Abstract 1090: Localization of the Mutually Interacting Regulatory Domains for Channel Gating within Ryanodine Receptor
Both CPVT and ARVC are mutation-linked diseases of cardiac ryanodine receptor (RyR2). DPc10, a synthetic peptide (Gly2460-Pro2495) of RyR2 (one of the mutable domains in CPVT), was found to mimic channel dysfunction in human CPVT, by acting competitively to reduce stabilizing interactions between the N-terminal1–600 and central2000–2500 domains of RyR2 (viz. domain unzipping). Here, we localized the mutually interacting domains within RyR2, and investigated the role of the inter-domain interactions on Ca2+ release function of RyR2. SR vesicles were isolated from dog LV muscles (n=5), then RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) using N-terminal or central domain peptide (DP163–195 or DP2460–2495), which includes single point mutation site in ARVC or CPVT, respectively, as a site-directing carrier. Both DP163–195 and DP2460–2495 mediated specific MCA fluorescence labeling of RyR2. After tryptic digestion, the DP163–195-MCA-labeled RyR2 fragment (170 kD) was detected by anti-central region antibody (Ab2163) but not by anti-N-terminal (Ab10) nor anti-C-terminal antibody (Ab5030), while DP2460–2495-MCA-labeled RyR2 fragment (150 kD) was detected only by the anti-N terminal antibody. To further localize the counter-domain of DP163–195, we synthesized human recombinant RyR2 fragments: 1245–1768, 1741–2270, 2234–2750. Of these fragments, only fragment 2234–2750 was specifically MCA-fluorescently labeled by using DP163–195 as a carrier. Incorporation of either DP163–195 or DP2460–2495 to normal cardiomyocytes (by protein delivery kit) induced diastolic Ca2+ spark, and delayed afterdeporalization-mediated Ca2+ transient. However, either Arg-to-Gln mutation (DP163–195mut) or Arg-to-Ser mutation (DP2460–2495mut) made in these domain peptides, mimicking the same human mutation in ARVC or CPVT, abolished all of these effects that would have been produced by these domain peptides. Thus, there is a tight interaction between N-terminal and central domains, in which several human ARVC or CPVT mutation sites are included. Either specific mutation of N-terminal or central domain leads to common defective inter-domain interaction, resulting in diastolic Ca2+ spark that may trigger ventricular arrhythmia.