A Cryo-EM Based Study of An Equivalent N Terminal Domain Mutation in Skeletal and Cardiac Ryanodine Receptor (RyR)

Abstract

Mutations in ryanodine receptors (RyRs), large intracellular Ca2+ channels on the sarcoplasmic reticulum, are linked to disorders such as malignant hyperthermia and central core disease in the case of skeletal RyR1 isoform and catecholaminergic polymorphic ventricular tachycardia (CPVT) in the case of cardiac RyR2 isoform. RyRs are complex molecular machines whose activity is under precise regulation of interdomain interactions between the various subdomains as well as external binding partners. We examined equivalent single point mutations in the N-terminal A (NTD-A) domain of skeletal and cardiac RyR isoforms using cryo-EM to study their effect on the channel conformation and shed light on the possible underlying mechanism of leakiness. We resolved the 3D structures of RyR1 R164C and RyR2 R176Q at 3.5 Å and 3.9 Å, respectively. 3D analysis suggests that mutation R164C results in a tilting of the rhomboid structure (composed of NTDs, SPRY1-3, P1, HD1 and HD2 domains) indicative of a “primed” state in the absence of any activator ligands (such as Ca2+ or caffeine) and could be a result of disruption of the NTD-A interaction with the neighboring central domain. The effects of mutation R176Q in RyR2 were milder, suggesting isoform-specific effects on channel conformation despite the mutation being a loss of positively charged residue, arginine, in both isoforms. Analysis of these and other long-range conformational changes provides insight into the leaky phenotype of RyR1 and RyR2 produced by mutations situated far away (>80 Å) from the pore. Supported by grants AHA 19POST34430178 (K.A.I) and AHA 14GRNT19660003, MDA352845, NIH R01 AR068431, and NIH U24GM116789 (M.S.).