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 (IpTx_A) binding but when IpTx_A bound first, it prevented ryanodol binding. Moreover, IpTx_A and PepA excluded each other from their sites. This suggests that IpTx_A 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 IpTx_A 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.