Disparate Ryanodine Receptor Association with the FK506-binding Proteins in Mammalian Heart

Abstract

The FK506-binding proteins (FKBP12 and FKBP12.6) are accessory subunits of the ryanodine receptor-calcium release channel (RyR). Aberrant RyR2-FKBP12.6 interactions have been proposed to be the underlying cause of channel dysfunction in acquired and inherited cardiac disease. However, the stoichiometric RyR2 association with FKBP12/12.6 in mammalian heart is currently unknown. Here, we describe detailed quantitative analysis of cardiac RyR2:FKBP12/12.6 stoichiometry using immunoblot analysis and [3H]ryanodine binding assays, revealing striking disparities between four mammalian species. In mouse and pig heart, RyR2 is found complexed with both FKBP12 and FKBP12.6, although the former is the most abundant isoform. In rat heart, RyR2 is predominantly associated with FKBP12.6, whereas in rabbit it is associated with FKBP12 only. Co-immunoprecipitation experiments demonstrate RyR2-specific interaction with both FKBP isoforms in native cardiac tissue. Assuming four FKBP binding sites per RyR2 tetramer, only a small proportion of available sites are occupied by endogenous FKBP12.6. FKBP interactions with RyR2 are very strong and resistant to drug (FK506, rapamycin, cyclic ADPribose) and redox (H2O2, diamide) treatment. In contrast, the RyR1-FKBP12 association in skeletal muscle is readily disrupted under oxidative conditions. This is the first study to directly assess endogenous FKBP12 and FKBP12.6 associated with RyR2 in native cardiac tissue. Our results challenge the widespread perception that RyR2 associates exclusively with FKBP12.6 to near saturation, with important implications for the role of the FK506-binding proteins in RyR2 pathophysiology and cardiac disease.