Ryanoids and imperatoxin affect the modulation of cardiac ryanodine receptors by dihydropyridine receptor Peptide A

Ca2+-entry via L-type Ca2+ channels (DHPR) is known to trigger ryanodine receptor (RyR)-mediated Ca2+-release from sarcoplasmic reticulum (SR). The mechanism that terminates SR Ca2+ release is still unknown. Previous reports showed evidence of Ca2+-entry independent inhibition of Ca2+ sparks by DHPR in cardiomyocytes. A peptide from the DHPR loop II-III (PepA) was reported to modulate isolated RyRs. We found that PepA induced voltage-dependent “flicker block” and transition to substates of fully-activated cardiac RyRs in planar bilayers. Substates had less voltage-dependence than block and did not represent occupancy of a ryanoid site. However, ryanoids stabilized PepA-induced events while PepA increased RyR2 affinity for ryanodol, which suggests cooperative interactions. Ryanodol stabilized Imperatoxin A (IpTxA) binding but when IpTxA bound first, it prevented ryanodol binding. Moreover, IpTxA and PepA excluded each other from their sites. This suggests that IpTxA generates a vestibular gate (either sterically or allosterically) that prevents access to the peptides and ryanodol binding sites. Inactivating gate moieties (“ball peptides”) from K+ and Na+ channels (ShakerB and KIFMK, respectively) induced well resolved slow block and substates, which were sensitive to ryanoids and IpTxA and allowed, by comparison, better understanding of PepA action. The RyR2 appears to interact with PepA or ball peptides through a two-step mechanism, reminiscent of the inactivation of voltage-gated channels, which includes binding to outer (substates) and inner (block) vestibular regions in the channel conduction pathway. Our results open the possibility that “ball peptide-like” moieties in RyR2-interacting proteins could modulate SR Ca2+ release in cells.