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(Circulation Research. 1996;78:990-997.)
© 1996 American Heart Association, Inc.

Articles

Effects of Rapamycin on Ryanodine Receptor/Ca²⁺-Release Channels From Cardiac Muscle

Edward Kaftan, Andrew R. Marks, Barbara E. Ehrlich

From the Departments of Physiology and Medicine (E.K., B.E.E.), University of Connecticut, Farmington, and the Department of Medicine and Brookdale Center for Molecular Biology (A.R.M.), Mount Sinai School of Medicine, New York, NY.

Correspondence to Dr Edward Kaftan, Laboratory of Molecular Hermeneutics, Department of Physiology, University of Connecticut, 263 Farmington Ave, Farmington, CT 06030-3505. E-mail ekaftan@neuron.uchc.edu.

	Abstract

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Abstract Ryanodine receptors (RyRs) are intracellular channels that regulate the release of Ca²⁺ from the endoplasmic reticulum of many cell types. The RyRs are physically associated with FK506-binding proteins (FKBPs); immunophilins, with cis-trans peptidyl-prolyl isomerase activity. FKBP12 copurifies with RyR1 (skeletal isoform) and modulates its gating. A different form of FKBP with a slightly higher molecular weight copurifies with RyR2 (cardiac isoform). Previous studies have demonstrated that FKBP stabilizes gating of the skeletal Ca²⁺-release channel. In the present study, we measured the activity of cardiac RyRs incorporated into planar lipid bilayers to show that rapamycin, a drug that inhibits the prolyl isomerase activity of FKBP and dissociates FKBP from the RyR, increases the open probability and reduces the current amplitude of cardiac muscle Ca²⁺-release channels. These experiments show for the first time that submicromolar concentrations of rapamycin can alter channel function. Our results provide support for the hypotheses that FKBP functionally associates with the RyR and that the immunosuppressant drug, rapamycin, alters the function of both cardiac and skeletal muscle isoforms of the Ca²⁺-release channel. Our findings suggest that FKBP-dependent modulation of channel function may be generally applicable to all members of the intracellular Ca²⁺-release channel family and that FKBPs may play important regulatory roles in many cell processes, ranging from long-term depression in neurons to contractility in cardiomyocytes.

Key Words: intracellular channels • immunosuppressive therapy • FK506 • FK506-binding protein

	Introduction

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The RyR, the major intracellular Ca²⁺-release channel in striated muscle,^{1 2 3} plays an essential role in excitation-contraction coupling by regulating the delivery of Ca²⁺ from the SR for binding to the contractile apparatus.^{1 4 5 6} The RyR is also found in many other tissue types, ranging from brain^{7 8} to sea urchin egg.^{9 10} Although the stimuli used to activate the RyR in many of these tissues is unknown, Ca²⁺ release via the RyR has been implicated in the regulation of a number of physiological processes, including long-term depression, secretion, and cell proliferation.

RyRs have been isolated from both skeletal and cardiac muscles,^{11 12 13} and Ca²⁺-release studies and single-channel measurements have shown that the properties of the native Ca²⁺-release channels from these two tissues are similar, but not identical.^{14 15 16 17 18} Primary structures of RyRs from skeletal^{19 20 21} and cardiac muscles^{22 23} have been deduced from cDNA cloning, and the sequences are similar but sufficiently different to designate them as isoforms encoded by separate genes, RyR1 (skeletal type) and RyR2 (cardiac type). A number of recent reviews provide a comprehensive description of the regulation of Ca²⁺-release channel function.^{2 3 7 24}

An accessory protein, FKBP12, copurifies with the RyR during sucrose density gradient centrifugation and colocalizes to the terminal cisternae of the SR, and an anti-FKBP12 antibody can immunoprecipitate the RyR.²⁵ The immunosuppressant drugs FK506 and rapamycin bind to FKBP12 and inhibit a cis-trans peptidyl-prolyl isomerase.²⁶ However, the prolyl isomerase activity of FKBP12 is not required for the immunosuppressant properties of the two drugs.²⁷ The rapamycin-FKBP12 complex blocks the cell cycle transition from G₁ to S^{25 28 29 30 31 32 33} and inhibits p70 S6 and p34cdc2 kinases.^{33 34 35} The target for the rapamycin-FKBP12 complex has been identified as a mammalian homologue of the yeast TOR proteins.^{36 37}

The physiological function of FKBP in the absence of the immunosuppressant drugs was unclear; the association between the RyR1 and FKBP12,²⁵ the demonstration that FKBP12 modulates channel gating,³⁸ and the potency of FKBP12 as a chemoattractant³⁹ have suggested important cellular functions. FKBP12 has also been shown to associate with the InsP₃ receptor, and addition of FK506 to cerebellar microsomes increased the potency of InsP₃ to induce Ca²⁺ release.⁴⁰ In addition, an FKBP with a different electrophoretic mobility is associated with the RyR2, the cardiac isoform of the Ca²⁺-release channel.⁴¹ A novel FKBP, designated FKBP12.6, has been cloned and shown to be 85% homologous with FKBP12.⁴² FKBP12.6 binds FK506 and rapamycin and is a candidate for the form associated with the cardiac RyR. The finding that FKBPs are found in virtually all cell types³⁰ suggests that these proteins are involved in a range of cellular processes.

We have recently shown that FKBP12 modulates the gating of the Ca²⁺-release channel from skeletal muscle.³⁸ Because a novel FKBP associates with the cardiac RyR,⁴¹ in the present study we have examined the effects of rapamycin on the activity of the Ca²⁺-release channel from cardiac muscle. We found that rapamycin altered channel activity by increasing the open probability of the channel and by decreasing the single-channel conductance of the Ca²⁺-release channel from cardiac muscle.

	Materials and Methods

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SR vesicles from canine heart were prepared as described previously.⁴³ FKBP12 was purchased from Sigma Chemical Co, and rapamycin was from Signal Transduction, Inc or Calbiochem.

Immunoprecipitation and Western Blot Analysis
The SR vesicles prepared above were solubilized (1% CHAPS, 100 mmol/L NaCl, and 25 mmol/L HEPES) at 1 mg/mL for 1 hour at 4°C. Unsolubilized material was removed by centrifugation, and the supernatant was precleared with protein A–Sepharose. Anti-RyR antibody (Affinity BioReagents) was added at 4 µg/mL, incubated for 1 hour, and collected with protein A–Sepharose, and the pellets were washed and resuspended in solubilization buffer. Rapamycin was added to the immunocomplex at 10 µmol/L and incubated for 1 hour at room temperature. The complex was collected, washed with solubilization buffer, and subjected to SDS-PAGE (4% to 20%) followed by Western transfer. Blots were probed with anti-FKBP12 (rabbit) antibody²⁵ and detected with horseradish peroxidase–conjugated secondary antibody (Kirkegaard and Perry Laboratories).

Single-Channel Experiments
Single-channel experiments were conducted under voltage-clamp conditions with a pair of Ag/AgCl electrodes contacting the solutions via CsCl junctions. Vesicles were added to the cis chamber and induced to fuse with planar lipid bilayers composed of phosphatidylethanolamine/phosphatidylcholine (3:1, 30 mg/mL in decane, Avanti Polar Lipids). Solutions used for channel analysis were as follows: trans solution containing 250 mmol/L HEPES and 53 mmol/L Ba(OH)₂, pH 7.35, and cis solution containing 250 mmol/L HEPES-Tris, pH 7.35. Free Ca²⁺ concentrations (cis) were adjusted by additions of Ca²⁺-EGTA, in which the free Ca²⁺ was calculated as described previously.⁴⁴ Initial experiments used rapamycin dissolved in dimethyl sulfoxide, but control experiments indicated that dimethyl sulfoxide alone altered channel behavior. Therefore, subsequent experiments were performed with rapamycin dissolved in methanol, a solvent found to have minimal effects on bilayer and channel characteristics. Additions of rapamycin were made directly to the cis chamber after a test period in which the effects of methanol alone were observed. Channel currents were amplified using a bilayer clamp amplifier (BC-525A, Warner Instruments) and recorded on VHS tape (Dagan Corp). Data were filtered with an eight-pole Bessel filter (Frequency Devices) to 500 Hz, digitized at 2 kHz, transferred to a personal computer, and analyzed with pClamp version 6.0 (Axon Instruments). Channel recordings are representative samples taken from a minimum of 3 minutes of continuous recording (at a holding potential of 0 mV) at each experimental condition. Single-channel recordings were selected from at least three similar experiments. In all cases, mean±SEM values are reported unless otherwise indicated.

Ryanodine Binding
Ryanodine binding to cardiac microsomes was performed using both standard assay conditions and assay conditions similar to those used in single-channel experiments. Specifically, assay solutions contained 40 µg SR vesicles and 1 to 160 nmol/L [³H]ryanodine. For standard conditions, solutions contained 50 mmol/L Tris, pH 7.3, 350 mmol/L KCl, 50 µmol/L CaCl₂, and 1 mmol/L ATP; for conditions similar to those used in single-channel experiments, solutions contained 250 mmol/L HEPES, 125 mmol/L Tris, pH 7.35, 1 mmol/L EGTA, and 1.01 mmol/L CaCl₂. Rapamycin was added to test samples (final concentration, 20 µmol/L), and equivalent amounts of methanol were included in control samples (final concentration, 0.2%). Nonspecific binding samples included 10 µmol/L unlabeled ryanodine. After a 2-hour incubation at 22°C, microsomes were pelleted by centrifugation (16000g, 10 minutes), the supernatant was removed, and the pellets were solubilized in Packard Soluene. Paired two-tailed P values were calculated using Instat (GraphPad Software).

	Results

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FKBP Associates With the Cardiac RyR
To confirm that the association of FKBP with the cardiac RyR was maintained after vesicle preparation, immunoprecipitation of the RyR was conducted, followed by SDS-PAGE and Western blot analysis (Fig 1). The top half of the gel was stained with Coomassie blue to show that equal amounts of RyR were loaded on the gel (Fig 1A, lanes 2 and 4). The Western blot was probed with anti-FKBP antibody (Fig 1B). The antibody to FKBP we used is an affinity-purified polyclonal antibody specific for amino acids 3 to 16 of the rabbit FKBP12.²⁵ Because all amino acids except the first one in this peptide are identical between FKBP12 and the cardiac isoform of FKBP,⁴² this antibody should recognize the cardiac isoform as well. Lanes 1 and 5 contain 12 ng of purified human FKBP12, which serves as a positive control and relative molecular weight marker. Lane 2 shows that the FKBP that coprecipitates with the cardiac RyR migrates slower than FKBP12, as shown previously.⁴¹ When 10 µmol/L rapamycin was added to the immunocomplex before electrophoresis and blotting, only a very faint band could be detected at the location expected for the cardiac FKBP, suggesting that the FKBP had dissociated from the RyR complex (Fig 1B, lane 4). The removal of FKBP could be observed when the incubation was conducted at 22°C, whereas dissociation was not observed with incubations at 4°C (data not shown).

View larger version (98K): [in this window] [in a new window]   Figure 1. SDS-PAGE (A) and Western blot analysis (B) of immunoprecipitated RyR from cardiac muscle. RyR was immunoprecipitated from solubilized cardiac SR vesicles and subjected to electrophoresis on 4% to 20% SDS-PAGE and transferred to nitrocellulose. Lanes 1 and 5 contain 12 ng purified FKBP12; lane 2, immunoprecipitated RyR; lane 4, immunoprecipitated RyR treated with 10 µmol/L rapamycin; and lane 3, 3 µg anti-RyR antibody. A, Coomassie blue staining of the top half of the gel showing equal amounts of RyR loaded. B, Western blot analysis of the lower half of the gel probed with anti-FKBP12 antibody. C, Western blot analysis of native microsomes from cardiac muscle. Lane 2 contains 10 µg of native microsomes; lane 3, 10 µg of native microsomes pretreated with 10 µmol/L rapamycin; and lanes 1 and 4, 3 ng purified FKBP12.

Because native SR microsomes were used in the single-channel and ryanodine-binding experiments, the ability of rapamycin to interact with FKBP in intact microsomes was also tested. Rapamycin (10 µmol/L) was added to native microsomes and incubated for 1 hour at room temperature. Pelleted microsomes were subjected to SDS-PAGE and Western blot analysis as above. In Fig 1C, lanes 1 and 4 contain 3 ng of purified human FKBP12. Lane 2 shows that FKBP is normally associated with the microsomes and that the addition of rapamycin (Fig 1C, lane 3) can remove the FKBP associated with the native microsomes.

Effects of Rapamycin on RyR Function
To examine the effects of rapamycin on the activity of single Ca²⁺-release channels, cardiac SR vesicles were incorporated into planar lipid bilayers. Channel activity was observed in the presence of 5 µmol/L free Ca²⁺ (Fig 2, upper two traces). Addition of rapamycin (2 µmol/L; Fig 2, middle two traces) induced an increase in channel activity that was followed by a decrease in channel amplitude. After 5 minutes, no full-amplitude (4-pA) channel openings could be observed. Considering the small current fluctuations (1 to 2 pA) as channel openings, the open probability increased from 1.3±0.6% (n=5) in the control condition to 19.8±8.2% (n=5) in the presence of 2 µmol/L rapamycin. Ruthenium red (2 µmol/L; Fig 2, bottom two traces) completely inhibited activity, confirming that the observed small currents were Ca²⁺-release channel openings. The mean open time of the channel was increased by the addition of rapamycin (compare left panels of Fig 3) from 3.1±0.4 milliseconds (n=4) in the control condition to 6.7±0.8 milliseconds (n=4) in the presence of 2 µmol/L rapamycin. In the absence of rapamycin, the all-points amplitude histogram generated from the cardiac SR channel activity shows full 4-pA openings (Fig 3, top right panel). The effect of rapamycin on cardiac channel amplitude is clearly demonstrated by the absence of a peak at 4 pA (the full-conductance level) and an increased number of points between 0.5 and 2 pA, indicating that the channel spends a large percentage of the time in subconductance states (Fig 3, bottom right panel). This result was obtained in five of seven experiments with 2 µmol/L rapamycin; the effect of rapamycin in the remaining two experiments was an increase in open probability (fivefold) with no change in channel amplitude.

View larger version (22K): [in this window] [in a new window]   Figure 2. Effects of rapamycin on the cardiac Ca2+-release channel. Channel activity was observed at 5 µmol/L free Ca2+ (upper two traces). Addition of rapamycin (2 µmol/L, middle two traces) caused a decrease in channel amplitude. Considering the small 1-pA currents as channel openings, 2 µmol/L rapamycin increased the open probability (19.8±8.2%, n=5) over the control condition (1.3±0.6%, n=5). Methanol was used as the vehicle for rapamycin addition at a final concentration of 0.2%. Ruthenium red (Ruth. Red, 2 µmol/L) completely inhibited activity (lower two traces), confirming that the observed small currents were Ca2+-release channel openings.

View larger version (23K): [in this window] [in a new window]   Figure 3. Properties of the cardiac muscle Ca2+-release channel. Open-time histograms are shown in the absence (top left panel) and presence (bottom left panel) of 2 µmol/L rapamycin. The curves were fit to 767 events in the control condition and 2653 events in the presence of rapamycin. The fitted curves shown were normalized to the maximum bin for comparison of the curves. The right panels are amplitude histograms of the cardiac muscle Ca2+-release channel. All-points amplitude histograms were generated from 5.5x104 points (top right panel) and 1.7x104 points (bottom right panel). In the absence of rapamycin (top right panel), a prominent peak is present at 0 pA (the closed state) and a less prominent peak is present at 4.0 pA (corresponding to the open state). Addition of rapamycin (2 µmol/L, bottom right panel) caused a decrease in channel amplitude.

The effect of rapamycin addition on single-channel behavior is not immediate and usually occurs in two steps—activation and then alteration of conductance. Channel activation occurs 2 to 10 minutes after addition of rapamycin (Fig 4) and is maintained even after removal of rapamycin from the bath. With prolonged exposure to rapamycin (>10 minutes), the channels open with a smaller current amplitude. This sequence of events may explain the lack of effect on channel amplitude in the two experiments described above, in which only channel activation was observed. In these two experiments, channel activity in the presence of rapamycin was observed for <10 minutes, due to premature rupture of the bilayer. Fig 4 shows a representative experiment conducted at 1 µmol/L free cytoplasmic Ca²⁺ in which channel activation occurred 3 minutes after the addition of 0.2 µmol/L rapamycin. Initially, the activated channels displayed full conductances. After 10 minutes, only smaller amplitude channels were observed. Subsequent addition of 1 µmol/L ruthenium red completely inhibited these smaller conductances.

View larger version (23K): [in this window] [in a new window]   Figure 4. Time course of rapamycin action. Cardiac RyRs were incorporated into the bilayer, and the cytoplasmic free Ca2+ was adjusted to 1 µmol/L. Open probabilities were calculated at 30-second intervals and plotted vs time. Rapamycin addition (0.2 µmol/L) is indicated by the arrow, and the letters mark the time at which the representative channel tracings were obtained. Note that the channel activity increases first; subsequently, the channel current amplitude decreases. Results shown are for one of three similar experiments.

The effects of rapamycin on channel function were observed over a wide concentration range. Previous studies have used 5 to 12 µmol/L rapamycin or FK506 to observe alterations in channel function.^{41 45 46} Both channel activation and openings to subconductance levels were observed in experiments with 5 to 10 µmol/L (data not shown), 2 µmol/L (Fig 2), and 0.2 µmol/L (Fig 4) rapamycin. The time between activation of the full-conductance channel and subconductance openings decreased as the rapamycin concentration increased. At concentrations of rapamycin above 2 µmol/L, the initial rapamycin-induced activation became sufficiently brief so that estimations of the open probability of the full conductance channel could not be calculated. In experiments with either 2 or 0.2 µmol/L rapamycin, the transition from full-conductance to subconductance openings occurred within 2 or 4 minutes, respectively.

The ability of rapamycin to alter channel activity was tested at several cytoplasmic Ca²⁺ concentrations. When the cytoplasmic Ca²⁺ was below 0.5 µmol/L free Ca²⁺, channel openings were rare, and increases in channel openings attributable to the addition of rapamycin were not observed. When cytoplasmic Ca²⁺ was above 50 µmol/L free Ca²⁺, the channel open probability was sufficiently elevated to make it difficult to quantify the effects of rapamycin addition on channel activation. Addition of rapamycin to the Ca²⁺-activated channel still induced subconductance openings (Fig 5) without further increase in the open probability (the open probability was 32±2.7% [n=3] in the control condition and 34±4.4% [n=3] in the presence of 2 µmol/L rapamycin). At intermediate concentrations of cytoplasmic Ca²⁺, rapamycin addition increased channel activity, but a significant shift in the Ca²⁺ dependence of channel activation was not observed.

View larger version (37K): [in this window] [in a new window]   Figure 5. Effects of rapamycin on the Ca2+-activated cardiac Ca2+-release channel. Channel activity was observed at 50 µmol/L free Ca2+ (upper two traces). Addition of rapamycin (2 µmol/L, middle two traces) caused a decrease in channel amplitude without further activation of the channel. Ruthenium red (Ruth. Red, 2 µmol/L) completely inhibited activity (lower two traces).

Effect of Rapamycin on Ryanodine Binding
The experiments using channels incorporated into bilayers demonstrate that the addition of rapamycin alters single-channel behavior of the cardiac RyR. In these studies, each experiment represents the behavior of a single-channel complex. To correlate the effects of rapamycin on single-channel properties with their effects on populations of channels, the effects of rapamycin on [³H]ryanodine binding were determined using cardiac microsomes under bilayer conditions (250 mmol/L HEPES, 125 mmol/L Tris, pH 7.35, and 10 µmol/L free Ca²⁺). Standard conditions generally used for ryanodine-binding experiments (50 mmol/L Tris, pH 7.3, 350 mmol/L KCl, 1 mmol/L ATP, and 50 µmol/L free Ca²⁺) are designed to maximize ryanodine binding. Under these standard conditions, the B_max for ryanodine binding was 5.0 pmol per milligram protein, and the K_d was 10.5 nmol/L (comparable to previously published values^{47 48} ). Addition of rapamycin to microsomes incubated under these conditions had a small effect (Fig 6, top). Rapamycin (20 µmol/L) decreased ryanodine binding (ryanodine concentration, 20 nmol/L; 12.1±3.0%; n=3), but this difference did not reach statistical significance (P<.06). Using solutions similar to bilayer conditions, the amount of ryanodine bound to the RyR was decreased relative to standard conditions (B_max, 2.9±0.9 pmol/mg; K_d, 25±7 nmol/L; n=3). Under bilayer conditions, the effect of rapamycin on [³H]ryanodine binding to cardiac microsomes was enhanced (compare top and bottom panels of Fig 6). In all experiments, the B_max was reduced (1.1±0.8 pmol/mg, n=3); in two preparations, binding was too low to allow for a reasonable determination of the K_d. Under bilayer conditions, rapamycin (20 µmol/L) decreased ryanodine binding by 37±8% (ryanodine concentration, 20 nmol/L; n=6; P<.0008). Similar effects of rapamycin on ryanodine binding were obtained when we used a lower free Ca²⁺ concentration (500 nmol/L); however, the maximum amount of binding in each sample was less. With the lower Ca²⁺ conditions, 2 µmol/L rapamycin decreased binding to cardiac microsomes by 45%. Alterations in ryanodine binding also have been observed in a series of experiments in which the ryanodine binding site was altered by treatment with trypsin.⁴⁹

View larger version (34K): [in this window] [in a new window]   Figure 6. Effects of rapamycin on [3H]ryanodine binding to cardiac SR microsomes. Ryanodine binding curves were generated by incubating microsomes in the presence of ryanodine (0 to 160 nmol/L). Each point is the mean of two determinations. Nonspecific binding was unaffected by the addition of rapamycin. Assay conditions were similar to standard conditions (top panel) or to those used in the planar lipid bilayer experiments (bottom panel). Standard conditions were as follows: 50 mmol/L Tris, pH 7.3, 350 mmol/L KCl, 1 mmol/L ATP, and 50 µmol/L free Ca2+; bilayer conditions were as follows: 250 mmol/L HEPES, 125 mmol/L Tris, pH 7.35, and 10 µmol/L free Ca2+. Rapamycin (20 µmol/L, ) decreased [3H]ryanodine binding to cardiac microsomes over control conditions ().

	Discussion

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In the present study, we have evaluated the effects of rapamycin on the Ca²⁺-release channel from cardiac muscle. Rapamycin binds to FKBP, a member of the immunophilin class of binding proteins.³⁰ The cardiac RyR is associated with an FKBP that can be distinguished from the FKBP that colocalizes with the skeletal RyR by its slower electrophoretic mobility. Despite the difference in FKBP from the two tissues, treatment of the cardiac RyR with rapamycin induced a loss of FKBP from the channel complex, as found with FK506 treatment of the skeletal RyR.⁵⁰ Similarly, addition of rapamycin to the cardiac channels induced a profound change in channel amplitude, as well as increasing the channel open probability fourfold, as found with FK506 treatment of the skeletal RyR.⁴⁶ In the present series of experiments, these effects were found to occur over a range of rapamycin concentrations from the submicromolar to the maximum soluble concentration of rapamycin (20 µmol/L). Rapamycin treatment also decreased ryanodine binding to the cardiac RyR. From these results, it is clear that this drug alters the function of the native Ca²⁺-release channel and could exert these effects at levels of the drug found in vivo.

To show that rapamycin acts on FKBP, rather than on the RyR itself, the effect of rapamycin was tested on the purified expressed RyR, a preparation devoid of FKBP12.³⁸ In the absence of drug, the expressed receptor displayed channel properties characteristic of the Ca²⁺-release channel.³⁸ Addition of 2 µmol/L rapamycin to the expressed channel that lacked FKBP had no effect on channel open probability or channel conductance.³⁸ These results support the observation that FKBP is an integral component of the Ca²⁺-release channel and that channel function depends on interactions between the RyR and FKBP.

Two distinct types of protein-protein associations have now been described for FKBP12. In one type, FKBP12 has been shown to associate with other proteins in a ligand-dependent manner. Thus, the complex of FK506 bound to FKBP12 interacts with and inhibits calcineurin,⁵¹ and the complex of rapamycin bound to FKBP12 associates with TOR in yeast⁵² and TOR homologues in mammals.^{36 37} The function of FKBP12 in the absence of ligand is unclear, whereas FKBP12 with ligand bound leads to inhibition of T-cell activation; these effects are examples of gain of function phenomena. In a second type of association, FKBP12 associates directly with intracellular Ca²⁺-release channels. A similar association between FKBP12 and the InsP₃ receptor has been proposed,⁴⁰ and an association between FKBP and the transforming growth factor-ß receptor (type 1) has been demonstrated using the yeast two-hybrid system.⁵³ Thus, it is highly probable that associations between FKBP and other transmembrane signaling molecules will be discovered.

The temporal separation of the rapamycin-induced alteration in channel activity and conductance may be because the two processes are mechanistically distinct. Channel activation may be the consequence of drug binding to FKBP, whereas changes in current amplitude may occur when FKBP and the RyR dissociate. Experiments with the bastadins, a class of compounds that are structurally related to the immunosuppressive drugs and that bind to FKBP,⁵⁴ support the hypothesis that multiple mechanisms are used. These authors showed in an elegant series of experiments that it is possible to alter channel activity without dissociating FKBP12 from the RyR.⁵⁴ Bastadin in the absence of FK506 increased single-channel activity and increased ryanodine binding to the channel complex without dissociating FKBP12 from the RyR and without alteration in the channel conductance.⁵⁴ The ability of bastadin to increase channel activity without alterations in channel conductance supports the possibility of independent mechanisms for the two processes.

Another model that could explain the multistep process in rapamycin-induced alteration in channel function relies on the stoichiometry of the FKBP-RyR interactions, in which four FKBPs bind to one RyR channel complex.^{25 41} If the dissociation of FKBP from the RyR occurs in a stepwise manner, then channel activation could be the consequence of the loss of the initial FKBP(s) from the channel complex, and the loss of additional FKBP molecules could lead to a reduction in channel conductance. In other words, the loss of the first FKBP affects the gate, and further losses of FKBP restructures the channel so that permeation is altered.

Although it is tempting to speculate that the proline isomerase activity of FKBP12 plays some role in changing the conformation of transmembrane signaling proteins, which, in turn, influences the function of the channels, there is no direct evidence to date for this hypothesis. In one report, however, it was suggested that the isomerase activity was unrelated to channel modulation.⁵⁵ If there truly are multiple processes that are altered by the immunosuppressive drugs, each with a distinct effect on channel function, then additional specific assays may be needed to determine the role of proline isomerase activity in channel modulation. Thus, at the present time, the molecular mechanism(s) underlying FKBP modulation of the Ca²⁺ release channel remains uncertain.

The dissociation of FKBP and RyR could lead to alterations in the interactions among the four subunits that constitute the ryanodine binding site. If the sole effect of rapamycin addition was to increase the open probability of the channel, ryanodine binding to the RyR (B_max) should increase, because ryanodine binds to the open channel.⁴⁸ In contrast, rapamycin decreased ryanodine binding to the RyR. This reduction may be related to the decrease in channel currents and increase in subconductance states of the channel. For example, ryanodine may have reduced access to the high-affinity ryanodine binding site because of the conformational changes in the channel structure after the loss of FKBP activity. A similar correlation between a decrease in channel currents and a decrease in ryanodine binding was observed after treatment of the RyR with trypsin.⁴⁹

The ability of the immunosuppressive agents to disrupt the interaction between FKBP and the RyR could lead to alterations in Ca²⁺ homeostasis in a variety of cell types when these drugs are administered to patients. These changes in intracellular Ca²⁺ could then induce side effects related to treatment with these immunosuppressive agents. Although there have been isolated reports of cardiac toxicity in patients and animals treated with FK506 and/or rapamycin, there has been no consistent toxicity related either to skeletal or cardiac muscles.⁵⁶ This is not surprising, however, because these drugs have been administered in doses that achieve intracellular levels sufficient to bind only a fraction of the cellular content of FKBP in all types of muscle,⁵⁶ which contains abundant levels of FKBP12.²⁵ Therefore, we would predict that only a small fraction of the cellular FKBP would be bound by these drugs in the muscle cells of treated patients and that the activity of FKBP associated with RyR would not necessarily be altered at therapeutic levels of drugs. Unlike the immunosuppressant effects of these drugs, which are gain-of-function phenomena, interference with FKBP12 stabilization of the Ca²⁺-release channels requires stoichiometric concentrations of FK506 or rapamycin.⁵⁰

When added to cardiac SR channels comprising RyR and FKBP12, rapamycin induced profound changes in channel gating manifested by activation of the channel (increased open probability) and the appearance of subconductance states. These effects of rapamycin on the cardiac muscle Ca²⁺-release channel were similar to those found with the skeletal muscle Ca²⁺-release channel (RyR1).^{46 50} Rapamycin added to the cloned expressed skeletal RyR, which does not include FKBP12,³⁸ had no effect on channel function. This finding indicates that the effects of these immunosuppressant drugs are dependent on the presence of FKBP12, not direct effects on the RyR itself. These experiments demonstrate that FKBP plays a functional role in the activity of both cardiac and skeletal muscle SR Ca²⁺-release channels.

	Selected Abbreviations and Acronyms

 

FKBP = FK506-binding protein InsP3 = inositol 1,4,5-tris-phosphate RyR = ryanodine receptor SR = sarcoplasmic reticulum TOR = target of rapamycin

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-33026 (Dr Ehrlich) and NS-29814 (Dr Marks), a Bristol Myers Squibb Established Investigatorship from the American Heart Association (Dr Marks), and a grant-in-aid from the Patrick and Catherine Weldon Donaghue Medical Research Foundation (Dr Ehrlich). We thank Dr Karol Ondrias for helpful discussions about the expressed RyR, Dr T. Jayaraman for providing the FKBP antibody, and Drs L. Flash Cohen, Beth Daro, Arnold Katz, Kerry Quinn, and Jim Watras for critical reading of the manuscript.

Received May 24, 1995; accepted February 16, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Fleischer S, Inui M. Biochemistry and biophysics of excitation-contraction coupling. Annu Rev Biophys Biophys Chem. 1989;18:333-364. [Medline] [Order article via Infotrieve]

2. Meissner G. Ryanodine receptor/Ca²⁺ release channels and their regulation by endogenous effectors. Annu Rev Physiol. 1994;56:485-508. [Medline] [Order article via Infotrieve]

3. Coronado R, Morrissette J, Sukhareva M, Vaughan DM. Structure and function of ryanodine receptors. Am J Physiol. 1994;266:C1485-C1504. [Abstract/Free Full Text]

4. Catterall WA. Excitation-contraction coupling in vertebrate skeletal muscle: a tale of two calcium channels. Cell. 1991;64:871-874. [Medline] [Order article via Infotrieve]

5. Rios E, Ma J, Gonzalez A. The mechanical hypothesis of excitation-contraction (EC) coupling in skeletal muscle. J Muscle Res Cell Motil. 1991;12:127-135. [Medline] [Order article via Infotrieve]

6. Franzini-Armstrong C, Jorgensen AO. Structure and development of E-C coupling units in skeletal muscle. Annu Rev Physiol. 1994;56:509-534. [Medline] [Order article via Infotrieve]

7. McPherson PS, Campbell KP. The ryanodine receptor/Ca²⁺ release channel. J Biol Chem. 1993;268:13765-13768. [Free Full Text]

8. Ellisman MH, Deerinck TJ, Ouyang Y, Beck CF, Tanksley SJ, Walton PD, Airey JA, Sutko JL. Identification and localization of ryanodine binding proteins in the avian central nervous system. Neuron. 1990;5:135-146. [Medline] [Order article via Infotrieve]

9. Galione A. Ca-induced Ca release and its modulation by cyclic ADP-ribose. Trends Pharmacol Sci. 1992;13:304-306. [Medline] [Order article via Infotrieve]

10. Lee HC, Aarhus R, Walseth TF. Calcium mobilization by dual receptors during fertilization of sea urchin eggs. Science. 1993;261:352-355. [Abstract/Free Full Text]

11. Inui M, Saito A, Fleischer S. Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle. J Biol Chem. 1987;262:1740-1747. [Abstract/Free Full Text]

12. Lai FA, Erickson HP, Rousseau E, Liu Q-Y, Meissner G. Purification and reconstitution of the calcium release channel from skeletal muscle. Nature. 1988;331:315-319. [Medline] [Order article via Infotrieve]

13. Smith JS, Imagawa T, Ma J, Fill M, Campbell KP, Coronado R. Purified ryanodine receptor from rabbit skeletal muscle is the calcium release-channel of sarcoplasmic reticulum. J Gen Physiol. 1988;92:1-26. [Abstract/Free Full Text]

14. Smith J, Coronado R, Meissner G. Single channel measurements of the calcium release channel from skeletal muscle sarcoplasmic reticulum: activation by Ca²⁺ and ATP and modulation by Mg²⁺. J Gen Physiol. 1986;88:573-588. [Abstract/Free Full Text]

15. Imagawa T, Smith J, Coronado R, Campbell K. Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca-permeable pore of the calcium release channel. J Biol Chem. 1987;262:16636-16643. [Abstract/Free Full Text]

16. Rousseau E, Smith JS, Henderson JS, Meissner G. Single channel and ⁴⁵Ca²⁺ flux measurements of the cardiac sarcoplasmic reticulum calcium channel. Biophys J. 1986;50:1009-1014. [Medline] [Order article via Infotrieve]

17. Ehrlich BE, Watras J. Inositol 1,4,5-trisphosphate activates a channel from smooth muscle sarcoplasmic reticulum. Nature. 1988;336:583-586. [Medline] [Order article via Infotrieve]

18. Lindsay ARG, Williams AJ. Functional characterisation of the ryanodine receptor purified from sheep cardiac muscle sarcoplasmic reticulum. Biochim Biophys Acta. 1991;1064:89-102. [Medline] [Order article via Infotrieve]

19. Marks AR, Tempst P, Hwang KS, Taubman MB, Inui M, Chadwick C, Fleischer S, Nadal-Ginard B. Molecular cloning and characterization of the ryanodine receptor/junctional channel complex cDNA from skeletal muscle sarcoplasmic reticulum. Proc Natl Acad Sci U S A. 1989;86:8683-8687. [Abstract/Free Full Text]

20. Takeshima H, Nishimura S, Matsumoto T, Ishida H, Kangawa K, Minamino N, Matsuo H, Ueda M, Hanaoka M, Hirose T, Numa S. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature. 1989;339:439-445. [Medline] [Order article via Infotrieve]

21. Zorzato F, Fujii J, Otsu K, Phillips M, Green NM, Lai FA, Meissner G, MacLennan DH. Molecular cloning of cDNA encoding human and rabbit forms of the Ca release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J Biol Chem. 1990;265:2244-2256. [Abstract/Free Full Text]

22. Nakai J, Imagawa T, Hakamata Y, Shigekawa M, Takeshima H, Numa H. Primary structure and functional expression from cDNA of the cardiac ryanodine receptor/calcium release channel. FEBS Lett. 1990;271:169-177. [Medline] [Order article via Infotrieve]

23. Otsu K, Willard HF, Khanna VK, Zorzato F, Green NM, MacLennan DH. Molecular cloning of cDNA encoding the Ca²⁺ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. J Biol Chem. 1990;265:13472-13483. [Abstract/Free Full Text]

24. Ehrlich BE, Kaftan E, Bezprozvannaya S, Bezprozvanny I. The pharmacology of intracellular Ca²⁺-release channels. Trends Pharmacol Sci. 1994;15:145-149. [Medline] [Order article via Infotrieve]

25. Jayaraman T, Brillantes AM, Timerman AP, Fleischer S, Erdjumentbromage H, Tempst P, Marks AR. FK506 binding protein associated with the calcium release channel (ryanodine receptor). J Biol Chem. 1992;267:9474-9477. [Abstract/Free Full Text]

26. Harding MW, Galat A, Uehling DE, Schreiber SL. A receptor for the immunosuppressant FK506 is a cis-trans peptidyl-prolyl isomerase. Nature. 1989;341:758-760. [Medline] [Order article via Infotrieve]

27. Bierer B, Somers PK, Wandless TJ, Burakoff SJ, Schreiber SL. Probing immunosuppressant action with a nonnatural immunophilin ligand. Science. 1990;250:556-559. [Abstract/Free Full Text]

28. Bierer BE, Mattilia PS, Standaert R, Herzenberg L, Burakoff S, Crabtree G, Schreiber S. Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin. Proc Natl Acad Sci U S A. 1990;87:9231-9235. [Abstract/Free Full Text]

29. Dumont FJ, Staruch MJ, Kooprak SL, Melino MR, Sigal NH. Distinct mechanisms of suppression of murine T-cell activation by the related macrolides FK-506 and rapamycin. J Immunol. 1990;144:251-258. [Abstract]

30. Schreiber S. Chemistry and biology of the immunophilins and their immunosuppressive ligands. Science. 1991;251:283-287. [Abstract/Free Full Text]

31. Chung J, Kuo CJ, Crabtree GR, Blenis J. Rapamycin-FKBP specifically blocks growth-dependent activation of and signalling the 70 kDa S6 protein kinases. Cell. 1992;69:1227-1236. [Medline] [Order article via Infotrieve]

32. Kuo CJ, Chung J, Fiorentino DF, Flanagan WM, Blenis J, Crabtree GR. Rapamycin selectively inhibits interleukin-2 activation and p70 S6 kinase 1. Nature. 1992;358:70-73. [Medline] [Order article via Infotrieve]

33. Price DJ, Grove JR, Calvo V, Avruch J, Bierer BE. Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science. 1992;257:973-977. [Abstract/Free Full Text]

34. Jayaraman T, Marks AR. Rapamycin-FKBP blocks proliferation and inhibits cdc2 kinase activity in a myogenic cell line. J Biol Chem. 1993;268:25385-25388. [Abstract/Free Full Text]

35. Morice WG, Brunn GJ, Wiederrecht G, Siekierka JJ, Abraham RT. Rapamycin-induced inhibition of p34cdc2 kinase activation is associated with G1/S-phase growth arrest in T lymphocytes. J Biol Chem. 1993;268:3734-3738. [Abstract/Free Full Text]

36. Brown E, Albers M, Shin T, Ichikawa K, Keith C, Lane W, Schreiber S. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature. 1994;369:756-758. [Medline] [Order article via Infotrieve]

37. Sabatini D, Erdjument-Bromage H, Lui M, Tempst P, Snyder S. RAFT1: a mammalian protein that binds FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell. 1994;78:35-43. [Medline] [Order article via Infotrieve]

38. Brillantes A-MB, Ondrias K, Scott A, Kobrinsky E, Ondriasova E, Moschella MC, Jayaraman T, Landers M, Ehrlich BE, Marks AR. Stabilization of calcium release channel (ryanodine receptor) function by FK-506 binding protein. Cell. 1994;77:513-523. [Medline] [Order article via Infotrieve]

39. Leiva MC, Lyttle CR. Leukocyte chemotactic activity of FKBP and inhibition by FK506. Biochem Biophys Res Commun. 1992;186:1178-1183. [Medline] [Order article via Infotrieve]

40. Cameron AM, Steiner JP, Sabatini DM, Kaplin AI, Walensky LD, Snyder SH. Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux. Proc Natl Acad Sci U S A. 1995;92:1784-1788. [Abstract/Free Full Text]

41. Timerman AP, Jayaraman T, Wiederrecht G, Onoue H, Marks AR, Fleischer S. The ryanodine receptor from canine heart sarcoplasmic reticulum is associated with a novel FK-506 binding protein. Biochem Biophys Res Commun. 1994;198:701-706. [Medline] [Order article via Infotrieve]

42. Sewell TJ, Lam E, Martin MM, Leszyk J, Weidner J, Calaycay J, Griffin P, Williams H, Hung S, Cryan J, Sigal NH, Wiederrecht GJ. Inhibition of calcineurin by a novel FK-506-binding protein. J Biol Chem. 1994;269:21094-21102. [Abstract/Free Full Text]

43. Ondrias K, Borgatta L, Kim DH, Ehrlich BE. Biphasic effects of doxorubicin on the calcium release channel from sarcoplasmic reticulum of cardiac muscle. Circ Res. 1990;67:1167-1174. [Abstract/Free Full Text]

44. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 1988;157:378-417. [Medline] [Order article via Infotrieve]

45. Mayrleitner M, Timerman AP, Wiederrecht G, Fleischer S. The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein: effect of FKBP-12 on single channel activity of the skeletal muscle ryanodine receptor. Cell Calcium. 1994;15:99-108. [Medline] [Order article via Infotrieve]

46. Ahern GP, Junankar PR, Dulhunty AF. Single channel activity of the ryanodine receptor calcium release channel is modulated by FK-506. FEBS Lett. 1994;352:369-374. [Medline] [Order article via Infotrieve]

47. Pessah IN, Waterhouse AL, Casida JE. The calcium-ryanodine receptor complex of skeletal and cardiac muscle. Biochem Biophys Res Commun. 1985;128:449-456. [Medline] [Order article via Infotrieve]

48. Anderson K, Lai FA, Liu Q-Y, Rousseau E, Erickson HP, Meissner G. Structural and functional characterization of the purified cardiac ryanodine receptor-Ca⁺² release channel complex. J Biol Chem. 1989;264:1329-1335. [Abstract/Free Full Text]

49. Hawkes MJ, Nelson TE, Hamilton SL. [³H]Ryanodine as a probe of changes in the functional state of the Ca²⁺-release channel in malignant hyperthermia. J Biol Chem. 1992;267:6702-6709. [Abstract/Free Full Text]

50. Timerman AP, Ogunbunmi E, Freund E, Wiederrecht G, Marks A, Fleischer S. The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein. J Biol Chem. 1993;268:22992-22999. [Abstract/Free Full Text]

51. Liu J, Farmer J, Lane W, Freidman J, Weissman I, Schreiber S. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991;66:807-815. [Medline] [Order article via Infotrieve]

52. Kunz J, Henriquez R, Schneider U, Deuter-Reinhard M, Movva NR, Hall MN. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell. 1993;73:585-596. [Medline] [Order article via Infotrieve]

53. Wang T, Donahoe PK, Zervos AS. Specific interaction of type 1 receptors of the TGF-beta family with the immunophilin FKBP-12. Science. 1994;265:674-676. [Abstract/Free Full Text]

54. Mack MM, Molinski TF, Buck ED, Pessah IN. Novel modulators of skeletal muscle FKBP12/calcium channel complex from Ianthella basta. J Biol Chem. 1994;269:23236-23249. [Abstract/Free Full Text]

55. Timerman AP, Wiederrecht G, Marcy A, Fleischer S. Characterization of an exchange reaction between soluble FKBP-12 and the FKBP-ryanodine receptor complex. J Biol Chem. 1995;270:2451-2459. [Abstract/Free Full Text]

56. Morris R. Rapamycins: antifungal, antiproliferative, and immunosuppressive macrolides. Transplant Rev. 1992;6:39-87.

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				S. Kanoh, M. Kondo, J. Tamaoki, H. Shirakawa, K. Aoshiba, S. Miyazaki, H. Kobayashi, N. Nagata, and A. Nagai Effect of FK506 on ATP-induced intracellular calcium oscillations in cow tracheal epithelium Am J Physiol Lung Cell Mol Physiol, June 1, 1999; 276(6): L891 - L899. [Abstract] [Full Text] [PDF]

				D. M Bers and E. Perez-Reyes Ca channels in cardiac myocytes: structure and function in Ca influx and intracellular Ca release Cardiovasc Res, May 1, 1999; 42(2): 339 - 360. [Abstract] [Full Text] [PDF]

				M. D. Stern, L.-S. Song, H. Cheng, J. S.K. Sham, H. T. Yang, K. R. Boheler, and E. Rios Local Control Models of Cardiac Excitation-Contraction Coupling: A Possible Role for Allosteric Interactions between Ryanodine Receptors J. Gen. Physiol., March 1, 1999; 113(3): 469 - 489. [Abstract] [Full Text] [PDF]

				K. S. Park, T. K. Kim, and D. H. Kim Cyclosporin A treatment alters characteristics of Ca2+-release channel in cardiac sarcoplasmic reticulum Am J Physiol Heart Circ Physiol, March 1, 1999; 276(3): H865 - H872. [Abstract] [Full Text] [PDF]

				W. H. duBell, S. T. Gaa, W. J. Lederer, and T. B. Rogers Independent inhibition of calcineurin and K+ currents by the immunosuppressant FK-506 in rat ventricle Am J Physiol Heart Circ Physiol, December 1, 1998; 275(6): H2041 - H2052. [Abstract] [Full Text] [PDF]

				M. B. Meyers, T. S. Puri, A. J. Chien, T. Gao, P.-H. Hsu, M. M. Hosey, and G. I. Fishman Sorcin Associates with the Pore-forming Subunit of Voltage-dependent L-type Ca2+ Channels J. Biol. Chem., July 24, 1998; 273(30): 18930 - 18935. [Abstract] [Full Text] [PDF]

				D.A Eisner, A.W Trafford, M.E Dnaz, C.L Overend, and S.C O'Neill The control of Ca release from the cardiac sarcoplasmic reticulum: regulation versus autoregulation Cardiovasc Res, June 1, 1998; 38(3): 589 - 604. [Abstract] [Full Text] [PDF]

				K. E. Quinn, L. Castellani, K. Ondrias, and B. E. Ehrlich Characterization of the ryanodine receptor/channel of invertebrate muscle Am J Physiol Regulatory Integrative Comp Physiol, February 1, 1998; 274(2): R494 - R502. [Abstract] [Full Text] [PDF]

				R. Zucchi and S. Ronca-Testoni The Sarcoplasmic Reticulum Ca2+ Channel/Ryanodine Receptor: Modulation by Endogenous Effectors, Drugs and Disease States Pharmacol. Rev., March 1, 1997; 49(1): 1 - 52. [Abstract] [Full Text] [PDF]

				E. McCall, L. Li, H. Satoh, T. R. Shannon, L. A. Blatter, and D. M. Bers Effects of FK-506 on Contraction and Ca2+ Transients in Rat Cardiac Myocytes Circ. Res., December 1, 1996; 79(6): 1110 - 1121. [Abstract] [Full Text]

				M. Gaburjakova, J. Gaburjakova, S. Reiken, F. Huang, S. O. Marx, N. Rosemblit, and A. R. Marks FKBP12 Binding Modulates Ryanodine Receptor Channel Gating J. Biol. Chem., May 11, 2001; 276(20): 16931 - 16935. [Abstract] [Full Text] [PDF]

				Y. Li, E. G. Kranias, G. A. Mignery, and D. M. Bers Protein Kinase A Phosphorylation of the Ryanodine Receptor Does Not Affect Calcium Sparks in Mouse Ventricular Myocytes Circ. Res., February 22, 2002; 90(3): 309 - 316. [Abstract] [Full Text] [PDF]

				S. O. Marx, J. Gaburjakova, M. Gaburjakova, C. Henrikson, K. Ondrias, and A. R. Marks Coupled Gating Between Cardiac Calcium Release Channels (Ryanodine Receptors) Circ. Res., June 8, 2001; 88(11): 1151 - 1158. [Abstract] [Full Text] [PDF]

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