Probing a putative dantrolene-binding site on the cardiac ryanodine receptor

Abstract

Dantrolene is an inhibitor of intracellular Ca2+ release from skeletal muscle SR (sarcoplasmic reticulum). Direct photoaffinity labelling experiments using [3H]azidodantrolene and synthetic domain peptides have demonstrated that this drug targets amino acids 590-609 [termed DP1 (domain peptide 1)] of RyR1 (ryanodine receptor 1), the skeletal muscle RyR isoform. Although the identical sequence exists in the cardiac isoform, RyR2 (residues 601-620), specific labelling of RyR2 by dantrolene has not been demonstrated, even though some functional studies show protective effects of dantrolene on heart function. Here we test whether dantrolene-active domains exist within RyR2 and if so, whether this domain can be modulated. We show that elongated DP1 sequences from RyR1 (DP1-2s; residues 590-628) and RyR2 (DP1-2c; residues 601-639) can be specifically photolabelled by [3H]azidodantrolene. Monoclonal anti-RyR1 antibody, whose epitope is the DP1 region, can recognize RyR1 but not RyR2 in Western blot and immunoprecipitation assays, yet it recognizes both DP1-2c and DP1-2s. This suggests that although the RyR2 sequence has an intrinsic capacity to bind dantrolene in vitro, this site may be poorly accessible in the native channel protein. To examine whether it is possible to modulate this site, we measured binding of [3H]dantrolene to cardiac SR as a function of free Ca2+. We found that > or =10 mM EGTA increased [3H]dantrolene binding to RyR2 by approximately 2-fold. The data suggest that the dantrolene-binding site on RyR2 is conformationally sensitive. This site may be a potential therapeutic target in cardiovascular diseases sensitive to dysfunctional intracellular Ca2+ release.

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

“…Dantrolene has also been found to improve intracellular Ca 2ϩ handling in hearts (25). It has been well established that the skeletal RyR1 and cardiac RyR2 are direct targets of dantrolene, as revealed by biochemical photoaffinity labeling experiments (34,35). Dantrolene binding appears to stabilize interactions between domains in the RyR1 molecule believed to be important for the functional regulation of the channel (20).…”

Dynamic, inter-subunit interactions between the N-terminal and central mutation regions of cardiac ryanodine receptor

“…Dantrolene and its more water-soluble analog, azumolene, have been shown to strongly inhibit DP4-induced unzipping of the domain-domain interactions, and stabilize the domain switch in the zipped configuration of RyR1 (Kobayashi et al, 2005). Dantrolene has little or no effect on normal RyR2 (Paul-Pletzer et al, 2001;PaulPletzer et al, 2005); however, it has an effect on diseased RyR2 (Paul-Pletzer et al, 2005;Tian et al, 1991). The dantrolene-binding site on RyR1 has been identified in an N-terminal region of the RyR1 sequence encompassing amino acids 590-609 (Paul-Pletzer et al, 2002).…”

“…Interestingly, this sequence is within the N-terminal mutation region. Since the dantrolene binding sequence is exactly the same in the two RyR isoforms (Kobayashi et al, 2009;PaulPletzer et al, 2005), a reasonable assumption is that the potentially active dantrolene binding site in RyR2 becomes accessible for drug binding only when the inter-domain interaction becomes defective (Paul-Pletzer et al, 2005). Consistent with this idea, we observed that both dantrolene and azumolene completely inhibited the FRET decrease in RyR2 S437-YFP/S2367-CFP that is caused by DPc10 (Fig.…”

Dantrolene prevents arrhythmogenic Ca2+release in heart failure

“…This stabilizing effect inhibits aberrant activation of the channel and prevents excessive Ca 2ϩ release from intracellular stores. Furthermore, dantrolene binds to the corresponding sequence (amino acids 601-620) of RyR2 (33,47) and has recently been proposed to have effects on cardiac RyR2 (24,50); however, no effect of the drug on the properties of native RyR2 channels has been observed in single channel analysis (12). Recent evidence has suggested that dantrolene may ameliorate abnormal RyR2-mediated Ca 2ϩ release associated with heart failure (HF) (24,31).…”