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Regulation of Ryanodine Receptors by FK506 Binding Proteins
Regulation of Ryanodine Receptors by FK506 Binding Proteins Mihail G. Chelu, Cristina I. Danila, Charles P. Gilman, and Susan L. Hamilton*
Ryanodine receptors (RyRs) are the major sarcoplasmic reticulum calcium-release channels required for excitation–contraction coupling in skeletal and cardiac muscle. Mutations in RyRs have been linked to several human diseases. Mutations in the cardiac isoform of RyR2 are associated with catecholaminergic polymorphic ventricular arrhythmias (CPVT), and arrhythmogenic right ventricular dysplasia type 2 (ARVD2), whereas mutations in the skeletal muscle isoform (RyR1) are linked to malignant hyperthermia (MH) and central core disease (CCD). RyRs are modulated by several other proteins, including the FK506 binding proteins (FKBPs), FKBP12 and FKBP12.6. These immunophilins appear to stabilize a closed state of the channel and are important for cooperative interactions among the subunits of RyRs. This review discusses the regulation of RyRs by FKBPs and the possibility that defective modulation of RyR2 by FKBP12.6 could play a role in heart failure, CPVT, and ARVD2. Also discussed are the consequences of FKBP12 depletion to skeletal muscle and the possibility of FKBP12 involvement in certain forms of MH or CCD. (Trends Cardiovasc Med 2004;14:227–234) D 2004, Elsevier Inc.
Ryanodine Receptors As Components of Macromolecular Complexes
Ryanodine receptors (RyRs) are members of a family of calcium (Ca2+)-release channels found on intracellular Ca2+ storage/release organelles (i.e., sarco/ endoplasmic reticulum). Three isoforms (RyR1, RyR2, and RyR3) share over 60%
Mihail G. Chelu, Cristina I. Danila, Charles P. Gilman, and Susan L. Hamilton are at the Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, USA. * Address correspondence to: Susan L. Hamilton, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel.: (+1) 713-798-3894; fax: (+1) 713798-5441; e-mail: [email protected]. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter
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amino acid sequence identity, but are encoded by separate genes: isoform 1 (RyR1) is highly enriched in skeletal muscle; isoform 2 (RyR2) is enriched in the cardiac muscle; and isoform 3 (RyR3), along with RyR1 and RyR2, is found at lower levels in a variety of tissues (for a review, see Fill and Copello 2002). RyRs are homotetramers with a total molecular mass greater than 2 106 Da. All isoforms have a large cytoplasmic region (~4/5 of the molecule) and a smaller hydrophobic membrane-spanning region (~1/5 of the molecule). RyRs in skeletal and cardiac tissue are known to form macromolecular complexes with a number of other proteins that may modulate channel activity. On the cytoplasmic side of the sarcoplasmic reticulum (SR) membrane, RyRs bind E-F hand proteins [e.g., calmodulin (H. Zhang et al. 2003) and sorcin (Meyers et al. 1995)],
scaffolding proteins [e.g., homer (Feng et al. 2002)], anchoring proteins [e.g., AKAPs (Ruehr et al. 2003)], enzymes [e.g., calcineurin (PP2B), protein kinase A (PKA), PP1, and PP2A (for a review, see Meissner 2002)], and immunophilins [e.g., FK506 binding proteins FKBP12 and FKBP12.6 (Lehnart et al. 2003)]. On the lumenal side of the SR membrane, RyRs interact either directly or indirectly with calsequestrin, triadin, and junctin (for a review, see Muller et al. 2002). The activity of the channel is also regulated by a number of small intracellular molecules such as Ca2+, Mg2+, and adenosine triphosphate (ATP), and can be regulated pharmacologically by ryanodine, caffeine, ruthenium red, and the immunosupressant drugs FK506 and rapamycin (for a review, see Fill and Copello 2002). To add to the complexity of RyR regulation, these channels are subject to covalent modifications such as oxidation, nitrosylation, and phosphorylation (for a review, see Meissner 2002).
Multiple Functional Roles for Immunophilins FKBP12 and FKBP12.6
FKBPs are a family of proteins that bind the immunosuppressive drugs FK506 and rapamycin and have cis-trans prolylisomerase activity (PPIase or rotamase). The FKBP family has more than 20 members, of which at least 8 are mammalian. These proteins are named according to their molecular mass (e.g., FKBP12, FKBP12.6, FKBP 13, FKBP 23, FKBP 25, FKBP 51, FKBP 52, FKBP 65, and so forth). FKBP12 (human) is a 108-amino-acid protein that shares 85% sequence identity with FKBP12.6. These two immunophilins differ by only 18 residues and most of these differences are conservative substitutions. Both FKBP12 and FKBP12.6 are amphiphilic h-sheet proteins containing five antiparallel strands (Figure 1). FKBP12.6 is found at lower concentrations than is FKBP12 in both skeletal and cardiac tissues (Timmerman et al. 1996). Originally, the FKBP12.6 isoform was thought to be the only isoform that binds RyR2 (Lam et al. 1995). Recent studies, however, have shown that the two FKBP isoforms bind both RyR1 and RyR2 (Jeyakumar et al. 2001). Although RyR1 has a higher affinity for FKBP12.6 than for FKBP12, the higher tissue con-
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Figure 1. FK506 binding protein (FKBP12) ribbon structure when bound to rapamycin at 1.7 2 resolution (adapted with Chimera software [Pettersen et al. 2004] from PDB ID-1FKB) (Van Duyne et al. 1993). Amino acids involved in forming the hydrophobic binding pocket (Tyr26, Phe46, Phe48, Trp59, Tyr82, and Phe99) are shown in red and rapamycin is shown in violet.
centrations of FKBP12 result in FKBP12 being the predominant form bound to RyR1. In cardiac muscle, the tissue concentration of FKBP12 is considerably higher than that of FKBP12.6, but the preferential interaction of FKBP12.6 with RyR2 is driven by the much higher affinity of RyR2 for FKBP12.6 compared with FKBP12 (Timerman et al. 1996). RyR3 has also been shown to bind FKBP12 (Bultynck et al. 2001). Each RyR1 tetramer binds four molecules of FKBP12 (i.e., one molecule per subunit). The binding of FK506 and rapamycin to FKBP12 displaces it from RyRs. Removal of FKBP12 from RyR1 increases both the probability of channel opening and the occurrence of subconductance states of the channel (Ahern et al. 1997). These findings suggest that FKBP12 stabilizes a closed state of the channel and it is important for cooperative interactions among the subunits of the tetramer (Brillantes et al. 1994). FKBP12 has also been suggested to regulate RyR1 coupled gating, a phenomenon whereby neighboring channels open simultaneously (Marx et al. 1998). The binding site in the three-dimensional (3D) structure of RyR1 appears to be between domain 9 and domain 5 (for a review, see Wagenknecht and Samso 2002), close to the clamp-like domain (Serysheva et al. 1995). The placement of FKBP12 and calmodulin in the 3D structure of RyR1 is shown in Figure 2. FKBP12/FKBP12.6 binding near domain
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Figure 2. Solid-body representations of three-dimensional reconstruction of ryanodine receptor 1 (RyR1) together with the differences attributed to CaM (yellow) and FKBP12 (pink). Selected RyR1 domains are indicated by numerals. (A) top-down view, (B) bottom-up view, and (C) side view. (Reproduced and adapted from Wagenknecht T, Radermacher M, Grassucci R, et al.:1997. Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor. J Biol Chem 272:32463–32471, with permission. Copyright 1997, The American Society for Biochemistry and Molecular Biology.)
9 is also supported by the mapping of divergent region 3 (DR3) to domain 9 in the 3D reconstruction of RyR1, coupled
with the observation that FKBP12.6 binding is abolished by a truncation of DR3 (J. Zhang et al. 2003). TCM Vol. 14, No. 6, 2004
A key residue in RyR1 involved in the binding of FKBP12 is Val2461. Mutation of the Val2461 residue in RyR1 to an isoleucine greatly reduces the affinity of RyR1 for FKBP12, but not FKBP12.6 (Gaburjakova et al. 2001). Similarly, Bultynck et al. (2001) reported that the mutation of Val2322 in RyR3 (comparable with Val2461 in RyR1) to a leucine or isoleucine reduced RyR3 affinity for FKBP12 and increased its selectivity for FKBP12.6. In the equivalent sequence of RyR2, this residue is replaced with an isoleucine (Ile2427). Marx et al. (2000) proposed that, analogous to RyR1, this residue plays a key role in FKBP12.6 binding to RyR2. However, the studies of Masumiya et al. (2003) and Zissimopolous and Lai (2002) challenge the importance of Ile2427 in FKBP12.6 binding to RyR2. Masumiya et al. (2003) mapped the binding site of FKBP12.6 to the NH2-terminal region of RyR2 (a large region between residues 305–1937 that includes the DR3 region). In contrast, Zissimopolous and Lai (2002) found that a large region in the C-terminal domain, which encompassed the transmembrane domain, interacts with FKBP12.6. Both studies suggest that multiple determinants from different regions of RyR2 contribute to a single FKBP12.6 binding site. Residues on FKBP12 and FKBP12.6, important for interactions with RyR1 and RyR2, have also been examined. Xin et al. (1999) have shown that the critical residues Gln31, Asn32, and Phe59 of FKBP12.6 account for the selective binding of FKBP12.6 to cardiac RyR2. Recently, Lee et al. (2004) reported that the three amino acids identified for binding of FKBP12.6 to RyR2 were not involved in the binding of FKBP12, despite its similarity in sequence and structure. Amino acids on FKBP12 thought to be involved in the distinctive binding to RyR1 were suggested to be Gln3, Arg18, and Met49 (Lee et al. 2004). The mutations of several amino acids on FKBP12 to the corresponding ones on FKBP12.6 (E31Q, D32N, and W59F) did not alter its ability to bind to RyR1 (Lee et al. 2004). Deivanayagam et al. (2000) determined the crystal structure of FKBP12.6 in complex with rapamycin at 2.0 2. The structures of FKBP12.6 and FKBP12 are nearly identical, except for displacement in the helical region of FKBP12.6 toward TCM Vol. 14, No. 6, 2004
the hydrophobic pocket. Deivanayagam et al. (2000) predicted that amino acids Ala63 and Val90 are candidates for amino acids that contribute to the differences in regulation of RyR1 and RyR2 by FKBP12 and FKBP12.6. Ala63 allows the helix shift to occur, whereas Val90 creates extra space on the edge of the binding pocket to accommodate the larger Ile-Pro in RyR2. The hydrophobic pocket on FKBP12/12.6—thought to be involved in the interaction with RyRs— is formed by Tyr26, Phe46, Phe48, Trp(Phe)59, Tyr82, and Phe99 (Figure 1). This pocket of FKBP12/12.6 also binds FK506 and rapamycin and is probably the site of interaction with Val2641 of RyR1 (Gaburjakova et al. 2001). FKBP12 and FKBP12.6 are also cistrans prolylisomerases that catalyze the cis-trans isomerization peptidylprolyl bonds by lowering the energy of the unstable twisted amide transition state intermediate (Rosen et al. 1990). Timerman et al. (1995) mutated F36Y, W59H, and F99Y and found substantial loss in the enzyme activity. These mutated FKBP12s still interact with RyR1, producing effects on Ca2+ fluxes from the SR similar to wild-type FKBP12. These findings suggest that the enzymatic activity of FKBP12 does not play a major role in modulation of the channels.
FKBPs and Regulation of RYR1 Function
FKBP12 is thought to stabilize a closed state of the channel (Brillantes et al. 1994). Expression of V2461G-RyR1 and V2461I-RyR1 (mutations that disrupt FKBP12 binding) in dyspedic myotubes decreased maximal voltage-gated Ca2+ release to half that of controls (Avila et al. 2003a). Voltage-gated Ca2+ release was restored by the coexpression of FKBP12.6, suggesting that FKBP12 plays a role in the gain of voltage-dependent, excitation–contraction (E-C) coupling. Mice deficient in FKBP12 displayed no gross morphologic or functional skeletal muscle abnormalities, but had severe dilated cardiomyopathy and ventricular septal defects (Shou et al. 1998). Most of the mice died between embryonic day 14 and birth, making analysis of skeletal muscle phenotypes difficult. The cardiac defects were unanticipated, because FKBP12.6 has higher affinity and/or selectivity for RyR2 than does
FKBP12. Both RyR1 and RyR2 from the FKBP12 knockout (KO) animals reconstituted into planar lipid bilayers showed increased open probability, but opened to a number of subconductance states, suggesting that FKBP12 can modulate the activity of both RyR1 and RyR2. Alternatively, the cardiac defects associated with the deficiency could produce altered phosphorylation of RyR2 and the altered single-channel properties could arise from the loss of FKBP12.6 binding, as suggested by Marx et al. (2000). The embryonic lethality associated with the FKBP12 KO makes it difficult to analyze the functional consequences of FKBP12 deficiency in skeletal muscle. We have recently generated skeletalmuscle-specific, FKBP12-deficient mice using Cre-loxP-mediated gene recombination (Tang et al. 2004). Similar to the findings with myotubes containing the RyR1 mutation that destroys the FKBP12 binding site (Avila et al. 2003a), primary myotubes from these mice showed no change in either Ca2+ stores or resting cytosolic Ca2+ levels, but displayed decreased voltage-gated intracellular Ca2+ release, suggesting that FKBP12 plays a role in E-C coupling gain. In addition, we found an increase in the L-type Ca 2+ currents without a change in dihydropyridine receptor expression. These findings suggest that FKBP12 also plays a role in retrograde coupling, which might have been obscured by the overexpression of the mutated RyR1 in the study of Avila et al. (2003a). Consistent with the decreased, voltage-gated, intracellular Ca2+ release, maximal tetanic force production was decreased and the force frequency curves were shifted to the right in extensor digitorum longus (EDL) muscles of the mutant mice. Compared with controls, there was no decrease in maximal tetanic force production in the mutant diaphragm or soleus muscle, but the force frequency curve was shifted to the left in the F KBP12-deficient diaphragm muscle. Of the three muscle groups (diaphragm, soleus, and EDL) only diaphragm muscle displayed an increased ratio of slow to fast myosin heavy chain isoforms. Consistent with this, calcineurin levels were increased in the diaphragm of the mutant mice, but not in the soleus or EDL. These studies suggested that FKBP12 deficiency alters
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both orthograde and retrograde coupling between the L-type Ca2+ channel and RyR1 and the consequences of these changes depend on muscle type and activity. Our findings do not support the hypothesis that the absence of FKBP12 increases Ca2+ leak from the SR, suggesting that either FKBP12-depleted RyR1 is not leaky in vivo or that other cellular events compensate or mask bCa2+ leakQ from FKBP12-depleted RyR1.
RyRs and FKBPs in Skeletal Muscle Function and Disease
The first link between RyR1 and a human disease was reported in 1993 (Quane et al. 1993, Zhang et al. 1993). Since then, over 40 mutations in RyR1 have been reported to segregate with several pathologic phenotypes, including malignant hyperthermia (MH) (Quane et al. 1993), central core disease (CCD) (Quane et al. 1993, Zhang et al. 1993), and multiminicore disease (MmD) (Guis et al. 2004). MH is a pharmacogenetic syndrome with variable penetrance, characterized by sustained contraction of skeletal muscle in response to volatile anesthetics (e.g., halothane) and depolarizing muscle relaxants (e.g., succinylcholine). CCD is a congenital myopathy, characterized by hypotonia, proximal muscle weakness in infants and children, frequent skeletal abnormalities (e.g., pes cavus, kyphoscoliosis), and susceptibility to malignant hyperthermia (MHS). This rare disease exhibits a broad clinical presentation ranging from asymptomatic to death in utero or fetal akinesia (floppy infant syndrome) (Romero et al. 2003). Muscle fibers from CCD patients frequently show a lack of mitochondria and oxidative enzyme activity at the center (i.e., at the core) (Shy and Magee 1956). These cores can be single or multiple, as well as central or peripheral (beccentricQ cores). Although CCD is usually diagnosed by the presence of central cores, family members with the same mutation can have no cores, minicores, or unevenness of oxidative staining. Type I fiber uniformity appears to be a consistent feature of the disease, however (Sewry et al. 2002). Frequently, the contractile apparatus, SR, and T tubules degenerate concomitantly (Engel 1961). Although autosomal-recessive, missense, mutations (Romero et al. 2003);
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in-frame deletions of one to three amino acids (Monnier et al. 2001); and an outof-frame mutation (Monnier et al. 2003) have also been identified, the majority of mutations affecting the RyR1 gene— which maps to chromosome 19q13.1— are inherited as autosomal-dominant missense mutations (for a review, see Dirksen and Avila 2002). The outof-frame mutation, if translated, would produce a channel truncated by 65 amino acids at the N terminus, which is not detected (Monnier et al. 2003). Instead, a dramatic depletion of RyR1 protein was reported, suggesting that the amount of RyR1 expressed also is crucial for muscle function (Monnier et al. 2003). In addition, a double substitution (R2676W and T2787S) on the same RYR1 allele has been identified in a family with MHS and MmD (Guis et al. 2004). The mutations that produce MH susceptibility and/or CCD are clustered in three regions: region A, amino acids 35–614; region B, amino acids 2129– 2458; and region C, amino acids 4136– 4973. Regions A and B are part of the putative cytoplasmic domain and region C is in the region containing the transmembrane segments. Mutations in regions A and B are most commonly associated with MHS, whereas mutations in regions C appear to be the most common mutations associated with CCD (for a review, see Dirksen and Avila 2002). When expressed transiently in HEK293 cells, mutations in region A or B make the channel more sensitive to agonists and/or increase channelmediated Ca2+ leak (Tong et al. 1997 and 1999). RyR1 with mutations associated with MHS but not CCD, expressed in HEK-293 cells (Tong et al. 1997 and 1999) or dyspedic myotubes (cells derived from the RyR1 KO mice) (Avila and Dirksen 2001), exhibit a heightened sensitivity to caffeine and increased calciuminduced calcium release (CICR). Cells expressing RyR1 with mutations in regions A and B that are associated with both MHS and CCD display high basal cytosolic Ca2+, supporting the hypothesis of leaky channels. How mutations in RYR1 produce leaky channels is not known. One possibility was suggested by Ikemoto and Yamamoto (2000), who proposed that an intramolecular interaction
between domains A and B regulates channel activation by stabilizing a closed state of the channel. They suggested that the MH/CCD mutations weaken this interaction, allowing the channel to open more frequently, producing a Ca2+ leak. Mutations in the cytoplasmic domains may alter RyR1 function by increasing Ca2+ leak, but some of the mutations in region C (Avila et al. 2001 and 2003b, Lynch et al. 1999) produce a completely distinct effect. Mutations in the region of RyR1 that is thought to line the pore (Zhao et al. 1999) decrease voltagedependent activation of the channel, an effect that has been termed E-C uncoupling (Avila et al. 2001). Dyspedic myotubes expressing RyR1 with the I4898T mutation do not release SR calcium following stimulation by membrane depolarization, but have normal resting cytosolic and SR Ca2+ concentrations (Avila et al. 2001). This loss of voltagegated release could alter the ability of the muscle to generate force; however, it is unclear how this could produce the central cores in the muscle. An extremely important question is how the changes in RyR1 lead to the formation of central cores in the muscle. Loke and MacLennan 1998 proposed that an increase in resting Ca2+ concentrations leads to central mitochondrial damage, muscle contracture, and activation of compensatory genes. Their hypothesis for the ability of leaky channels to produce cores in CCD is that Ca2+ in the central part of the fiber overloads mitochondria, leading to their destruction. At the periphery, Ca2+ handling is more efficient and the peripheral mitochondria are spared. The activation of compensatory genes may contribute to the type 1, fiber-type predominance. Although this hypothesis may explain central cores, it is unlikely to explain either the formation of eccentric cores or the presence of cores in patients with region C mutations that produce E-C uncoupling. Mutations in RyR1 could also alter its interactions with its modulators. One cluster of MH/CCD mutations present in region B occurs close to or within the FKBP12 binding site and could increase the Ca2+ leak by destabilizing a closed state of the channel. Mutational analysis of RyR1 and/or animal models with the domain B mutations are needed to TCM Vol. 14, No. 6, 2004
determine whether FKBP12 binding to RyR1 is altered in CCD or MH.
RyR Regulation by FKBPs in Heart Disease
Recently, mutations in the cardiac isoform of RyR (RyR2) have been linked to human disease. To date, 21 RyR2 missense mutations have been shown to be associated with two congenital arrhythmias: familial/catecholaminergic polymorphic ventricular tachycardia (FCVT/ CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2) (for a review, see Wehrens and Marks 2003). These mutations are clustered in three regions similar to the MH/CCD mutations of RyR1, reinforcing the importance of these domains in both RyR1 and RyR2 function. CPVT manifests as exercise-induced bidirectional ventricular tachycardia, leading to sudden death. The hearts of affected individuals, however, lack gross anatomic defects. Functional studies in cell-culture-expressing RyR2 channels with the CPVT mutations showed that both CICR and caffeine sensitivity were increased. Also, the open time for the channel at low Ca2+ concentrations and spontaneous Ca 2+ oscillations were increased (D. Jiang et al. 2002). One possible mechanism for the exercise-induced arrhythmias is RyR2 hyperphosphorylation with subsequent displacement of FKBP12.6 from its binding site, leading to Ca2+ leak. The phenotype of the FKBP12.6 KO mice is consistent with this model. In response to strenuous exercise followed by epinephrine injection (conditions that mimic CPVT-inducing exercise in humans), FKBP12.6 KO mice display progression from episodes of polymorphic ventricular arrhythmias to sustained ventricular arrhythmias (ending in death) (Wehrens et al. 2003). RyR2 channels isolated from the FKBP12.6 KO mouse and phosphorylated CPVT mutant channels have similar singlechannel behavior in vitro (Wehrens et al. 2003). Wehrens et al. (2003) proposed that exercise activates RyR2anchored PKA, which phosphorylates RyR2 and—in the case of the CPVT mutant RyR2—leads to FKBP12.6 dissociation. FKBP12.6 release from RyR2 could produce leaky channels during diastole, which trigger delayed afterdeTCM Vol. 14, No. 6, 2004
polarizations (DADs) and cardiac arrhythmias (Wehrens et al. 2003). It should be noted, however, that studies from other laboratories using the CPVT mutant and wild-type RyR2 channels expressed in HL-1 cardiomyocytes found no difference in either the phosphorylation status of RyR2 or its interaction with FKBP12.6 (George et al. 2003), despite augmented Ca2+ release on hadrenergic receptor (h-AR) stimulation. Studies on the role of the cardiac isoform of calsequestrin (CASQ2) in SR Ca 2+ release led to the proposal of another mechanism for the genesis of CPVT. Terentyev et al. (2003) used adenoviral infection to manipulate the levels of CASQ2 expression in dissociated rat cardiomyocytes. Overexpression of CASQ2 resulted in increased magnitude and duration of Ca 2+ transients in response to depolarizations at various membrane potentials. Ca2+ sparks were brighter and had longer rise times. Also, repetitive Ca2+ sparks were less frequent when compared with control cardiomyocytes. The opposite was observed for cardiomyocytes with reduced CASQ2 expression levels. PKA stimulation induced increased spark frequency in cardiomyocytes underexpressing CASQ2, whereas cyclical electrical stimulation combined with isoproterenol resulted in extrasystolic elevations of Ca2+. In the same system, overexpression of a mutant CASQ2 (D307H), which manifests as a recessive CPVT syndrome in humans (Lahat et al. 2001), resulted in findings similar to those obtained with the CASQ2 underexpressing cardiomyocytes (Viatchenko-Karpinski et al. 2004). Terentyev et al. (2003) proposed that mutations in CASQ2 that impair the Ca2+ buffering inside SR and/or disrupt CASQ2-RyR2 interactions result in altered regulation of Ca2+ release by the lumenal calcium. Adrenergic stimulation promotes accelerated Ca2+ recharging of SR by enhancing SR Ca 2+ -ATPase (SERCA) intake, which promotes premature restoration of function for RyR2 and triggers DADs. Viatchenko-Karpinski et al. (2004) speculated that mutations in junctin, triadin, or RyR2 (compromising its ability to sense lumenal Ca2+) could produce CPVT. Much less is known about ARVD2, which is characterized by structural alterations of the myocardium. The hearts of ARVD2 patients exhibit
progressive degeneration (fibrofatty infiltration) of the right ventricular myocardium, electrical instability (effortinduced ventricular arrhythmias), and sudden death (Tiso et al. 2001). The dominant involvement of the right ventricle is particularly striking because it is under a much lower pressure load than is the left ventricle. Tiso et al. (2002) noted that the ARVD2 mutations are clustered in the A region, whereas CPVT mutations are clustered in the C region. Mutations that produce both diseases are found in the B region that includes the putative FKBP12.6 binding site. Tiso et al. (2002) found that the ARVD mutations decreased the affinity of RyR2 for FKBP12.6, whereas the CPVT mutations actually increased affinity. Based on these observations, Tiso et al. (2002) suggested that the CPVT mutations interfere with E-C coupling by decreasing channel opening and generating arrhythmias. On the other hand, ARVD2 mutations produce Ca2+ leak, leading to arrhythmias and—via Ca2+driven apoptosis/necrosis processes— myocardial damage. The hyperphosphorylation hypothesis has been proposed to explain the relative E-C coupling inefficiency in heart failure (HF) (for a review, see Wehrens and Marks 2003). Marx et al. (2000) have proposed that PKA phosphorylation at Ser2808 in human RyR2 induces dissociation of FKBP12.6, leading to diastolic calcium leak and cardiac dysfunction. The authors (Marx et al. 2000) found increased phosphorylation of RyR2 from humans with HF, as well as a canine model for HF. They also found that RyR2 phosphorylation in vitro and in vivo (failing hearts) resulted in decreased FKBP12.6 binding, causing increased open probability and long-lasting subconductance states. h-AR antagonists restored single-channel properties, phosphatase-to-PKA ratios, and associated FKBP12.6 levels to those indistinguishable from nonfailing hearts (for a review, see Wehrens and Marks 2003). Several laboratories have challenged the RyR2 hyperphosphorylation hypothesis in HF. Labeling experiments identified phosphorylation sites in addition to Ser2809 (Stange et al. 2003). Also, an S2809D substitution, which should mimic constitutively phosphorylated channels, neither released FKBP12.6 nor changed the intrinsic
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channel properties of the channel (Stange et al. 2003, Xiao et al. 2004). In addition, M.T. Jiang et al. 2002 failed to detect a change in the RyR2 phosphorylation or decreased binding of FKBP12.6 in humans and canine HF models. Instead, a significant decrease in SERCA2a levels was identified. To simplify the system and identify Ca2+ signaling abnormalites, Li et al. (2002) used freshly dissociated ventricular myocytes derived from mice, which expressed only mutant nonphosphorylatable phospholambam (PLB-DM), an inhibitor of SERCA. In response to PKA activation, control myocytes displayed increased SR Ca2+ concentrations and increased spark amplitude and frequency. This response was not detected in PLB-KO or PLB-DM cultures, despite an increase in RyR2 phosphorylation. In a subsequent study, Shannon et al. (2003) measured an increased SR Ca2+ leak compared with controls in a rabbit model of HF. The data from the first study and the mathematic simulations of the latter suggested that PKA-induced phosphorylation of RyR2 alone is not sufficient to explain altered SR Ca2+ handling in HF. Shannon et al. (2003) also suggested that PKA effects on Na+/ K+ exchange, SR Ca2+ uptake, and SR Ca2+ content may be more important than the SR Ca2+ leak in HF. Benitah et al. (2002) suggested that the reduction of SR Ca2+ release in failing hearts might be a consequence of a spatial or functional reorganization of the T tubules.
Conclusions
FKBPs bind with high affinity to RyRs and appear to stabilize a closed state of the channel. Thus, removal of FKBP could contribute to Ca2+ leak from the SR. Because the FKBP binding site includes a region close to one of the mutation clusters that produce MH/CCD in skeletal muscle or CPVT/ARVD2 in cardiac muscle, these mutations may alter FKBPs binding to RyRs. One intriguing possibility is that FKBP deficiency could underlie some recessive forms of these diseases. There are many unanswered questions about the role of FKBPs in skeletal/cardiac muscle E-C coupling. How does FKBP stabilize a closed state of the channel? How does this type of mechanism explain the decreased E-C coupling gain with chan-
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nels that do not bind FKBP12? Because FKBP12 does not bind to synthetic peptides matching sequences around amino acid 2461 of RYR1, what other determinants contribute to its binding site? What is the role of the cis-trans prolylisomerase activity of the FKBPs? Can FKBP deficiency or mutation produce mammalian disease? Future research into animal models of the diseases associated with RyR1/RyR2 mutations and higher resolution structures of the channels should help to answer some of the questions.
Acknowledgments
The authors thank Dr. Terence Wagenknecht for the permission to use Figure 2 and the members of the Hamilton laboratory for their many helpful comments on this manuscript.
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Tiso N, Salamon M, Bagattin A, et al.: 2002. The binding of the RyR2 calcium channel to its gating protein FKBP12.6 is oppositely affected by ARVD2 and VTSIP mutations. Biochem Biophys Res Commun 299:594 – 598. Tong J, Oyamada H, Demaurex N, et al.: 1997. Caffeine and halothane sensitivity of intracellular Ca2+ release is altered by 15 calcium release channel (ryanodine receptor) mutations associated with malignant hyperthermia and/or central core disease. J Biol Chem 272:26,332 – 26,339. Tong J, McCarthy TV, MacLennan DH: 1999. Measurement of resting cytosolic Ca2+ concentrations and Ca2+ store size in HEK-293 cells transfected with malignant hyperthermia or central core disease mutant Ca2+ release channels. J Biol Chem 274:693 – 702. Van Duyne GD, Standaert RF, Karplus PA, et al.: 1993. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J Mol Biol 229: 105–124. Viatchenko-Karpinski S, Terentyev D, Gyorke I, et al.: 2004. Abnormal calcium signaling and sudden cardiac death associated with mutation of calsequestrin. Circ Res 94: 471 – 477. Wagenknecht T, Radermacher M, Grassucci R, et al.: 1997. Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor. J Biol Chem 272: 32,463–32,471. Wagenknecht T, Samso M: 2002. Threedimensional reconstruction of ryanodine receptors. Front Biosci 7:1464 – 1474. Wehrens XH, Lehnart SE, Huang F, et al.: 2003. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell 113:829 – 840. Wehrens XH, Marks AR: 2003. Altered function and regulation of cardiac ryanodine receptors in cardiac disease. Trends Biochem Sci 28:671 – 678. Xiao B, Sutherland C, Walsh MP, Chen SR: 2004. Protein kinase A phosphorylation at serine-2808 of the cardiac Ca2+-release channel (ryanodine receptor) does not dissociate 12.6-kDa FK506-binding protein (FKBP12.6). Circ Res 94:487 – 495.
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Zhao M, Li P, Li X, et al.: 1999. Molecular identification of the ryanodine receptor pore-forming segment. J Biol Chem 274: 25,971 – 25,974. Zissimopoulos S, Lai FA: 2002. Evidence for a FKBP12.6 binding site at the C-terminus of the cardiac ryanodine receptor. Biophys J 82:59. PII S1050-1738(04)00088-X
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Ryanodine receptors (RyRs) are the major sarcoplasmic reticulum calcium-release channels required for excitation–contraction coupling in skeletal and cardiac muscle. Mutations in RyRs have been linked to several human diseases. Mutations in the cardiac isoform of RyR2 are associated with catecholaminergic polymorphic ventricular arrhythmias (CPVT), and arrhythmogenic right ventricular dysplasia type 2 (ARVD2), whereas mutations in the skeletal muscle isoform (RyR1) are linked to malignant hyperthermia (MH) and central core disease (CCD). RyRs are modulated by several other proteins, including the FK506 binding proteins (FKBPs), FKBP12 and FKBP12.6. These immunophilins appear to stabilize a closed state of the channel and are important for cooperative interactions among the subunits of RyRs. This review discusses the regulation of RyRs by FKBPs and the possibility that defective modulation of RyR2 by FKBP12.6 could play a role in heart failure, CPVT, and ARVD2. Also discussed are the consequences of FKBP12 depletion to skeletal muscle and the possibility of FKBP12 involvement in certain forms of MH or CCD. (Trends Cardiovasc Med 2004;14:227–234) D 2004, Elsevier Inc.
Ryanodine Receptors As Components of Macromolecular Complexes
Ryanodine receptors (RyRs) are members of a family of calcium (Ca2+)-release channels found on intracellular Ca2+ storage/release organelles (i.e., sarco/ endoplasmic reticulum). Three isoforms (RyR1, RyR2, and RyR3) share over 60%
Mihail G. Chelu, Cristina I. Danila, Charles P. Gilman, and Susan L. Hamilton are at the Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas, USA. * Address correspondence to: Susan L. Hamilton, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel.: (+1) 713-798-3894; fax: (+1) 713798-5441; e-mail: [email protected]. D 2004, Elsevier Inc. All rights reserved. 1050-1738/04/$-see front matter
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amino acid sequence identity, but are encoded by separate genes: isoform 1 (RyR1) is highly enriched in skeletal muscle; isoform 2 (RyR2) is enriched in the cardiac muscle; and isoform 3 (RyR3), along with RyR1 and RyR2, is found at lower levels in a variety of tissues (for a review, see Fill and Copello 2002). RyRs are homotetramers with a total molecular mass greater than 2 106 Da. All isoforms have a large cytoplasmic region (~4/5 of the molecule) and a smaller hydrophobic membrane-spanning region (~1/5 of the molecule). RyRs in skeletal and cardiac tissue are known to form macromolecular complexes with a number of other proteins that may modulate channel activity. On the cytoplasmic side of the sarcoplasmic reticulum (SR) membrane, RyRs bind E-F hand proteins [e.g., calmodulin (H. Zhang et al. 2003) and sorcin (Meyers et al. 1995)],
scaffolding proteins [e.g., homer (Feng et al. 2002)], anchoring proteins [e.g., AKAPs (Ruehr et al. 2003)], enzymes [e.g., calcineurin (PP2B), protein kinase A (PKA), PP1, and PP2A (for a review, see Meissner 2002)], and immunophilins [e.g., FK506 binding proteins FKBP12 and FKBP12.6 (Lehnart et al. 2003)]. On the lumenal side of the SR membrane, RyRs interact either directly or indirectly with calsequestrin, triadin, and junctin (for a review, see Muller et al. 2002). The activity of the channel is also regulated by a number of small intracellular molecules such as Ca2+, Mg2+, and adenosine triphosphate (ATP), and can be regulated pharmacologically by ryanodine, caffeine, ruthenium red, and the immunosupressant drugs FK506 and rapamycin (for a review, see Fill and Copello 2002). To add to the complexity of RyR regulation, these channels are subject to covalent modifications such as oxidation, nitrosylation, and phosphorylation (for a review, see Meissner 2002).
Multiple Functional Roles for Immunophilins FKBP12 and FKBP12.6
FKBPs are a family of proteins that bind the immunosuppressive drugs FK506 and rapamycin and have cis-trans prolylisomerase activity (PPIase or rotamase). The FKBP family has more than 20 members, of which at least 8 are mammalian. These proteins are named according to their molecular mass (e.g., FKBP12, FKBP12.6, FKBP 13, FKBP 23, FKBP 25, FKBP 51, FKBP 52, FKBP 65, and so forth). FKBP12 (human) is a 108-amino-acid protein that shares 85% sequence identity with FKBP12.6. These two immunophilins differ by only 18 residues and most of these differences are conservative substitutions. Both FKBP12 and FKBP12.6 are amphiphilic h-sheet proteins containing five antiparallel strands (Figure 1). FKBP12.6 is found at lower concentrations than is FKBP12 in both skeletal and cardiac tissues (Timmerman et al. 1996). Originally, the FKBP12.6 isoform was thought to be the only isoform that binds RyR2 (Lam et al. 1995). Recent studies, however, have shown that the two FKBP isoforms bind both RyR1 and RyR2 (Jeyakumar et al. 2001). Although RyR1 has a higher affinity for FKBP12.6 than for FKBP12, the higher tissue con-
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Figure 1. FK506 binding protein (FKBP12) ribbon structure when bound to rapamycin at 1.7 2 resolution (adapted with Chimera software [Pettersen et al. 2004] from PDB ID-1FKB) (Van Duyne et al. 1993). Amino acids involved in forming the hydrophobic binding pocket (Tyr26, Phe46, Phe48, Trp59, Tyr82, and Phe99) are shown in red and rapamycin is shown in violet.
centrations of FKBP12 result in FKBP12 being the predominant form bound to RyR1. In cardiac muscle, the tissue concentration of FKBP12 is considerably higher than that of FKBP12.6, but the preferential interaction of FKBP12.6 with RyR2 is driven by the much higher affinity of RyR2 for FKBP12.6 compared with FKBP12 (Timerman et al. 1996). RyR3 has also been shown to bind FKBP12 (Bultynck et al. 2001). Each RyR1 tetramer binds four molecules of FKBP12 (i.e., one molecule per subunit). The binding of FK506 and rapamycin to FKBP12 displaces it from RyRs. Removal of FKBP12 from RyR1 increases both the probability of channel opening and the occurrence of subconductance states of the channel (Ahern et al. 1997). These findings suggest that FKBP12 stabilizes a closed state of the channel and it is important for cooperative interactions among the subunits of the tetramer (Brillantes et al. 1994). FKBP12 has also been suggested to regulate RyR1 coupled gating, a phenomenon whereby neighboring channels open simultaneously (Marx et al. 1998). The binding site in the three-dimensional (3D) structure of RyR1 appears to be between domain 9 and domain 5 (for a review, see Wagenknecht and Samso 2002), close to the clamp-like domain (Serysheva et al. 1995). The placement of FKBP12 and calmodulin in the 3D structure of RyR1 is shown in Figure 2. FKBP12/FKBP12.6 binding near domain
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Figure 2. Solid-body representations of three-dimensional reconstruction of ryanodine receptor 1 (RyR1) together with the differences attributed to CaM (yellow) and FKBP12 (pink). Selected RyR1 domains are indicated by numerals. (A) top-down view, (B) bottom-up view, and (C) side view. (Reproduced and adapted from Wagenknecht T, Radermacher M, Grassucci R, et al.:1997. Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor. J Biol Chem 272:32463–32471, with permission. Copyright 1997, The American Society for Biochemistry and Molecular Biology.)
9 is also supported by the mapping of divergent region 3 (DR3) to domain 9 in the 3D reconstruction of RyR1, coupled
with the observation that FKBP12.6 binding is abolished by a truncation of DR3 (J. Zhang et al. 2003). TCM Vol. 14, No. 6, 2004
A key residue in RyR1 involved in the binding of FKBP12 is Val2461. Mutation of the Val2461 residue in RyR1 to an isoleucine greatly reduces the affinity of RyR1 for FKBP12, but not FKBP12.6 (Gaburjakova et al. 2001). Similarly, Bultynck et al. (2001) reported that the mutation of Val2322 in RyR3 (comparable with Val2461 in RyR1) to a leucine or isoleucine reduced RyR3 affinity for FKBP12 and increased its selectivity for FKBP12.6. In the equivalent sequence of RyR2, this residue is replaced with an isoleucine (Ile2427). Marx et al. (2000) proposed that, analogous to RyR1, this residue plays a key role in FKBP12.6 binding to RyR2. However, the studies of Masumiya et al. (2003) and Zissimopolous and Lai (2002) challenge the importance of Ile2427 in FKBP12.6 binding to RyR2. Masumiya et al. (2003) mapped the binding site of FKBP12.6 to the NH2-terminal region of RyR2 (a large region between residues 305–1937 that includes the DR3 region). In contrast, Zissimopolous and Lai (2002) found that a large region in the C-terminal domain, which encompassed the transmembrane domain, interacts with FKBP12.6. Both studies suggest that multiple determinants from different regions of RyR2 contribute to a single FKBP12.6 binding site. Residues on FKBP12 and FKBP12.6, important for interactions with RyR1 and RyR2, have also been examined. Xin et al. (1999) have shown that the critical residues Gln31, Asn32, and Phe59 of FKBP12.6 account for the selective binding of FKBP12.6 to cardiac RyR2. Recently, Lee et al. (2004) reported that the three amino acids identified for binding of FKBP12.6 to RyR2 were not involved in the binding of FKBP12, despite its similarity in sequence and structure. Amino acids on FKBP12 thought to be involved in the distinctive binding to RyR1 were suggested to be Gln3, Arg18, and Met49 (Lee et al. 2004). The mutations of several amino acids on FKBP12 to the corresponding ones on FKBP12.6 (E31Q, D32N, and W59F) did not alter its ability to bind to RyR1 (Lee et al. 2004). Deivanayagam et al. (2000) determined the crystal structure of FKBP12.6 in complex with rapamycin at 2.0 2. The structures of FKBP12.6 and FKBP12 are nearly identical, except for displacement in the helical region of FKBP12.6 toward TCM Vol. 14, No. 6, 2004
the hydrophobic pocket. Deivanayagam et al. (2000) predicted that amino acids Ala63 and Val90 are candidates for amino acids that contribute to the differences in regulation of RyR1 and RyR2 by FKBP12 and FKBP12.6. Ala63 allows the helix shift to occur, whereas Val90 creates extra space on the edge of the binding pocket to accommodate the larger Ile-Pro in RyR2. The hydrophobic pocket on FKBP12/12.6—thought to be involved in the interaction with RyRs— is formed by Tyr26, Phe46, Phe48, Trp(Phe)59, Tyr82, and Phe99 (Figure 1). This pocket of FKBP12/12.6 also binds FK506 and rapamycin and is probably the site of interaction with Val2641 of RyR1 (Gaburjakova et al. 2001). FKBP12 and FKBP12.6 are also cistrans prolylisomerases that catalyze the cis-trans isomerization peptidylprolyl bonds by lowering the energy of the unstable twisted amide transition state intermediate (Rosen et al. 1990). Timerman et al. (1995) mutated F36Y, W59H, and F99Y and found substantial loss in the enzyme activity. These mutated FKBP12s still interact with RyR1, producing effects on Ca2+ fluxes from the SR similar to wild-type FKBP12. These findings suggest that the enzymatic activity of FKBP12 does not play a major role in modulation of the channels.
FKBPs and Regulation of RYR1 Function
FKBP12 is thought to stabilize a closed state of the channel (Brillantes et al. 1994). Expression of V2461G-RyR1 and V2461I-RyR1 (mutations that disrupt FKBP12 binding) in dyspedic myotubes decreased maximal voltage-gated Ca2+ release to half that of controls (Avila et al. 2003a). Voltage-gated Ca2+ release was restored by the coexpression of FKBP12.6, suggesting that FKBP12 plays a role in the gain of voltage-dependent, excitation–contraction (E-C) coupling. Mice deficient in FKBP12 displayed no gross morphologic or functional skeletal muscle abnormalities, but had severe dilated cardiomyopathy and ventricular septal defects (Shou et al. 1998). Most of the mice died between embryonic day 14 and birth, making analysis of skeletal muscle phenotypes difficult. The cardiac defects were unanticipated, because FKBP12.6 has higher affinity and/or selectivity for RyR2 than does
FKBP12. Both RyR1 and RyR2 from the FKBP12 knockout (KO) animals reconstituted into planar lipid bilayers showed increased open probability, but opened to a number of subconductance states, suggesting that FKBP12 can modulate the activity of both RyR1 and RyR2. Alternatively, the cardiac defects associated with the deficiency could produce altered phosphorylation of RyR2 and the altered single-channel properties could arise from the loss of FKBP12.6 binding, as suggested by Marx et al. (2000). The embryonic lethality associated with the FKBP12 KO makes it difficult to analyze the functional consequences of FKBP12 deficiency in skeletal muscle. We have recently generated skeletalmuscle-specific, FKBP12-deficient mice using Cre-loxP-mediated gene recombination (Tang et al. 2004). Similar to the findings with myotubes containing the RyR1 mutation that destroys the FKBP12 binding site (Avila et al. 2003a), primary myotubes from these mice showed no change in either Ca2+ stores or resting cytosolic Ca2+ levels, but displayed decreased voltage-gated intracellular Ca2+ release, suggesting that FKBP12 plays a role in E-C coupling gain. In addition, we found an increase in the L-type Ca 2+ currents without a change in dihydropyridine receptor expression. These findings suggest that FKBP12 also plays a role in retrograde coupling, which might have been obscured by the overexpression of the mutated RyR1 in the study of Avila et al. (2003a). Consistent with the decreased, voltage-gated, intracellular Ca2+ release, maximal tetanic force production was decreased and the force frequency curves were shifted to the right in extensor digitorum longus (EDL) muscles of the mutant mice. Compared with controls, there was no decrease in maximal tetanic force production in the mutant diaphragm or soleus muscle, but the force frequency curve was shifted to the left in the F KBP12-deficient diaphragm muscle. Of the three muscle groups (diaphragm, soleus, and EDL) only diaphragm muscle displayed an increased ratio of slow to fast myosin heavy chain isoforms. Consistent with this, calcineurin levels were increased in the diaphragm of the mutant mice, but not in the soleus or EDL. These studies suggested that FKBP12 deficiency alters
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both orthograde and retrograde coupling between the L-type Ca2+ channel and RyR1 and the consequences of these changes depend on muscle type and activity. Our findings do not support the hypothesis that the absence of FKBP12 increases Ca2+ leak from the SR, suggesting that either FKBP12-depleted RyR1 is not leaky in vivo or that other cellular events compensate or mask bCa2+ leakQ from FKBP12-depleted RyR1.
RyRs and FKBPs in Skeletal Muscle Function and Disease
The first link between RyR1 and a human disease was reported in 1993 (Quane et al. 1993, Zhang et al. 1993). Since then, over 40 mutations in RyR1 have been reported to segregate with several pathologic phenotypes, including malignant hyperthermia (MH) (Quane et al. 1993), central core disease (CCD) (Quane et al. 1993, Zhang et al. 1993), and multiminicore disease (MmD) (Guis et al. 2004). MH is a pharmacogenetic syndrome with variable penetrance, characterized by sustained contraction of skeletal muscle in response to volatile anesthetics (e.g., halothane) and depolarizing muscle relaxants (e.g., succinylcholine). CCD is a congenital myopathy, characterized by hypotonia, proximal muscle weakness in infants and children, frequent skeletal abnormalities (e.g., pes cavus, kyphoscoliosis), and susceptibility to malignant hyperthermia (MHS). This rare disease exhibits a broad clinical presentation ranging from asymptomatic to death in utero or fetal akinesia (floppy infant syndrome) (Romero et al. 2003). Muscle fibers from CCD patients frequently show a lack of mitochondria and oxidative enzyme activity at the center (i.e., at the core) (Shy and Magee 1956). These cores can be single or multiple, as well as central or peripheral (beccentricQ cores). Although CCD is usually diagnosed by the presence of central cores, family members with the same mutation can have no cores, minicores, or unevenness of oxidative staining. Type I fiber uniformity appears to be a consistent feature of the disease, however (Sewry et al. 2002). Frequently, the contractile apparatus, SR, and T tubules degenerate concomitantly (Engel 1961). Although autosomal-recessive, missense, mutations (Romero et al. 2003);
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in-frame deletions of one to three amino acids (Monnier et al. 2001); and an outof-frame mutation (Monnier et al. 2003) have also been identified, the majority of mutations affecting the RyR1 gene— which maps to chromosome 19q13.1— are inherited as autosomal-dominant missense mutations (for a review, see Dirksen and Avila 2002). The outof-frame mutation, if translated, would produce a channel truncated by 65 amino acids at the N terminus, which is not detected (Monnier et al. 2003). Instead, a dramatic depletion of RyR1 protein was reported, suggesting that the amount of RyR1 expressed also is crucial for muscle function (Monnier et al. 2003). In addition, a double substitution (R2676W and T2787S) on the same RYR1 allele has been identified in a family with MHS and MmD (Guis et al. 2004). The mutations that produce MH susceptibility and/or CCD are clustered in three regions: region A, amino acids 35–614; region B, amino acids 2129– 2458; and region C, amino acids 4136– 4973. Regions A and B are part of the putative cytoplasmic domain and region C is in the region containing the transmembrane segments. Mutations in regions A and B are most commonly associated with MHS, whereas mutations in regions C appear to be the most common mutations associated with CCD (for a review, see Dirksen and Avila 2002). When expressed transiently in HEK293 cells, mutations in region A or B make the channel more sensitive to agonists and/or increase channelmediated Ca2+ leak (Tong et al. 1997 and 1999). RyR1 with mutations associated with MHS but not CCD, expressed in HEK-293 cells (Tong et al. 1997 and 1999) or dyspedic myotubes (cells derived from the RyR1 KO mice) (Avila and Dirksen 2001), exhibit a heightened sensitivity to caffeine and increased calciuminduced calcium release (CICR). Cells expressing RyR1 with mutations in regions A and B that are associated with both MHS and CCD display high basal cytosolic Ca2+, supporting the hypothesis of leaky channels. How mutations in RYR1 produce leaky channels is not known. One possibility was suggested by Ikemoto and Yamamoto (2000), who proposed that an intramolecular interaction
between domains A and B regulates channel activation by stabilizing a closed state of the channel. They suggested that the MH/CCD mutations weaken this interaction, allowing the channel to open more frequently, producing a Ca2+ leak. Mutations in the cytoplasmic domains may alter RyR1 function by increasing Ca2+ leak, but some of the mutations in region C (Avila et al. 2001 and 2003b, Lynch et al. 1999) produce a completely distinct effect. Mutations in the region of RyR1 that is thought to line the pore (Zhao et al. 1999) decrease voltagedependent activation of the channel, an effect that has been termed E-C uncoupling (Avila et al. 2001). Dyspedic myotubes expressing RyR1 with the I4898T mutation do not release SR calcium following stimulation by membrane depolarization, but have normal resting cytosolic and SR Ca2+ concentrations (Avila et al. 2001). This loss of voltagegated release could alter the ability of the muscle to generate force; however, it is unclear how this could produce the central cores in the muscle. An extremely important question is how the changes in RyR1 lead to the formation of central cores in the muscle. Loke and MacLennan 1998 proposed that an increase in resting Ca2+ concentrations leads to central mitochondrial damage, muscle contracture, and activation of compensatory genes. Their hypothesis for the ability of leaky channels to produce cores in CCD is that Ca2+ in the central part of the fiber overloads mitochondria, leading to their destruction. At the periphery, Ca2+ handling is more efficient and the peripheral mitochondria are spared. The activation of compensatory genes may contribute to the type 1, fiber-type predominance. Although this hypothesis may explain central cores, it is unlikely to explain either the formation of eccentric cores or the presence of cores in patients with region C mutations that produce E-C uncoupling. Mutations in RyR1 could also alter its interactions with its modulators. One cluster of MH/CCD mutations present in region B occurs close to or within the FKBP12 binding site and could increase the Ca2+ leak by destabilizing a closed state of the channel. Mutational analysis of RyR1 and/or animal models with the domain B mutations are needed to TCM Vol. 14, No. 6, 2004
determine whether FKBP12 binding to RyR1 is altered in CCD or MH.
RyR Regulation by FKBPs in Heart Disease
Recently, mutations in the cardiac isoform of RyR (RyR2) have been linked to human disease. To date, 21 RyR2 missense mutations have been shown to be associated with two congenital arrhythmias: familial/catecholaminergic polymorphic ventricular tachycardia (FCVT/ CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2) (for a review, see Wehrens and Marks 2003). These mutations are clustered in three regions similar to the MH/CCD mutations of RyR1, reinforcing the importance of these domains in both RyR1 and RyR2 function. CPVT manifests as exercise-induced bidirectional ventricular tachycardia, leading to sudden death. The hearts of affected individuals, however, lack gross anatomic defects. Functional studies in cell-culture-expressing RyR2 channels with the CPVT mutations showed that both CICR and caffeine sensitivity were increased. Also, the open time for the channel at low Ca2+ concentrations and spontaneous Ca 2+ oscillations were increased (D. Jiang et al. 2002). One possible mechanism for the exercise-induced arrhythmias is RyR2 hyperphosphorylation with subsequent displacement of FKBP12.6 from its binding site, leading to Ca2+ leak. The phenotype of the FKBP12.6 KO mice is consistent with this model. In response to strenuous exercise followed by epinephrine injection (conditions that mimic CPVT-inducing exercise in humans), FKBP12.6 KO mice display progression from episodes of polymorphic ventricular arrhythmias to sustained ventricular arrhythmias (ending in death) (Wehrens et al. 2003). RyR2 channels isolated from the FKBP12.6 KO mouse and phosphorylated CPVT mutant channels have similar singlechannel behavior in vitro (Wehrens et al. 2003). Wehrens et al. (2003) proposed that exercise activates RyR2anchored PKA, which phosphorylates RyR2 and—in the case of the CPVT mutant RyR2—leads to FKBP12.6 dissociation. FKBP12.6 release from RyR2 could produce leaky channels during diastole, which trigger delayed afterdeTCM Vol. 14, No. 6, 2004
polarizations (DADs) and cardiac arrhythmias (Wehrens et al. 2003). It should be noted, however, that studies from other laboratories using the CPVT mutant and wild-type RyR2 channels expressed in HL-1 cardiomyocytes found no difference in either the phosphorylation status of RyR2 or its interaction with FKBP12.6 (George et al. 2003), despite augmented Ca2+ release on hadrenergic receptor (h-AR) stimulation. Studies on the role of the cardiac isoform of calsequestrin (CASQ2) in SR Ca 2+ release led to the proposal of another mechanism for the genesis of CPVT. Terentyev et al. (2003) used adenoviral infection to manipulate the levels of CASQ2 expression in dissociated rat cardiomyocytes. Overexpression of CASQ2 resulted in increased magnitude and duration of Ca 2+ transients in response to depolarizations at various membrane potentials. Ca2+ sparks were brighter and had longer rise times. Also, repetitive Ca2+ sparks were less frequent when compared with control cardiomyocytes. The opposite was observed for cardiomyocytes with reduced CASQ2 expression levels. PKA stimulation induced increased spark frequency in cardiomyocytes underexpressing CASQ2, whereas cyclical electrical stimulation combined with isoproterenol resulted in extrasystolic elevations of Ca2+. In the same system, overexpression of a mutant CASQ2 (D307H), which manifests as a recessive CPVT syndrome in humans (Lahat et al. 2001), resulted in findings similar to those obtained with the CASQ2 underexpressing cardiomyocytes (Viatchenko-Karpinski et al. 2004). Terentyev et al. (2003) proposed that mutations in CASQ2 that impair the Ca2+ buffering inside SR and/or disrupt CASQ2-RyR2 interactions result in altered regulation of Ca2+ release by the lumenal calcium. Adrenergic stimulation promotes accelerated Ca2+ recharging of SR by enhancing SR Ca 2+ -ATPase (SERCA) intake, which promotes premature restoration of function for RyR2 and triggers DADs. Viatchenko-Karpinski et al. (2004) speculated that mutations in junctin, triadin, or RyR2 (compromising its ability to sense lumenal Ca2+) could produce CPVT. Much less is known about ARVD2, which is characterized by structural alterations of the myocardium. The hearts of ARVD2 patients exhibit
progressive degeneration (fibrofatty infiltration) of the right ventricular myocardium, electrical instability (effortinduced ventricular arrhythmias), and sudden death (Tiso et al. 2001). The dominant involvement of the right ventricle is particularly striking because it is under a much lower pressure load than is the left ventricle. Tiso et al. (2002) noted that the ARVD2 mutations are clustered in the A region, whereas CPVT mutations are clustered in the C region. Mutations that produce both diseases are found in the B region that includes the putative FKBP12.6 binding site. Tiso et al. (2002) found that the ARVD mutations decreased the affinity of RyR2 for FKBP12.6, whereas the CPVT mutations actually increased affinity. Based on these observations, Tiso et al. (2002) suggested that the CPVT mutations interfere with E-C coupling by decreasing channel opening and generating arrhythmias. On the other hand, ARVD2 mutations produce Ca2+ leak, leading to arrhythmias and—via Ca2+driven apoptosis/necrosis processes— myocardial damage. The hyperphosphorylation hypothesis has been proposed to explain the relative E-C coupling inefficiency in heart failure (HF) (for a review, see Wehrens and Marks 2003). Marx et al. (2000) have proposed that PKA phosphorylation at Ser2808 in human RyR2 induces dissociation of FKBP12.6, leading to diastolic calcium leak and cardiac dysfunction. The authors (Marx et al. 2000) found increased phosphorylation of RyR2 from humans with HF, as well as a canine model for HF. They also found that RyR2 phosphorylation in vitro and in vivo (failing hearts) resulted in decreased FKBP12.6 binding, causing increased open probability and long-lasting subconductance states. h-AR antagonists restored single-channel properties, phosphatase-to-PKA ratios, and associated FKBP12.6 levels to those indistinguishable from nonfailing hearts (for a review, see Wehrens and Marks 2003). Several laboratories have challenged the RyR2 hyperphosphorylation hypothesis in HF. Labeling experiments identified phosphorylation sites in addition to Ser2809 (Stange et al. 2003). Also, an S2809D substitution, which should mimic constitutively phosphorylated channels, neither released FKBP12.6 nor changed the intrinsic
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channel properties of the channel (Stange et al. 2003, Xiao et al. 2004). In addition, M.T. Jiang et al. 2002 failed to detect a change in the RyR2 phosphorylation or decreased binding of FKBP12.6 in humans and canine HF models. Instead, a significant decrease in SERCA2a levels was identified. To simplify the system and identify Ca2+ signaling abnormalites, Li et al. (2002) used freshly dissociated ventricular myocytes derived from mice, which expressed only mutant nonphosphorylatable phospholambam (PLB-DM), an inhibitor of SERCA. In response to PKA activation, control myocytes displayed increased SR Ca2+ concentrations and increased spark amplitude and frequency. This response was not detected in PLB-KO or PLB-DM cultures, despite an increase in RyR2 phosphorylation. In a subsequent study, Shannon et al. (2003) measured an increased SR Ca2+ leak compared with controls in a rabbit model of HF. The data from the first study and the mathematic simulations of the latter suggested that PKA-induced phosphorylation of RyR2 alone is not sufficient to explain altered SR Ca2+ handling in HF. Shannon et al. (2003) also suggested that PKA effects on Na+/ K+ exchange, SR Ca2+ uptake, and SR Ca2+ content may be more important than the SR Ca2+ leak in HF. Benitah et al. (2002) suggested that the reduction of SR Ca2+ release in failing hearts might be a consequence of a spatial or functional reorganization of the T tubules.
Conclusions
FKBPs bind with high affinity to RyRs and appear to stabilize a closed state of the channel. Thus, removal of FKBP could contribute to Ca2+ leak from the SR. Because the FKBP binding site includes a region close to one of the mutation clusters that produce MH/CCD in skeletal muscle or CPVT/ARVD2 in cardiac muscle, these mutations may alter FKBPs binding to RyRs. One intriguing possibility is that FKBP deficiency could underlie some recessive forms of these diseases. There are many unanswered questions about the role of FKBPs in skeletal/cardiac muscle E-C coupling. How does FKBP stabilize a closed state of the channel? How does this type of mechanism explain the decreased E-C coupling gain with chan-
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nels that do not bind FKBP12? Because FKBP12 does not bind to synthetic peptides matching sequences around amino acid 2461 of RYR1, what other determinants contribute to its binding site? What is the role of the cis-trans prolylisomerase activity of the FKBPs? Can FKBP deficiency or mutation produce mammalian disease? Future research into animal models of the diseases associated with RyR1/RyR2 mutations and higher resolution structures of the channels should help to answer some of the questions.
Acknowledgments
The authors thank Dr. Terence Wagenknecht for the permission to use Figure 2 and the members of the Hamilton laboratory for their many helpful comments on this manuscript.
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