Abstract 794: Identification Of Target Domains Of The Cardiac Ryanodine Receptor To Correct Channel Disorder In Failing Hearts
We previously demonstrated that K201(JTV519) inhibits the Ca2+ leak by correcting the defective inter-domain interaction between N-terminal (0 – 600) and central regions (2000–2500) of the ryanodine receptor (RyR2) in failing hearts. Here, we identified the K201-binding domain and characterized the role of this novel domain on RyR2 channel gating. An assay using a quartz-crystal microbalance technique revealed that K201 specifically bound to recombinant RyR2 fragment:1741–2270, but not to other RyR2 fragments (∼500 amino acid residues) from the 1–2750 region. By further analysis of the fragment1741–2270, K201 was found to specifically bind to its sub-fragment2114 –2149. Using the peptide matching this sub-fragment (DP2114 –2149) as a carrier, the RyR2 was specifically labeled with methylcoumarin acetate (MCA). Moreover, of several recombinant RyR2 fragments, only fragment2234 –2750 was specifically MCA-labeled; this suggests that the K201 binding domain2114 –2149 binds with domain2234 –2750. Addition of DP2114 –2149 to the MCA-labeled SR interfered with the interaction between domain2114 –2149 and domain2234 –2750 causing domain unzipping, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. In failing cardiomyocytes (F; n=15), the frequency of spontaneous Ca2+ spark (CaSF:s−1·100μm−1) was markedly increased compared with normal (N; n=15) cardiomyocytes (F:2.7±0.4, N:0.9±0.3; p<0.01 vs N), whereas incorporation of DP2114 –2149 markedly decreased CaSF (1.3±0.3, p<0.01 vs F), the same effect as that produced by K201. In conclusion, we first identified the K201-binding site as domain2114 –2149 of RyR2. Interruption of the inter-domain interaction between the domain2114 –2149 and central domain2234 –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.