The calcium release channels (CRC)/ryanodine receptors of skeletal (Sk) and cardiac (C) muscle sarcoplasmic reticulum (SR) are hetero-oligomeric complexes with the structural formulas (ryanodine recepter (RyR)1 protomer) 4 (FKBP12) 4 and (RyR2 protomer) 4 (FKBP12.6) 4 , respectively, where FKBP12 and FKBP12.6 are isoforms of the 12-kDa receptor for the immunosuppressant drug FK506. The sequence similarity between the RyR protomers and FKBP12 isoforms is 63 and 85%, respectively. Using 35 S-labeled FKBP12 and 35 S-labeled FKBP12.6 as probes to study the interaction with CRC, we find that: 1) analogous to its action in skeletal muscle sarcoplasmic reticulum (SkMSR), FK506 (or analog FK590) dissociates FKBP12.6 from CSR; 2) both FKBP isoforms bind to FKBP-stripped SkMSR and exchange with endogenously bound FKBP12 of SkMSR; and 3) by contrast, only FKBP12.6 exchanges with endogenously bound FKBP12.6 or rebinds to FKBP-stripped CSR. This selective binding appears to explain why the cardiac CRC is isolated as a complex with FKBP12.6, whereas the skeletal muscle CRC is isolated as a complex with FKBP12, although only FKBP12 is detectable in the myoplasm of both muscle types. Also, in contrast to the activation of the channel by removal of FKBP from skeletal muscle, no activation is detected in CRC activity in FKBPstripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.FK506 is a potent immunosuppressive drug that binds to a family of related intracellular receptors termed FK506-binding proteins, varying in size from 12 to 54 kDa. Among these FKBPs, 1 FKBP12 is the most abundant and is involved in mediating the immunosuppressive action of FK506 in T lymphocytes. All of the known FKBP family members display cis-trans peptidyl-prolyl isomerase (PPIase) activity that is inhibited by FK506 and a structurally related compound, rapamycin (1). However, neither the immunosuppressive nor toxic side effects (including severe neuro-and nephrotoxicity) associated with FK506 therapy result from the inhibition of PPIase activity. Rather, the action of FK506 results from the specific inhibition of calcineurin by the FKBP12⅐drug complex. Calcineurin is a calcium-dependent protein phosphatase involved in the activation of T-lymphocytes (2). FKBP12 is a mere bystander protein during T-cell activation that becomes involved in blocking activation after it binds FK506. To date, the only physiological function directly associated with FKBP12 is its tight binding to (3, 4) and modulation of the ryanodine receptor (RyR-1) or calcium release channel of skeletal muscle SR (5-10). The purified SkM CRC 2 isolated from the CHAPS-solubilized TC is a hetero-oligomeric complex, with the structural formula of (RyR-1 protomer) 4 (FKBP12) 4 (5). Both FK506 and rapamycin bind to and dissociate FKBP12 from the SkM CRC (5). Both the native and FKBP-stripped CRCs have similar unitary conductance and sensitivity to ruthenium red (6). However, in co...

Abstract. The calcium release channel (CRC) from skeletal muscle is an unusually large tetrameric ion channel of the sarcoplasmic reticulum, and it is a major component of the triad junction, the site of excitation contraction coupling. The three-dimensional architecture of the CRC was determined from a random conical tilt series of images extracted from electron micrographs of isolated detergent-solubilized channels prepared in a frozen-hydrated state. Three major classes of fourfold symmetric images were identified, and three-dimensional reconstructions were determined for two of these. The two independent reconstructions were almost identical, being related to each other by a 180 ° rotation about an axis in the plane of the specimen grid.The CRC consists of a large cytoplasmic assembly (29 x 29 x 12 nm) and a smaller transmembrane assembly that protrudes 7 nm from one of its faces. A cylindrical low-density region, 2-3 nm in apparent diameter, extends down the center of the transmembrane assembly, and possibly corresponds to the transmembrane Ca2+-conducting pathway. At its cytoplasmic end this channel-like feature appears to be plugged by a globular mass of density. The cytoplasmic assembly is apparently constructed from 10 or more domains that are loosely packed together such that greater than 50% of the volume enveloped by the assembly is occupied by solvent. The cytoplasmic assembly is suggestive of a scaffolding and seems well adapted to maintain the structural integrity of the triad junction while allowing ions to freely diffuse to and away from the transmembrane assembly.I N striated muscle the calcium release channel (CRC) ~, also known as the ryanodine receptor, plays a key role in excitation-contraction coupling, the process by which neuronal-induced depolarization of the sarcolemma leads to release of calcium from the lumen of the sarcoplasmic reticulum (SR). The CRC is an intracellular integral membrane protein of the SR (for reviews see Fleischer and Inui, 1989;McPherson and Campbell, 1993). Depolarization of the sarcolemma causes the CRC to open by mechanisms (apparently different in cardiac and skeletal muscle) whose elucidation represents the central remaining problem in understanding excitation-contraction coupling.Several groups succeeded in purifying the CRC from skeletal muscle, and in reconstituting Ca 2+ channel activity in lipid bilayers (Inui et al

FK506, an immunosuppressant that prolongs allograft survival, is a co-drug with its intracellular receptor, FKBP12. The FKBP12⅐FK506 complex inhibits calcineurin, a critical signaling molecule during T-cell activation. FKBP12 was, until recently, the sole FKBP known to mediate calcineurin inhibition at clinically relevant FK506 concentrations. The best characterized cellular function of FKBP12 is the modulation of ryanodine receptor isoform-1, a component of the calcium release channel of skeletal muscle sarcoplasmic reticulum.Recently, a novel protein, FKBP12.6, was found to inhibit calcineurin at clinically relevant FK506 concentrations. We have cloned the cDNA encoding human FKBP12.6 and characterized the protein. In transfected Jurkat cells, FKBP12.6 is equivalent to FKBP12 at mediating the inhibitory effects of FK506. Upon binding rapamycin, FKBP12.6 complexes with the 288-kDa mammalian target of rapamycin. In contrast to FKBP12, FKBP12.6 is not associated with ryanodine receptor isoform-1 but with the distinct ryanodine receptor isoform-2 in cardiac muscle sarcoplasmic reticulum. Our results suggest that FKBP12.6 has both a unique physiological role in excitation-contraction coupling in cardiac muscle and the potential to contribute to the immunosuppressive and toxic effects of FK506 and rapamycin.FK506 (tacrolimus) is a powerful immunosuppressive drug for treating graft rejection and autoimmune disorders. Rapamycin (RAP, 1 sirolimus) is an immunosuppressant structurally-related to FK506 but with a distinct mechanism of action. Both drugs bind to a family of intracellular receptors, the FK506 binding proteins (FKBPs), whose members include FKBPs 12, 12.6, 13, 25, 51, and 52 (for review, see Ref. 1). All FKBPs are peptidyl-prolyl isomerases, catalyzing the cis-trans isomerization of peptidyl-prolyl bonds in peptides and proteins, an activity inhibited by both FK506 and RAP.Peptidyl-prolyl isomerase inhibition is unrelated to immunosuppression. FK506 and RAP gain function upon binding FKBP12. The FKBP12⅐FK506 and FKBP12⅐RAP complexes are the actual immunosuppressive species whose targets are calcineurin (CaN) and the mammalian target of RAP (mTOR), respectively (for review, see Refs. 1 and 2). CaN is a Ca 2ϩ -dependent, serine-threonine phosphatase required during the commitment phase (G 0 3 G 1 ) of T-cell activation (3). Inhibition of CaN blocks the nuclear translocation of transcription factors such as nuclear factor of activated T-cells and NF-B, controlling the expression of cytokine genes whose products are required for immune response coordination (for review, see Ref.2). RAP, unlike FK506, does not block lymphokine production but inhibits the T-cell proliferative response to cytokines by blocking G 1 3 S-phase progression. The function of mTOR, a 288-kDa protein related to phosphatidylinositol kinases, is unknown.CaN is a ubiquitous protein, and its inhibition at unwanted sites is most responsible for the toxicity associated with FK506 therapy (4). That immunosuppression and toxicity are mechanistica...

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