Abstract
The ryanodine receptor/calcium release channel (CRC) of rabbit skeletal muscle terminal cisternae (TC) of sarcoplasmic reticulum (SR) has been found to be tightly associated with FK-506 binding protein (FKBP-12), the cytosolic receptor (immunophilin) for the immunosuppressant drug FK-506 (Jayaraman, T., Brillantes, A. M., Timerman, A. P., Fleischer, S., Erdjument-Bromage, H., Tempst, P., and Marks, A. (1992) J. Biol. Chem. 267, 9474-9477). In this study, a procedure is described to dissociate FKBP from TC and reconstitute human recombinant FKBP-12 back to the ryanodine receptor so that the role of the immunophilin on CRC activity can be assessed. Titration of TC vesicles with FK-506 dissociates FKBP from the ryanodine receptor. Sedimentation of FK-506-treated vesicles effectively separates the TC from the soluble FKBP-FK506 complex which remains in the supernatant. The FKBP-deficient TC vesicles have altered functional characteristics: 1) the ATP-stimulated calcium uptake rate of TC vesicles is reduced 2-fold; and 2) the threshold concentration of caffeine required to induce calcium release from TC vesicles is decreased. These changes appear to reflect modification of the calcium release channel since: 1) severalfold higher concentrations of FK-506 do not alter the calcium uptake rate of either longitudinal tubules of SR, or TC vesicles in the presence of ruthenium red; 2) human recombinant FKBP reassociates with FKBP-deficient TC but not with control TC or longitudinal tubules of SR; and 3) the reduced Ca2+ uptake rate in FKBP-deficient TC is restored to control values in the FKBP-reconstituted TC. These studies demonstrate that FKBP-12 modulates the CRC of rabbit skeletal muscle sarcoplasmic reticulum.
Highlights
From the Department of Molecular Biology, Vanderbilt University, Nashville Tennessee37235, the SMerck Research Laboratories, Rahway,New Jersey 07065-0900, and the TBrookdale Center for Molecular Biology and Molecular Medicine, Mount Sinai School of Medicine, New York, New York10021
The longitudinal tubules are of sarcoplasmic reticulum (SR) has been found to be composedlargely of calcium pumpmembrane involved in tightlayssociatewd itFhK-50b6indinpgrotein calcium uptake enabling muscles to relax [2]
FKBP-deficient TC vesicles have altered functional the receptor isa homotetramer which displays distinct 4-fold characteristics: 1)the ATP-stimulated calcium uptake symmetry asviewed by electron microscopy [11].The purified rate of TC vesicles is reduced 2-fold;and 2) the thresh-receptor reconstituted into planar lipid bilayers displays calold concentration of caffeine required to induce cal- cium ion channel activity which is modulated by drugs in a ciumreleasefrom TC vesicles is decreased.These manner that is consistent with the modulaotifocnalcium ion changes appear to reflect modification of the calcium fluxes observed in isolated SRvesicles [6, 12, 13]. release channelsince: 1)severalfold higher concentra- Cyclophilin andFKbindingprotein(FKBP-12)arethe tions of FK-506 do notalter the calcium uptake rate of cytosolic receptors for the potent immunosuppressant drugs either longitudinal tubulesof SR, orTC vesicles in the cyclosporin A and FK-506, respectively
Summary
0 1993 hy The American Society for Biochemistry and Molecular Biology, Inc. Vol 268,No 31,Issue of November 5,pp. 22992-22999,1993 Printed in U.S.A. Release channelsince: 1)severalfold higher concentra- Cyclophilin andFKbindingprotein(FKBP-12)arethe tions of FK-506 do notalter the calcium uptake rate of cytosolic receptors for the potent immunosuppressant drugs either longitudinal tubulesof SR, orTC vesicles in the cyclosporin A and FK-506, respectively. These receptor propresence of rutheniumred;2)humanrecombinant teins are referred to as immunophilins [14, 15]. The finding that FKBP-12 is tightly associated with the calcium release channel of rabbit skeletal muscle sarcoplasmic reticulum provides a system to study the role of this immunophilin in skeletal muscle excitation-contraction coupling. As shown in this report, thipsrocedure is useful to assess the role of FKBP12 on ryanodine receptor function
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