The mechanism of Ca2' release from sarcoplasmic reticulum, which triggers contraction in skeletal muscle, remains the key unresolved problem in excitation-contraction coupling. Recently, we have described the isolation of purified fractions referable to terminal and longitudinal cisternae of sarcoplasmic reticulum. Junctional terminal cisternae are distinct in that they have a low net energized Ca2+ loading, which can be enhanced 5-fold or more by addition of ruthenium red. The loading rate, normalized for calcium pump protein content, then approaches that of longitudinal cisternae of sarcoplasmic reticulum. We now find that the ruthenium red-enhaniced Ca2+ loading rate can be blocked by the previous addition of ryanodine. The inhibition constant is in the nanomolar range (20-180 nM). Ryanodine and ruthenium red have no effect on the Ca21 loading rate of longitudinal cisternae. Direct binding studies with [3HJryano-dine localized the receptors to the terminal cisternae and not to longitudinal cisternae. Scatchard analysis of the binding data gives a dissociation constant for ryanodine in the range of the drug action on the terminal cisternae ("100 nM range) with approximately 4 to 20 pmol bound per mg of protein.Ryanodine is known to be toxic in animals, leading to irreversible muscle contractures. These studies provide evidence on the mode of action of ryanodine and its localization to the terminal cisternae. The low concentration at which the drug is effective appears to account for its toxicity. Ryanodine locks the Ca2+ release channels in the "open state," so that Ca2+ is not reaccumulated and the muscle fiber cannot relax.

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The ryanodine receptor/calcium release channel (RyR1) of sarcoplasmic reticulum from rabbit skeletal muscle terminal cisternae (TC) contains four tightly associated FK506-binding proteins (FKBP12). Dissociation and reconstitution studies have shown that RyR1 can be modulated by FKBP12, which helps to maintain the channel in the quiescent state. In this study, we found that the association of FKBP with RyR1 of skeletal muscle is common to each of the five classes of vertebrates. TC from skeletal muscle representing animals from different vertebrates, i.e. mammals (rabbit), birds (chicken), reptiles (turtle), fish (salmon and rainbow trout), and amphibians (frog), were isolated. For each, we find the following: 1) FKBP12 is localized to the TC (there are four FKBP binding sites/ryanodine receptor); 2) soluble FKBP exchanges with the bound form on RyR1 of TC; 3) release of FKBP from terminal cisternae by drug (FK590) treatment leads to a significant reduction in the net calcium loading rate, consistent with channel activation (the calcium loading rate is restored to the control value by reconstitution with FKBP12); and 4) RyR1 of skeletal muscle TC can bind to and exchange with either FKBP12 or FKBP12.6 (FKBP12.6 is the novel FKBP isoform found selectively associated with RyR2 of dog cardiac sarcoplasmic reticulum). We conclude that FKBP is an integral part of the RyR1 of skeletal muscle in each of the classes of vertebrate animals. The studies are consistent with a role for FKBP in skeletal muscle excitation-contraction coupling.FK506 is a powerful immunosuppressive drug that prevents T-cell activation and is thereby used to prevent allograft rejection following transplant surgery. FK506-binding protein (FKBP12), 1 the cytosolic receptor for FK506, has a molecular mass of 11.8 kDa and is widely expressed in eukaryotic cells and tissues, predominantly in the cytosol. The sequence is highly conserved throughout eukaryotic phylogeny (1, 2).In rabbit skeletal muscle, FK-binding protein is bound to the ryanodine receptor of terminal cisternae of SR (3-10), in a stoichiometry of four FKBP/ryanodine receptor (4, 8); i.e. for mammalian skeletal muscle, the ryanodine receptor is a hetero-oligomer with a structural formula of (RyR1 protomer) 4 (FKBP12) 4 . Although FKBP is tightly bound to the RyR of terminal cisternae of SR, soluble FKBP readily exchanges with FKBP12 on the RyR in TC (6). The ryanodine receptor of heart SR is also associated with FKBP (7) in a stoichiometry of 4 (8), albeit with a novel isoform, i.e. FKBP12.6. The latter differs from FKBP12 by 18 of 108 amino acids (8). Recently, we found that the ryanodine receptor from skeletal muscle binds to and exchanges with both FKBP12 and FKBP12.6, whereas the RyR2 of heart binds to and exchanges only with FKBP12.6 (9). In vitro reconstitution studies indicate that FKBP12 modulates the channel function of the ryanodine receptor of skeletal muscle (RyR1) (4, 5, 10). In this regard, FKBP stabilizes the closed conformation of the skeletal muscle ryanodine receptor. Im...

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