Identification of target domains of the cardiac ryanodine receptor to correct channel disorder in failing hearts

Abstract

BACKGROUND - We previously demonstrated that defective interdomain interaction between N-terminal (0 to 600) and central regions (2000 to 2500) of ryanodine receptor 2 (RyR2) induces Ca leak in failing hearts and that K201 (JTV519) inhibits the Ca leak by correcting the defective interdomain interaction. In the present report, we identified the K201-binding domain and characterized the role of this novel domain in the regulation of the RyR2 channel. METHODS AND RESULTS - An assay using a quartz-crystal microbalance technique (a very sensitive mass-measuring technique) revealed that K201 specifically bound to recombinant RyR2 fragments 1741 to 2270 and 1981 to 2520 but not to other RyR2 fragments from the 1 to 2750 region (1 to 610, 494 to 1000, 741 to 1260, 985 to 1503, 1245 to 1768, 2234 to 2750). By further analysis of the fragment, K201 was found to specifically bind to its subfragment. With the use of the peptide matching this subfragment (DP) as a carrier, the RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) in a site-directed manner. After tryptic digestion, the major MCA-labeled fragment of RyR2 (155 kDa) was detected by an antibody raised against the central region (Ab). Moreover, of several recombinant RyR2 fragments, only fragment was specifically MCA labeled; this suggests that the K201-binding domain binds with domain. Addition of DP to the MCA-labeled sarcoplasmic reticulum interfered with the interaction between domain and domain, causing domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In failing cardiomyocytes, the frequency of spontaneous Ca spark was markedly increased compared with normal cardiomyocytes, whereas incorporation of DP markedly decreased the frequency of spontaneous Ca spark. CONCLUSIONS - We first identified the K201-binding site as domain of RyR2. Interruption of the interdomain interaction between the domain and central domain seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure and perhaps lethal arrhythmia. © 2008 American Heart Association, Inc.