Binding of RyR2 "Unzipping" Peptide in Cardiomyocytes Activates RyR2 and Reciprocally Inhibits Calmodulin Binding
Rationale: One hypothesis for elevated Ca2+ leak through cardiac ryanodine receptors (RyR2) in heart failure (HF) is interdomain "unzipping" that can enhance aberrant channel activation. A peptide (DPc10) corresponding to RyR2 central domain 2460-2495 recapitulates this arrhy-thmogenic RyR2 leakiness by unzipping N- and central-domains. Calmodulin (CaM) and FK-506 binding protein (FKBP12.6) bind to RyR2 and stabilize the closed channel. Little is known about DPc10 binding to the RyR2 and how that may interact with binding (and effects) of CaM and FKBP12.6 with RyR2.
Objective: Measure, directly in cardiac myocytes, the kinetics and binding affinity of DPc10 to RyR2 and how that affects RyR2 interaction with FKBP12.6 and CaM.
Methods and Results: We used permeabilized rat ventricular myocytes, and fluorescently-labeled DPc10, FKBP12.6, and CaM. DPc10 access to its binding site is extremely slow in resting RyR2, but accelerated by promoting RyR opening or unzipping (by unlabeled DPc10). RyR2-bound CaM (but not FKBP12.6) drastically slowed DPc10 binding. Conversely, DPc10 binding significantly reduced CaM (but not FKBP12.6) binding to the RyR2. Fluorescence resonance energy transfer measurements indicate that DPc10 and CaM binding sites are separate and allow triangulation of the structural DPc10 binding locus on RyR2 vs. FKBP12.6 and CaM binding sites.
Conclusions: DPc10-RyR2 binding is sterically limited by the resting zipped RyR2 state. CaM binding to RyR2 stabilizes this zipped state, while RyR2 activation or prebound DPc10 enhances DPc10 access. DPc10 and CaM binding sites are distinct but allosterically interacting RyR2 sites. Neither DPc10 nor FKBP12.6 influences RyR2 binding of the other.
- Received October 11, 2012.
- Revision received November 21, 2012.
- Accepted December 11, 2012.
- Copyright © 2012, Circulation Research