In situ modulation of the human cardiac ryanodine receptor (hRyR2) by FKBP12.6.
The ryanodine receptor complex (RyR), a large oligomeric assembly that functions as a Ca(2+)-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) (CHO(hRyR2)). Confocal microscopy of CHO(hRyR2) 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 Ca(2+) release in CHO(hRyR2) cells in response to stimulation by 4-chloro- m -cresol was in direct proportion to the expression levels of hRyR2. However, in CHO(hRyR2) cells co-expressing FKBP12.6, Ca(2+) release triggered by the addition of 4-chloro- m -cresol was markedly decreased. In contrast, co-expression of FKBP12 did not affect agonist-induced Ca(2+) release in CHO(hRyR2) cells. Resting cytoplasmic [Ca(2+)] in CHO(hRyR2) remained unaltered after co-expression of FKBP12 or FKBP12.6, but estimation of the ER Ca(2+) load status showed that co-expression of FKBP12.6, but not FKBP12, promoted superfilling of the ER Ca(2+) store which could not be released by RyR2 after agonist activation. The effects of FKBP12.6 on hRyR2-mediated intracellular Ca(2+) handling could be antagonized using rapamycin (5 microM). These results suggest that FKBP12.6 associates with hRyR2 in situ to modulate precisely the functionality of hRyR2 Ca(2+)-release channel.
George
Christopher H
CH
Department of Cardiology, Wales Heart Research Institute, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK. GeorgeCH@cardiff.ac.uk
Sorathia
Rina
R
Bertrand
Benedicte M A
BM
Lai
F Anthony
FA
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005455:Fluorescent Antibody Technique; D006801:Humans; D009206:Myocardium / Q000378:metabolism*; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins / Q000235:genetics / Q000378:metabolism*
D020033:Protein Isoforms; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D022061:Tacrolimus Binding Protein 1A; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B