FKBP12 binding to RyR1 modulates excitation-contraction coupling in mouse skeletal myotubes

The skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel or ryanodine receptor (RyR1) binds four molecules of FKBP12, and the interaction of FKBP12 with RyR1 regulates both unitary and coupled gating of the channel. We have characterized the physiologic effects of previously identified mutations in RyR1 that disrupt FKBP12 binding (V2461G and V2461I) on excitation-contraction (EC) coupling and intracellular Ca2+ homeostasis following their expression in skeletal myotubes derived from RyR1-knockout (dyspedic) mice. Wild-type RyR1-, V246I-, and V2461G-expressing myotubes exhibited similar resting Ca2+ levels and maximal responses to caffeine (10 mm) and cyclopiazonic acid (30 microm). However, maximal voltage-gated Ca2+ release in V2461G-expressing myotubes was reduced by approximately 50% compared with that attributable to wild-type RyR1 (deltaF/Fmax = 1.6 +/- 0.2 and 3.1 +/- 0.4, respectively). Dyspedic myotubes expressing the V2461I mutant protein, that binds FKBP12.6 but not FKBP12, exhibited a comparable reduction in voltage-gated SR Ca2+ release (deltaF/Fmax = 1.0 +/- 0.1). However, voltage-gated Ca2+ release in V2461I-expressing myotubes was restored to a normal level (deltaF/Fmax = 2.9 +/- 0.6) following co-expression of FKBP12.6. None of the mutations that disrupted FKBP binding to RyR1 significantly affected RyR1-mediated enhancement of L-type Ca2+ channel activity (retrograde coupling). These data demonstrate that FKBP12 binding to RyR1 enhances the gain of skeletal muscle EC coupling.     

PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation-contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are "leaky." RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.     

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.     

FKBP12 activates the cardiac ryanodine receptor Ca2+-release channel and is antagonised by FKBP12.6

Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca(2+)-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca(2+)-induced Ca(2+)-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca(2+), whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 μM) increased Ca(2+)-wave frequency and decreased the SR Ca(2+)-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12.We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, 'leaky' RyR2 channels and Ca(2+)-dependent arrhythmias.     

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12_{E31Q/D32N/W59F}) where the residues Glu³¹, Asp³², and Trp⁵⁹ were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12_{E31Q/D32N/W59F} mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12_{E31Q/D32N/W59F} activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu³¹, Asp³², and Trp⁵⁹ in FKBP12 and Gln³¹, Asn³², and Phe⁵⁹ in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.     

FKBP12.6 Activates RyR1: Investigating the Amino Acid Residues Critical for Channel Modulation

We have previously shown that FKBP12 associates with RyR2 in cardiac muscle and that it modulates RyR2 function differently to FKBP12.6. We now investigate how these proteins affect the single-channel behavior of RyR1 derived from rabbit skeletal muscle. Our results show that FKBP12.6 activates and FKBP12 inhibits RyR1. It is likely that both proteins compete for the same binding sites on RyR1 because channels that are preactivated by FKBP12.6 cannot be subsequently inhibited by FKBP12. We produced a mutant FKBP12 molecule (FKBP12_{E31Q/D32N/W59F}) where the residues Glu³¹, Asp³², and Trp⁵⁹ were converted to the corresponding residues in FKBP12.6. With respect to the functional regulation of RyR1 and RyR2, the FKBP12_{E31Q/D32N/W59F} mutant lost all ability to behave like FKBP12 and instead behaved like FKBP12.6. FKBP12_{E31Q/D32N/W59F} activated RyR1 but was not capable of activating RyR2. In conclusion, FKBP12.6 activates RyR1, whereas FKBP12 activates RyR2 and this selective activator phenotype is determined within the amino acid residues Glu³¹, Asp³², and Trp⁵⁹ in FKBP12 and Gln³¹, Asn³², and Phe⁵⁹ in FKBP12.6. The opposing but different effects of FKBP12 and FKBP12.6 on RyR1 and RyR2 channel gating provide scope for diversity of regulation in different tissues.     

FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death

Arrhythmias, a common cause of sudden cardiac death, can occur in structurally normal hearts, although the mechanism is not known. In cardiac muscle, the ryanodine receptor (RyR2) on the sarcoplasmic reticulum releases the calcium required for muscle contraction. The FK506 binding protein (FKBP12.6) stabilizes RyR2, preventing aberrant activation of the channel during the resting phase of the cardiac cycle. We show that during exercise, RyR2 phosphorylation by cAMP-dependent protein kinase A (PKA) partially dissociates FKBP12.6 from the channel, increasing intracellular Ca(2+) release and cardiac contractility. FKBP12.6(-/-) mice consistently exhibited exercise-induced cardiac ventricular arrhythmias that cause sudden cardiac death. Mutations in RyR2 linked to exercise-induced arrhythmias (in patients with catecholaminergic polymorphic ventricular tachycardia [CPVT]) reduced the affinity of FKBP12.6 for RyR2 and increased single-channel activity under conditions that simulate exercise. These data suggest that "leaky" RyR2 channels can trigger fatal cardiac arrhythmias, providing a possible explanation for CPVT.     

Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor.

FK506-binding protein (FKBP12) has been found to be associated with the skeletal muscle ryanodine receptor (RyR1) (calcium release channel), whereas FKBP12.6, a novel isoform of FKBP, is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To assess the amino acid residues of FKBP12.6 that are critical for selective binding to RyR2, the residues of FKBP12.6 that differ with FKBP12 were mutated to the respective residues of FKBP12. RyR2 of cardiac sarcoplasmic reticulum, prelabeled by exchange with [35S]FKBP12.6, was used as assay system for binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W) of FKBP12.6 was found to lack selective binding to the cardiac RyR2, comparable with that of FKBP12.0. In complementary studies, mutations of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three amino acid residues (Gln31, Asn32, and Phe59) of human FKBP12.6 account for the selective binding to cardiac RyR2.     

Interaction of FKBP12.6 with the cardiac ryanodine receptor C-terminal domain

The ryanodine receptor-calcium release channel complex (RyR) plays a pivotal role in excitation-contraction coupling in skeletal and cardiac muscle. RyR channel activity is modulated by interaction with FK506-binding protein (FKBP), and disruption of the RyR-FKBP association has been implicated in cardiomyopathy, cardiac hypertrophy, and heart failure. Evidence for an interaction between RyR and FKBP is well documented, both in skeletal muscle (RyR1-FKBP12) and in cardiac muscle (RyR2-FKBP12.6), however definition of the FKBP-binding site remains elusive. Early reports proposed interaction of a short RyR central domain with FKBP12/12.6, however this site has been questioned, and recently an alternative FKBP12.6 interaction site has been identified within the N-terminal half of RyR2. In this study, we report evidence for the human RyR2 C-terminal domain as a novel FKBP12.6-binding site. Using competition binding assays, we find that short C-terminal RyR2 fragments can displace bound FKBP12.6 from the native RyR2, although they are unable to exclusively support interaction with FKBP12.6. However, expression of a large RyR2 C-terminal construct in mammalian cells encompassing the pore-forming transmembrane domains exhibits rapamycin-sensitive binding specifically to FKBP12.6 but not to FKBP12. We also obtained some evidence for involvement of the RyR2 N-terminal, but not the central domain, in FKBP12.6 interaction. Our studies suggest that a novel interaction site for FKBP12.6 may be present at the RyR2 C terminus, proximal to the channel pore, a sterically appropriate location that would enable this protein to play a central role in the modulation of this critical ion channel.     

The binding of the RyR2 calcium channel to its gating protein FKBP12.6 is oppositely affected by ARVD2 and VTSIP mutations

Arrhythmogenic right ventricular dysplasia/cardiomyopathy type 2 (ARVD2, OMIM 600996) and stress-induced polymorphic ventricular tachycardia (VTSIP, OMIM 604772) are two cardiac diseases causing juvenile sudden death, both associated with mutations in the RyR2 calcium channel. By using a quantitative yeast two-hybrid system, we show that VTSIP- and ARVD2-associated point mutations influence positively and negatively, respectively, the binding of RyR2 to its gating protein FKBP12.6. These findings suggest that ARVD2 mutations increase RyR2-mediated calcium release to cytoplasm, while VTSIP mutations do not affect significantly cytosolic calcium levels, thereby explaining the clinical differences between the two diseases. The present two-hybrid system appears to be an efficient molecular tool to assay the binding of FKBP12s proteins to both cardiac RyR2 and skeletal muscle RyR1 isoforms, circumventing the full-length expression of this class of giant channels. We also provide evidence of the suitability of this system to test new drugs that target RyRs-FKBP12s interactions and do not affect yeast growth.     

N-terminal region of FKBP12 is essential for binding to the skeletal ryanodine receptor

It is known that the two types of FK506-binding proteins FKBP12 and FKBP12.6 are tightly associated with the skeletal (RyR1) and cardiac ryanodine receptors (RyR2), respectively, and their interactions are important for channel functions of the RyR. In the case of cardiac muscle, three amino acid residues (Gln-31, Asn-32, and Phe-59) of FKBP12.6 could be essential for the selective binding to RyR2 (Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Fleischer, S. (1999) J. Biol. Chem. 274, 15315-15319). In this study to identify amino acid residues of FKBP12 that are important for the selective binding to RyR1, we mutated 9 amino acid residues of FKBP12 that differ from the counterparts of FKBP12.6 (Q3E, R18A, E31Q, D32N, M49R, R57A, W59F, H94A, and K105A), and we examined binding properties of these mutants to RyR1 by in vitro binding assay by using glutathione S-transferase-fused proteins of the mutants and Triton X-100-solubilized, FKBP12-depleted rabbit skeletal sarcoplasmic reticulum vesicles. Among the nine mutants tested, only Q3E and R18A lost their selective binding ability to RyR1. Furthermore, co-immunoprecipitation of RyR1 with 33 various mutants for the 9 positions produced by introducing different size, charge, and hydrophobicity revealed that an integration of the hydrogen bonds by the irreplaceable Gln-3 and the hydrophobic interactions by the residues Arg-18 and Met-49 could be a possible mechanism for the binding of FKBP12 to RyR1. Therefore, these results suggest that the N-terminal regions of FKBP12 (Gln-3 and Arg-18) and Met-49 are essential and unique for binding of FKBP12 to RyR1 in skeletal muscle.     

Ryanodine receptor oxidation causes intracellular calcium leak and muscle weakness in aging

Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24?months) rodents was oxidized, cysteine-nitrosylated, and depleted of the channel-stabilizing subunit calstabin1, compared to RyR1 from younger (3-6?months) adults. This RyR1 channel complex remodeling resulted in "leaky" channels with increased open probability, leading to intracellular calcium leak in skeletal muscle. Similarly, 6-month-old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month-old wild-type mice. Treating aged mice with S107 stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca(2+) release, muscle-specific force, and exercise capacity. Taken together, these data indicate that leaky RyR1 contributes to age-related loss of muscle function.     

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

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

Redox sensitivity of the ryanodine receptor interaction with FK506-binding protein

The ryanodine receptor (RyR) calcium release channel functions as a redox sensor that is sensitive to channel modulators. The FK506-binding protein (FKBP) is an important regulator of channel activity, and disruption of the RyR2-FKBP12.6 association has been implicated in cardiac disease. In the present study, we investigated whether the RyR-FKBP association is redox-regulated. Using co-immunoprecipitation assays of solubilized native RyR2 from cardiac muscle sarcoplasmic reticulum (SR) with recombinant [(35)S]FKBP12.6, we found that the sulfydryl-oxidizing agents, H(2)O(2) and diamide, result in diminished RyR2-FKBP12.6 binding. Co-sedimentation experiments of cardiac SR vesicles with [(35)S]FKBP12.6 also demonstrated that oxidizing reagents decreased FKBP binding. Matching results were obtained with skeletal muscle SR. Notably, H(2)O(2) and diamide differentially affected the RyR2-FKBP12.6 interaction, decreasing binding to approximately 75 and approximately 50% of control, respectively. In addition, the effect of H(2)O(2) was negligible when the channel was in its closed state or when applied after FKBP binding had occurred, whereas diamide was always effective. A cysteine-null mutant FKBP12.6 retained redox-sensitive interaction with RyR2, suggesting that the effect of the redox reagents is exclusively via sites on the ryanodine receptor. K201 (or JTV519), a drug that has been proposed to prevent FKBP12.6 dissociation from the RyR2 channel complex, did not restore normal FKBP binding under oxidizing conditions. Our results indicate that the redox state of the RyR is intimately connected with FKBP binding affinity.     

Subconductance states in single-channel activity of skeletal muscle ryanodine receptors after removal of FKBP12.

FKBP12 was removed from ryanodine receptors (RyRs) by incubation of rabbit skeletal muscle terminal cisternae membranes with rapamycin. The extent of FKBP12 removal was estimated by immunostaining Western blots of terminal cisternae proteins. Single FKBP12-depleted RyR channels, incorporated into planar lipid bilayers, were modulated by Ca2+, ATP, ryanodine, and ruthenium red in the cis chamber and opened frequently to the normal maximum conductance of approximately 230 pS and to substate levels of approximately 0.25, approximately 0.5, and approximately 0.75 of the maximum conductance. Substate activity was rarely seen in native RyRs. Ryanodine did not after the number of conductance levels in FKBP12-depleted channels, but, at a membrane potential of +40 mV, reduced both the maximum and the substate conductances by approximately 50%. FKBP12-stripped channels were activated by a 10-fold-lower [Ca2+] and inhibited by a 10-fold-higher [Ca2+], than RyRs from control-incubated and native terminal cisternae vesicles. The open probability (Po) of these FKBP12-deficient channels was greater than that of control channels at 0.1 microM and 1 mM cis Ca2+ but no different at 10 microM cis Ca2+, where channels showed maximal Ca2+ activation. The approximately 0.25 substate was less sensitive than the maximum conductance to inhibition by Ca2+ and was the dominant level in channels inhibited by 1 mM cis Ca2+. The results show that FKBP12 coordinates the gating of channel activity in control and ryanodine-modified RyRs.     

Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line

Of the three known ryanodine receptor (RyR) isoforms expressed in muscle, RyR1 and RyR2 have well-defined roles in contraction. However, studies on mammalian RyR3 have been difficult because of low expression levels relative to RyR1 or RyR2. Using the herpes simplex virus 1 (HSV-1) helper-free amplicon system, we expressed either RyR1 or RyR3 in 1B5 RyR-deficient myotubes. Western blot analysis revealed that RyR1- or RyR3-transduced cells expressed the appropriate RyR isoform of the correct molecular mass. Although RyR1 channels exhibited the expected unitary conductance for Cs(+) in bilayer lipid membranes, 74 of 88 RyR3 channels exhibited pronounced subconductance behavior. Western blot analysis with an FKBP12/12.6-selective antibody reveals that differences in gating behavior exhibited by RyR1 and RyR3 may be, in part, the result of lower affinity of RyR3 for FKBP12. In calcium imaging studies, RyR1 restored skeletal-type excitation-contraction coupling, whereas RyR3 did not. Although RyR3-expressing myotubes were more sensitive to caffeine than those expressing RyR1, they were much less sensitive to 4-chloro-m-cresol (CMC). In RyR1-expressing cells, regenerative calcium oscillations were observed in response to caffeine and CMC but were never seen in RyR3-expressing 1B5 cells. In [(3)H]ryanodine binding studies, only RyR1 exhibited sensitivity to CMC, but both RyR isoforms responded to caffeine. These functional differences between RyR1 and RyR3 expressed in a mammalian muscle context may reflect differences in association with accessory proteins, especially FKBP12, as well as structural differences in modulator binding sites.     

Characterization of the binding sites for the interactions between FKBP12 and intracellular calcium release channels

FKBP12, an FK506 binding protein, interacts with type 1 ryanodine receptor (RyR1) and modulates its calcium channel activity. However, there are many opposing reports of FKBP12's interaction with other related calcium channels, such as type 1 IP(3) receptor and type 3 ryanodine receptor (IP(3)R1 and RyR3). In addition, the involvement of the prolyl-dipeptide motif in the calcium channels and the corresponding binding residues in FKBP12 remain controversial. Through pulldown assays with recombinant proteins, we provide biochemical evidence of the interaction between FKBP12 and RyR1, RyR3 and IP(3)R1. Using NMR chemical shift mapping, we show that the important binding residues in FKBP12 are located in its hydrophobic FK506 binding region. Consistently, we demonstrate that FK506 can competitively inhibit the interaction between FKBP12 and the dipeptide motifs of the calcium channels. We believe our results shed lights on the binding mechanism of calcium channel-FKBP12 interaction.     

Structural characterization of the RyR1-FKBP12 interaction

The 12 kDa FK506-binding protein (FKBP12) constitutively binds to the calcium release channel RyR1. Removal of FKBP12 using FK506 or rapamycin causes an increased open probability and an increase in the frequency of sub-conductance states in RyR1. Using cryo-electron microscopy and single-particle image processing, we have determined the 3D difference map of FKBP12 associated with RyR1 at 16 A resolution that can be fitted with the atomic model of FKBP12 in a unique orientation. This has allowed us to better define the surfaces of close apposition between FKBP12 and RyR1. Our results shed light on the role of several FKBP12 residues that had been found critical for the specificity of the RyR1-FKBP12 interaction. As predicted from previous immunoprecipitation studies, our results suggest that Gln3 participates directly in this interaction. The orientation of RyR1-bound FKBP12, with part of its FK506 binding site facing towards RyR1, allows us to propose how FK506 is involved in the dissociation of FKBP12 from RyR1.     

Central domain of the human cardiac muscle ryanodine receptor does not mediate interaction with FKBP12.6

The immunophilin, FK506-binding protein (FKBP12), is an essential component of the ryanodine receptor channel complex of skeletal muscle (RyR1) and modulates intracellular calcium signaling from the nedoplasmic reticulum. The cardiac muscle RyR isoform (RyR2) specifically associates with a distinct FKBP isoform, FKBP12.6. Previous studies have led to the proposal that the central domain of RyR1 exclusively mediates the interaction with FKBP12. To characterize the topography of the FKBP 12.6 binding site on the human cardiac RyR2, we have applied complementary protein-protein interaction methods using both in vivo yeast two-hybrid analysis and in vitro immunoprecipitation experiments. Our results indicate an absence of interaction of FKBP12/12.6 with fragments containin the central domain of either RyR1, RyR2, or RyR3. Furthermore, no interaction was detected between FKBP12.6 with a series of overlapping fragments encompassing the entire RyR2, either individually or in multiple combination. We also found that a distinct, alternatively spliced variant of FKBP12.6 was unable to interact with RyR. In contrast, we successfully demonstrated a robust association between the cytoplasmic domain of transforming growth factor-β receptor type I and both FKBP12 and FKBP12.6 in parallel positive control experiments, as well as between native RyR2 and FKBP12.6. These results suggest that the specific interaction of FKBP12.6 with RyR2, and generally of FKBPs with any RyR isoform, is not readily reconstituted by peptide fragments corresponding to central RyR domains. Further structural analysis will be necessary to unravel this intricate signaling system and the current model of FKBP-12-RyR interaction via a single, central RyR, epitope may therefore require revision.     

Three-dimensional visualization of FKBP12.6 binding to an open conformation of cardiac ryanodine receptor

The cardiac isoform of the ryanodine receptor (RyR2) from dog binds predominantly a 12.6-kDa isoform of the FK506-binding protein (FKBP12.6), whereas RyR2 from other species binds both FKBP12.6 and the closely related isoform FKBP12. The role played by FKBP12.6 in modulating calcium release by RyR2 is unclear at present. We have used cryoelectron microscopy and three-dimensional (3D) reconstruction techniques to determine the binding position of FKBP12.6 on the surface of canine RyR2. Buffer conditions that should favor the "open" state of RyR2 were used. Quantitative comparison of 3D reconstructions of RyR2 in the presence and absence of FKBP12.6 reveals that FKBP12.6 binds along the sides of the square-shaped cytoplasmic region of the receptor, adjacent to domain 9, which forms part of the four clamp (corner-forming) structures. The location of the FKBP12.6 binding site on "open" RyR2 appears similar, but slightly displaced (by 1-2 nm) from that found previously for FKBP12 binding to the skeletal muscle ryanodine receptor that was in the buffer that favors the "closed" state. The conformation of RyR2 containing bound FKBP12.6 differs considerably from that depleted of FKBP12.6, particularly in the transmembrane region and in the clamp structures. The x-ray structure of FKBP12.6 was docked into the region of the 3D reconstruction that is attributable to bound FKBP12.6, to show the relative orientations of amino acid residues (Gln-31, Asn-32, Phe-59) that have been implicated as being critical in interactions with RyR2. A thorough understanding of the structural basis of RyR2-FKBP12.6 interaction should aid in understanding the roles that have been proposed for FKBP12.6 in heart failure and in certain forms of sudden cardiac death.