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FK506 binding protein associated with the calcium release channel (ryanodine receptor)
T Jayaraman1, A M Brillantes, A P Timerman
1Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029.
Insights
A 12-kDa FK506-binding protein (FKBP12) tightly associates with the calcium release channel (CRC)/ryanodine receptor (RyRec) in skeletal muscle. This interaction suggests FKBP12 is a component of the RyRec complex, crucial for muscle function.
Area of Science:
- Muscle physiology
- Molecular biology
- Biochemistry
Background:
- The calcium release channel (CRC)/ryanodine receptor (RyRec) is essential for skeletal muscle excitation-contraction coupling.
- RyRec forms the foot structure connecting the sarcoplasmic reticulum (SR) to the T-tubules.
- Previous studies indicated RyRec is composed of four identical large protomers.
Purpose of the Study:
- To identify and characterize proteins associated with purified RyRec from rabbit skeletal muscle SR.
- To investigate the functional significance of any identified associated proteins in muscle physiology.
Main Methods:
- Protein purification of RyRec from rabbit skeletal muscle SR.
- N-terminal amino acid sequencing and cDNA cloning.
- Co-purification, co-immunoprecipitation, and subcellular localization studies.
Main Results:
- A 12-kDa protein, identified as FK506-binding protein (FKBP12), was found tightly associated with purified RyRec.
- FKBP12 and RyRec co-purified through multiple chromatography steps.
- Co-immunoprecipitation and subcellular localization confirmed the association of FKBP12 with RyRec in the SR terminal cisternae.
- The molar ratio suggests one FKBP12 molecule per RyRec complex (1:4 ratio).
Conclusions:
- FKBP12 is a tightly associated component of the skeletal muscle calcium release channel (RyRec).
- This interaction is significant for the structure and function of the RyRec complex.
- The findings provide new insights into the molecular composition of the excitation-contraction coupling machinery.
Abstract:
The calcium release channel (CRC)/ryanodine receptor (RyRec) has been identified as the foot structure of the sarcoplasmic reticulum (SR) and provides the pathway for calcium efflux required for excitation-contraction coupling in skeletal muscle. The CRC has previously been reported to consist of four identical 565-kDa protomers. We now report the identification of a 12-kDa protein which is tightly associated with highly purified RyRec from rabbit skeletal muscle SR. N-terminal amino acid sequencing and cDNA cloning demonstrates that the 12-kDa protein from fast twitch skeletal muscle is the binding protein for the immunosuppressant drug FK506. In humans, FK506 binds to the 12-kDa FK506-binding protein (FKBP12) and blocks calcium-dependent T cell activation. We find that FKBP12 and the RyRec are tightly associated in skeletal muscle SR on the basis of: 1) co-purification through sequential heparin-agarose, hydroxylapatite, and size exclusion chromatography columns; 2) coimmunoprecipitation of the RyRec and FKBP12 with anti-FKBP12 antibodies; and 3) subcellular localization of both proteins to the terminal cisternae of the SR, and not in the longitudinal tubules of SR, in fast twitch skeletal muscle. The molar ratio of FKBP12 to RyRec in highly purified RyRec preparations is approximately 1:4, indicating that one FKBP12 molecule is associated with each calcium release channel/foot structure.