THE CALCIUM-RELEASE CHANNEL OF SARCOPLASMIC-RETICULUM IS MODULATED BYFK-506-BINDING PROTEIN - DISSOCIATION AND RECONSTITUTION OF FKBP-12 TO THE CALCIUM-RELEASE CHANNEL OF SKELETAL-MUSCLE SARCOPLASMIC-RETICULUM

The ryanodine receptor/calcium release channel (CRC) of rabbit skeleta l muscle terminal cisternae (TC) of sarcoplasmic reticulum (SR) has be en found to be tightly associated with FK-506 binding protein (FKBP-12 ), the cytosolic receptor (immunophilin) for the immunosuppressant dru g 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 dis sociate FKBP from TC and reconstitute human recombinant FKBP-12 back t o the ryanodine receptor so that the role of the immunophilin on CRC a ctivity can be assessed. Titration of TC vesicles with FK-506 dissocia tes FKBP from the ryanodine receptor. Sedimentation of FK-506-treated vesicles effectively separates the TC from the soluble FKBP-FK506 comp lex which remains in the supernatant. The FKBP-deficient TC vesicles h ave altered functional characteristics: 1) the ATP-stimulated calcium uptake rate of TC vesicles is reduced 2-fold; and 2) the threshold con centration of caffeine required to induce calcium release from TC vesi cles is decreased. These changes appear to reflect modification of the calcium release channel since: 1) severalfold higher concentrations o f FK-506 do not alter the calcium uptake rate of either longitudinal t ubules of SR, or TC vesicles in the presence of ruthenium red; 2) huma n recombinant FKBP reassociates with FKBP-deficient TC but not with co ntrol 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-r econstituted TC. These studies demonstrate that FKBP-12 modulates the CRC of rabbit skeletal muscle sarcoplasmic reticulum.