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 Ca²⁺ leak in failing hearts and that K201 (JTV519) inhibits the Ca²⁺ 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^1741–2270, K201 was found to specifically bind to its subfragment^2114–2149. With the use of the peptide matching this subfragment (DP^2114–2149) 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 (Ab²¹³²). Moreover, of several recombinant RyR2 fragments, only fragment^2234–2750 was specifically MCA labeled; this suggests that the K201-binding domain^2114–2149 binds with domain^2234–2750. Addition of DP^2114–2149 to the MCA-labeled sarcoplasmic reticulum interfered with the interaction between domain^2114–2149 and domain^2234–2750, 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 Ca²⁺ spark was markedly increased compared with normal cardiomyocytes, whereas incorporation of DP^2114–2149 markedly decreased the frequency of spontaneous Ca²⁺ spark.

Conclusions—We first identified the K201-binding site as domain^2114–2149 of RyR2. Interruption of the interdomain interaction between the domain^2114–2149 and central domain^2234–2750 seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure and perhaps lethal arrhythmia.