Pathological conformations of disease mutant Ryanodine Receptors revealed by cryo-EM

Kellie A Woll,Filip Van Petegem,Omid Haji-Ghassemi

doi:10.1038/s41467-021-21141-3

Kellie A Woll, Filip Van Petegem + Show 1 more

Open Access

https://doi.org/10.1038/s41467-021-21141-3

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Journal: Nature Communications	Publication Date: Feb 5, 2021
Citations: 37	License type: open-access

Affiliation: University of British Columbia

Abstract

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Hundreds of mutations are linked to malignant hyperthermia (MH), myopathies, and arrhythmias. Here, we explore the first MH mutation identified in humans by providing cryo-EM snapshots of the pig homolog, R615C, showing that it affects an interface between three solenoid regions. We also show the impact of apo-calmodulin (apoCaM) and how it can induce opening by bending of the bridging solenoid, mediated by its N-terminal lobe. For R615C RyR1, apoCaM binding abolishes a pathological ‘intermediate’ conformation, distributing the population to a mixture of open and closed channels, both different from the structure without apoCaM. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions.

Highlights

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum
We sought to answer basic questions around the pathophysiology of RyRs: do disease mutations alter the distribution between previously known conformations, or do they result in distinct pathological conformations? Previous experiments with short peptides have suggested the existence of pathological conformations, induced by disease mutations or abnormal conditions in the cell, resulting in altered interactions between different domains[13,14,15,16]
We capitalized on the availability of homozygous R615C pigs to obtain large quantities of R615C pig RyR1 for cryo-EM studies, as a representative for N-terminal disease hot spot mutations in RyR1 and RyR2

Summary

Introduction

Ryanodine Receptors (RyRs) are massive channels that release Ca2+ from the endoplasmic and sarcoplasmic reticulum. Comparisons show that the mutation primarily affects the closed state, inducing partial movements linked to channel activation. This shows that disease mutations can cause distinct pathological conformations of the RyR and facilitate channel opening by disrupting interactions between different solenoid regions. RyRs have mostly been studied in the context of cardiac and skeletal muscle tissue, where they release Ca2+ required for muscle contraction Their importance is underscored by the hundreds of sequence variants that have been linked to disease[2,3,4,5,6]. Most mutations cluster in three or four disease hot spots across the sequence Despite their different locations, functional investigation shows that the majority of mutations induce a gain-of-function phenotype, with increased opening and/or sensitivity to activating ligands such as caffeine and Ca2+. By virtue of adding Calmodulin, a well-known regulator of RyRs, we are able to obtain detailed comparisons of wild-type and disease mutant RyR1 in both the open and closed states

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