The N-Terminal Disease Hot Spot of Ryanodine Receptors Forms a Cytoplasmic Vestibule

Abstract

Ryanodine receptors (RyR) are ion channels that govern the release of Ca2+ from the endoplasmic reticulum. They thus regulate the contraction of skeletal and cardiac muscle. Mutations in RyR can lead to severe genetic conditions, including (but not limited to) malignant hyperthermia (MH) and catecholaminergic polymorphic ventricular tachycardia (CPVT). Despite the detailed investigation of the functional effects of the mutations, locating their position in the full-length channel structure has traditionally proven to be difficult. Here we present the 2.5 Ångström resolution crystal structure of a region spanning most of the N-terminal disease hot spot (residues 1-559), containing over 50 disease mutations in RyR1 and RyR2. The hot spot consists of three domains that interact through a predominantly hydrophilic interface. We have been able to dock the position of this hot spot into various RyR1 cryo electron microscopy maps, allowing its unambiguous positioning in the cytoplasmic portion of the channel, forming a 240-kDa ring around the fourfold symmetry axis. The disease mutations can be grouped into three different categories, either destabilizing the interfaces between the three N-terminal domains, affecting the folding of individual domains, or affecting one of six interfaces with other RyR parts. We propose a model whereby the opening of RyR coincides with allosterically coupled motions within the N-terminal domains. This can be affected by mutations that target various interfaces within and across subunits. We also propose a mechanism whereby RyRs are activated by redox modification through the destabilization of observed domain-domain interfaces. The structure provides a framework to understand the many disease mutations that have been studied using functional methods.