Localization of the 12.6-kDa FK506-binding Protein (FKBP12.6) Binding Site to the NH2-terminal Domain of the Cardiac Ca2+ Release Channel (Ryanodine Receptor)

Abstract

The 12.6-kDa FK506-binding protein (FKBP12.6) interacts with the cardiac ryanodine receptor (RyR2) and modulates its channel function. However, the molecular basis of FKBP12.6-RyR2 interaction is poorly understood. To investigate the significance of the isoleucineproline (residues 2427-2428) dipeptide epitope, which is thought to form an essential part of the FKBP12.6 binding site in RyR2, we generated single and double mutants, P2428Q, I2427E/P2428A, and P2428A/L2429E, expressed them in HEK293 cells, and assessed their ability to bind GST-FKBP12.6. None of these mutations abolished GST-FKBP12.6 binding, indicating that this isoleucine-proline motif is unlikely to form the core of the FKBP12.6 binding site in RyR2. To systematically define the molecular determinants of FKBP12.6 binding, we constructed a series of internal and NH 2 -and COOHterminal deletion mutants of RyR2 and examined the effect of these deletions on GST-FKBP12.6 binding. These deletion analyses revealed that the first 305 NH 2 -terminal residues and COOH-terminal residues 1937-4967 are not essential for GST-FKBP12.6 binding, whereas multiple sequences within a large region between residues 305 and 1937 are required for GST-FKBP12.6 interaction. Furthermore, an NH 2 -terminal fragment containing the first 1937 residues is sufficient for GST-FKBP12.6 binding. Co-expression of overlapping NH 2 and COOH-terminal fragments covering the entire sequence of RyR2 produced functional channels but did not restore GST-FKBP12.6 binding. These data suggest that FKBP12.6 binding is likely to be conformationdependent. Binding of FKBP12.6 to the NH 2 -terminal domain may play a role in stabilizing the conformation of this region.

Residue Gln4863 within a Predicted Transmembrane Sequence of the Ca2+ Release Channel (Ryanodine Receptor) Is Critical for Ryanodine Interaction

“…The proteins bound to the Sepharose beads were solubilized by the addition of 20 l of 2ϫ Laemmli sample buffer (24) plus 5% ␤-mercaptoethanol and boiled at 100°C for 5 min. The solubilized proteins (20 l) were separated by 6% SDS-PAGE (31). The SDS-PAGE-resolved proteins were transferred to nitrocellulose membranes at 45 V for 18 -20 h at 4°C in the presence of 0.01% SDS according to Towbin et al (32).…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…FKBP12 and FKBP12.6 (also known as calstabin 1 and 2, respectively) physically interact with all three isoforms of RyR but have different expression levels and binding affinity in different tissues (Chelu et al 2004). FKBP12 copurifies with RyR1 (Jayaraman et al 1992;Brillantes et al 1994) and FKBP12.6 copurifies with RyR2 (Timerman et al 1995;Timerman et al 1996;Barg et al 1997;Jeyakumar et al 2001;Masumiya et al 2003). Although somewhat controversial, a component of the FKBP12 binding site appears to be located between amino acids 2458 and 2468 of RyR1 (Rabbit sequence, SwissProt accession #P11716).…”

Modeling a Ryanodine Receptor N-terminal Domain Connecting the Central Vestibule and the Corner Clamp Region

“…The valine 2461 to proline 2462 motif in RyR1 (corresponding to isoleucine 2427 to proline 2428 in RyR2) has been identified as being required for FKBP binding by site-directed mutagenesis (33). However, the corresponding isoleucine 2427 to proline 2428 motif in RyR2 is not required for FKBP12.6 binding for RyR2 (34). Protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and affects the RyR2 channel open probability, and phosphorylation of RyR2 at a single residue serine 2808 (serine 2843 in RyR1) activates the channel by releasing FKBP (35,36).…”