Structural Insights into the Nature of the Unique Anion Binding Site within the Cardiac Ryanodine Receptor N-Terminal Region and Associated Disease Mutations

Abstract

Ryanodine receptors (RyRs) are large intracellular calcium-release channels of the endo/sarcoplasmic reticulum that play a critical role in mediating excitation-contraction coupling. Mutations within the skeletal muscle isoform (RyR1) are associated with the pharmacogenetic disorder known as Malignant Hyperthermia (MH) as well as congenital myopathies. Concurrently, mutations within the cardiac (RyR2) isoform have been linked to life-threatening arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT), a potential precursor of sudden cardiac death. While recent high-resolution structures have provided some mechanistic insights as to how mutations may impact receptor function, this subject requires more work. The RyR1 amino-terminal region contains an elaborate ionic interaction network embedded between its three domains which is key to the protein's stability. Despite all the ionic interaction partners being conserved within RyR2, the replacement of two histidine residues with Tyr125 and Arg420 within RyR2 is enough to abolish the ionic network and introduce a unique central anion binding site within RyR2. Here, we present structural insights into the nature of the RyR2 anion binding site. A 2.6A crystal structure has been solved of the amino-terminal RyR2 region (residues 1-547) into which two histidines, Y125H and R420H, were introduced which abolished chloride binding but which failed to restore the ionic network of its counterpart in RyR1. The amino-terminal intrasubunit domain-domain interfaces of both RyR1 and RyR2 are each targeted by over twenty disease-associated mutations. Here, we provide the biochemical and structural characterization of a novel MH RyR1 mutation, H113Q, and a potential CPVT-causing mutant, RyR2 R420W, both of which reside at a domain-domain interface.