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 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) (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 Ca²⁺ 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, 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 CHO^hRyR2 cells. Resting cytoplasmic [Ca²⁺] in CHO^hRyR2 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.