Type 2 Ryanodine Receptor Domain A Contains a Unique and Dynamic α-Helix That Transitions to a β-Strand in a Mutant Linked with a Heritable Cardiomyopathy

d Ryanodine receptors (RyRs) are large tetrameric calcium (Ca2+) release channels found on the sarcoplasmic reticulum that respond to dihydropyridine receptor activity through a direct conformational interaction and/or indirect Ca2+ sensitivity, propagating sarcoplasmic reticulum lumina! Ca2+ release in the process of excitation contraction coupling. There are three human RyR subtypes, and several debilitating diseases are linked to heritable mutations in RyR1 and RyR2 including malignant hypothermia, central core disease, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Despite the recent appreciation that many disease-associated mutations within the N-terminal RyRABC domains (i.e., residues 1-559) are located in the putative interfaces mediating tetrameric channel assembly, the precise structural and dynamical consequences of the mutations are not well understood. We used solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography to examine the effect of ARVD2-associated (i.e., R176Q) and CPVT-associated [i.e., P164S, R1 69Q and delta exon 3 (8,3)] mutations on the structure and dynamics of RyR2A. Our solution NMR data exposed a mobile a-helix, unique to type 2; further, this a2 helix rescues the 13-strand lost in RyR2A A3 but remains dynamic in the hot-spot loop (HS-loop) P1 64S, R1 69Q and R1 76Q mutant proteins. Docking of our X-ray crystal/NMR hybrid structure into the RyR1 cryo-electron microscopy map revealed that this RyR2A a2 helix is in close proximity to dense "columns" projecting toward the channel pore. This is in contrast to the HS-loop mutations that cause structural changes largely localized to the intersubunit interface between adjacent ABC domains. Taken together, our data suggest that ARVD2 and CPVT mutations have at least two distinct structural consequences linked to channel dysfunction: perturbation of the HS-loop (i.e., domain A):domain B intersubunit interface and disruption of the communication between the N-terminal region and the channel domain. (C) 2013 Elsevier Ltd. All rights reserved.