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


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 (Ab(2132)). 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.
Yamamoto
Takeshi
T
Department of Medicine and Clinical Science, Division of Cardiology, Yamaguchi University Graduate School of Medicine, 1-1-1 Minamikogushi, Ube, Yamaguchi, 755-8505, Japan.
Yano
Masafumi
M
Xu
XiaoJuan
X
Uchinoumi
Hitoshi
H
Tateishi
Hiroki
H
Mochizuki
Mamoru
M
Oda
Tetsuro
T
Kobayashi
Shigeki
S
Ikemoto
Noriaki
N
Matsuzaki
Masunori
M
D000595:Amino Acid Sequence; D000818:Animals; D017304:Annexin A5 / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004195:Disease Models, Animal; D004285:Dogs; D006333:Heart Failure / Q000378:metabolism; D016014:Linear Models; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum; D017386:Sequence Homology, Amino Acid; D013841:Thiazepines / Q000378:metabolism
D017304:Annexin A5; D019837:Ryanodine Receptor Calcium Release Channel; D013841:Thiazepines; C109183:K201 compound; D002118:Calcium