The ryanodine receptor complex (RyR), a large oligomeric assembly that functions as a Ca2+-release channel in the sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER), comprises four RyR subunits and four FK506-binding proteins (FKBP). The precise mode of interaction and modulation of the cardiac RyR (RyR2) channel by FKBP12/FKBP12.6 remains to be fully defined. We have generated a series of Chinese-hamster ovary (CHO) cell lines stably expressing discrete levels of recombinant human RyR2 (hRyR2) (CHOhRyR2). Confocal microscopy of CHOhRyR2 cells co-expressing either FKBP12 or FKBP12.6 demonstrated that FKBP12.6 was sequestered from the cytoplasm to ER membranes as the cellular levels of hRyR2 increased. There was negligible hRyR2-induced subcellular redistribution of FKBP12. The magnitude of Ca2+ release in CHOhRyR2 cells in response to stimulation by 4-chloro-m-cresol was in direct proportion to the expression levels of hRyR2. However, in CHOhRyR2 cells co-expressing FKBP12.6, Ca2+ release triggered by the addition of 4-chloro-m-cresol was markedly decreased. In contrast, co-expression of FKBP12 did not affect agonist-induced Ca2+ release in CHOhRyR2 cells. Resting cytoplasmic [Ca2+] in CHOhRyR2 remained unaltered after co-expression of FKBP12 or FKBP12.6, but estimation of the ER Ca2+ load status showed that co-expression of FKBP12.6, but not FKBP12, promoted superfilling of the ER Ca2+ store which could not be released by RyR2 after agonist activation. The effects of FKBP12.6 on hRyR2-mediated intracellular Ca2+ handling could be antagonized using rapamycin (5μM). These results suggest that FKBP12.6 associates with hRyR2 in situ to modulate precisely the functionality of hRyR2 Ca2+-release channel.
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March 2003
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Research Article|
March 01 2003
In situ modulation of the human cardiac ryanodine receptor (hRyR2) by FKBP12.6
Christopher H. GEORGE;
Christopher H. GEORGE
1
Department of Cardiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K.
1To whom correspondence should be addressed (e-mail GeorgeCH@cardiff.ac.uk).
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Rina SORATHIA;
Rina SORATHIA
Department of Cardiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K.
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Benedicte M.A. BERTRAND;
Benedicte M.A. BERTRAND
Department of Cardiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K.
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F. Anthony LAI
F. Anthony LAI
Department of Cardiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, U.K.
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Publisher: Portland Press Ltd
Received:
September 12 2002
Revision Received:
November 06 2002
Accepted:
November 21 2002
Accepted Manuscript online:
November 21 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 370 (2): 579–589.
Article history
Received:
September 12 2002
Revision Received:
November 06 2002
Accepted:
November 21 2002
Accepted Manuscript online:
November 21 2002
Citation
Christopher H. GEORGE, Rina SORATHIA, Benedicte M.A. BERTRAND, F. Anthony LAI; In situ modulation of the human cardiac ryanodine receptor (hRyR2) by FKBP12.6. Biochem J 1 March 2003; 370 (2): 579–589. doi: https://doi.org/10.1042/bj20021433
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