Selective binding of FKBP12.6 by the cardiac ryanodine receptor.

Anthony P Timerman,Hong-Bo Xin,Hitoshi Onoue,Greg Wiederrecht,Sebastian Barg,Julio Copello,Sidney Fleischer

doi:10.1074/jbc.271.34.20385

Abstract

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 35S-labeled FKBP12 and 35S-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 FKBP-stripped CSR. This differential action of FKBP may reflect a fundamental difference in the modulation of excitation-contraction coupling in heart versus skeletal muscle.

Highlights

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
We have reported that the cardiac calcium release channel/ ryanodine receptor (RyR-2) isolated from CHAPS-solubilized canine heart SR vesicles is tightly associated with FKBP-C (11)
Soluble FKBP12.6 Exchanges with FKBP Bound to RyR-2 of Cardiac and Skeletal Muscle—Cardiac microsomes (CSR)

Summary

EXPERIMENTAL PROCEDURES

Materials—L-683,590, a structural and functional analogue of FK506 was obtained from Merck Research Laboratories. The free FKBP was separated from the bound 35S-labeled FKBP isoform by diluting a 50-␮l sample (100 ␮g protein) into 200 ␮l of ice-cold buffer and immediately sedimenting the vesicles in a Beckman TL100.1 rotor at 95,000 rpm for 15 min at 4 °C. The RyR1⁄7GST/FKBP12.6 complex bound to the matrix was eluted by incubation for 10 min at room temperature with 1 bed volume of elution buffer (50 mM Tris-Cl, pH 8.0, 0.5 M KCl, 0.5% CHAPS, 2 ␮g/ml leupeptin, 2 mM dithiothreitol, and 0.1 M glutathione). The control (ethanol-treated vesicles) and drug-treated vesicles were sedimented and resuspended in buffer containing 150 mM KCl, 200 mM HEPES/KOH (pH ϭ 7.0), 60 nM of [3H]ryanodine, 1 mM HEDTA, and variable amounts of CaCl2 to obtain free [Ca2ϩ] ranging from 0.05 ␮M to 1 mM, as calculated by the IONS program (18). The Ca2ϩ uptake rate from each experiment was calculated from the average uptake rate of two pulses of Ca2ϩ

RESULTS AND DISCUSSION

Competition Binding Studies

Wiederrecht and Sidney Fleischer