FRET-based Mapping of Regions involved in FKBP12.6 Binding to the Type 1 Ryanodine Receptor

Abstract

The type 1 ryanodine receptor (RyR1) is an intracellular calcium channel that plays an integral role in skeletal muscle excitation-contraction coupling. The functional properties of this protein are regulated by numerous associated proteins, including FKBP12, a skeletal muscle protein that stabilizes full RyR1 channel gating. While the binding site of FKBP12 was originally identified at position 2458 of RyR1, subsequent reports have localized additional binding determinants within the N-terminal domain of RyR1. In this study, we used fluorescence resonance energy transfer (FRET) to determine the orientation and proximity of FKBP relative to sites either within the N-terminal domain or near amino acid position 2458. We utilized the cardiac muscle isoform FKBP12.6 (which binds to RyR1 at the same site as FKBP12 but with higher affinity and stability) labeled with the FRET donor Alexa Fluor 488 at either position 14, 44, 49, or 85. Specific binding of each labeled FKBP to RyR was confirmed in permeabilized HEK-293T cells expressing wtRyR1. Weak energy transfer was observed from FKBP12.6 to the FRET acceptor Cy3NTA, which was targeted to poly-histidine “tags” placed in the N-terminal domain. This result suggested that the acceptors were not proximal (∼90A away) to FKBP12.6 bound to RyR1. Next, we measured FRET from FKBP12.6 to Cy3NTA targeted to positions 2157, 2341, 2502 and 2777 of RyR1. FKBP12.6 binding to constructs containing His-tags at positions 2157 and 2502 was significantly reduced relative to binding to wtRyR1. In contrast, we detected strong and weak FRET efficiencies from FKBP12.6 to positions 2341 and 2777, respectively. These data suggest that FKBP12.6 binds near amino acid position 2341 but far from the N-terminal domain of RyR1. Supported by NIH grant R01AR059124 (to JDF and TG) and R01HL092097 (to RLC).