FRET Detection of Calmodulin Binding and Structural Rearrangements Within the Cardiac RyR2 Calcium Release Channel

Abstract

Calmodulin (CaM) binds to a conserved domain of the ryanodine receptor isoforms expressed in skeletal muscle (RyR1) and cardiac muscle (RyR2) to evoke isoform-specific changes in channel gating. To better understand CaM's interactions with the RyR2 isoform, we are using fluorescence resonance energy transfer (FRET) to define the orientation and kinetics of CaM binding, and to resolve structural rearrangements linked to channel regulation. A FRET donor was targeted to the RyR2 cytoplasmic assembly by preincubating cardiac sarcoplasmic reticulum membranes with a fluorescent-labeled FKBP12.6 (F-FKBP). An acceptor fluorophore was attached within the N-lobe of CaM (F-CaM). A decrease in F-FKBP fluorescence upon addition of F-CaM provided a specific, real-time readout of CaM binding to the RyR2, despite the presence of additional non-RyR CaM targets in the cardiac membranes. FRET demonstrated that the affinity of F-CaM binding to RyR2 was greater in 100 μM than in 30 nM Ca2+. The maximal FRET observed in the presence of saturating [F-CaM] increased as a function of [Ca2+] (30 nM to 1 mM). The Ca2+ dependence of this increase in FRET was similar to the Ca2+ dependence of [3H]ryanodine binding to RyR2 assayed in equivalent media (KCa ∼5 μM). A marked decrease in FRET between FKBP12.6 and CaM was observed when the acceptor was shifted from CaM's N-lobe to CaM's C-lobe. We conclude that CaM binds to the RyR2 in an extended conformation, with its N-lobe oriented nearest to the FKBP12.6 subunit. CaM's conformation and orientation when bound to the RyR2 are therefore similar to what has been demonstrated previously for the RyR1 isoform (Cornea et al., 2009). Ca2+ dependent changes in FRET between FKBP12.6 and CaM may reflect structural changes within the RyR2 linked to channel activation by Ca2+.