation by cytoplasmic Ca2+ and regulation by ryanodine. Unlike RyR-wt, which exhibits a linear current-voltage relationship and inactivates at millimolar Ca2+, the channels formed by RyR-C display significant inward rectification and fail to close at high cytoplasmic Ca2+. Our results show that the C-terminal portion of RyR contains structures sufficient to form a functional Ca2+ release channel, but the N-terminal portion of RyR also affects the ion-conduction and calcium-dependent regulation of the Ca2+ release channel.     

Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor

Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.     

Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle

Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca²⁺ channels in the surface/transverse tubular membrane and ryanodine receptor Ca²⁺ release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation–contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein–protein interaction remain unknown. The trigger for Ca²⁺ release through ryanodine receptors in cardiac muscle is a Ca²⁺ influx through the L-type Ca²⁺ channel. The Ca²⁺ entering through the surface membrane Ca²⁺ channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.     

Activation of the calcium release channel (ryanodine receptor) by heparin and other polyanions is calcium dependent.

Heparin has been used as a potent competitive inhibitor of inositol 1,4,5-trisphosphate (IP3)-binding to IP3 receptors and to block IP3-gated calcium channels in bilayer experiments. In contrast to the effect on the IP3-gated channel, heparin (0.1-1 micrograms/ml) opened the Ca release channel (ryanodine receptor). Other polyanions such as pentosan polysulfate and polyvinyl sulfate also activated the Ca release channel. The effect of polyanions on the Ca release channel was Ca dependent. Polyanion addition activated the Ca release channel when free Ca was > 80 nM, but was ineffective when free Ca was < 20 nM. The level of channel activation could be altered by manipulating the free Ca concentration. These results suggest that the polyanions act by increasing the local concentration of Ca near regulatory sites on the channel complex. As most cells have both types of intracellular channels, the opposite effects of the polyanions on the two channel types suggests that addition of polyanions to intact cells may produce multiple effects.     

Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.     

Regulation of ryanodine receptors by calsequestrin: effect of high luminal Ca2+ and phosphorylation

Calsequestrin, the major calcium sequestering protein in the sarcoplasmic reticulum of muscle, forms a quaternary complex with the ryanodine receptor calcium release channel and the intrinsic membrane proteins triadin and junctin. We have investigated the possibility that calsequestrin is a luminal calcium concentration sensor for the ryanodine receptor. We measured the luminal calcium concentration at which calsequestrin dissociates from the ryanodine receptor and the effect of calsequestrin on the response of the ryanodine receptor to changes in luminal calcium. We provide electrophysiological and biochemical evidence that: 1), luminal calcium concentration of >/=4 mM dissociates calsequestrin from junctional face membrane, whereas in the range of 1-3 mM calsequestrin remains attached; 2), the association with calsequestrin inhibits ryanodine receptor activity, but amplifies its response to changes in luminal calcium concentration; and 3), under physiological calcium conditions (1 mM), phosphorylation of calsequestrin does not alter its ability to inhibit native ryanodine receptor activity when the anchoring proteins triadin and junctin are present. These data suggest that the quaternary complex is intact in vivo, and provides further evidence that calsequestrin is involved in the sarcoplasmic reticulum calcium signaling pathway and has a role as a luminal calcium sensor for the ryanodine receptor.