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

Abstract

SummaryThe FK506-binding proteins (FKBP12 and FKBP12.6; also known as FKBP1A and FKBP1B, respectively) are accessory subunits of the ryanodine receptor (RyR) Ca 2+ release channel. Aberrant RyR2-FKBP12.6 interactions have been proposed to be the underlying cause of channel dysfunction in acquired and inherited cardiac disease. However, the stoichiometry of the RyR2 association with FKBP12 or FKBP12.6 in mammalian heart is currently unknown. Here, we describe detailed quantitative analysis of cardiac stoichiometry between RyR2 and FKBP12 or FKBP12.6 using immunoblotting and [3 H]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 and cyclic ADPribose) and redox (H 2 O 2 and diamide) treatment. By contrast, the RyR1-FKBP12 association in skeletal muscle is readily disrupted under oxidative conditions. This is the first study to directly assess association of endogenous FKBP12 and FKBP12.6 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.

Non-ventricular, Clinical, and Functional Features of the RyR2R420Q Mutation Causing Catecholaminergic Polymorphic Ventricular Tachycardia

“…[20][21][22] Protein fractions (100 mg) were resuspended in SDS-PAGE loading buffer, and proteins were separated in a 4% SDS-PAGE gel strengthened with 0.5% agarose (for RyR). 21 Proteins from 4% gels were electrophoretically transferred to a polyvinylidenedifluoride membrane (Immobilon-P, Millipore) using a semi-dry transfer system (Trans-Blot SD, Bio-Rad) at 22 V for 4 h. Primary antibody specific for the RyR2 isoform (Ab1093, rabbit polyclonal antibody raised to RyR2 residues 4454-4474) was applied at 1:1000 dilution overnight at 4 8C. Immunoreactive protein bands were visualized negativo del 93%), todos ellos portadores de una mutació n heterocigota RyR2 R420Q , que estaba presente tambié n en el caso probando y en una chica joven sin prueba de esfuerzo, lo que corresponde a una penetrancia del 91% al final del seguimiento.…”

Effect of exercise training on Ca2+ release units of left ventricular myocytes of spontaneously hypertensive rats

“…The normal functioning of CRUs is essential to local control of Ca 2+ release during excitation-contraction coupling (ECC) (2) and is influenced by several factors including tight junction protein-protein interactions. One of these, the RyR2-FKBP12.6 interaction is important (3) because it occurs in around 45% of the available sites in the rat RyR2 (4). FKBP12.6 is anchored in the corner of the RyR2 homotetramer, forming a structural complex that stabilizes and regulates the closed state of these channels, preventing intracellular Ca 2+ leak and arrhythmogenesis (5).…”

Genetic and Biochemical Approaches for <em>In Vivo</em> and <em>In Vitro</em> Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

“…We routinely carry out co-IP experiments (Figure 3) following transient expression in a mammalian cell line (HEK293), as an independent biochemical assay to reinforce the Y2H findings [2][3][4][12][13][14][21][22][23][24] . To verify RyR2 N-terminus self-interaction, two separate plasmids encoding for RyR2 residues 1 -906 tagged with either the cMyc or HA peptide epitope (BT4L and AD4L, respectively), were co-transfected in HEK293 cells using the calcium phosphate precipitation method 3 .…”

“…Thus, recombinant proteins are often tagged with a peptide epitope, e.g., influenza hemagglutinin (YPYDVPDYA) or human cMyc (EQKLISEEDL), for which high-affinity specific antibodies are commercially available. If desired, the immunoprecipitating antibody can be chemically conjugated onto the Protein-A resin to avoid its elution and detection during the immunoblotting stage that may obscure the co-precipitated protein 16 ; to achieve this, we have successfully used the chemical cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) 24 . It is imperative that an appropriate detergent is included in the IP buffer and the insoluble material of homogenized cells is removed by centrifugation to minimize non-specific binding (step 3.1.3).…”