Investigation of Mutant Ryanodine Receptor Channel Activity using Functional Analysis and Molecular Dynamics

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Membrane depolarization is translated into intracellular Ca2+ signals, and ryanodine receptors (RyRs), located in the sarcoplasmic/endoplasmic reticulum membrane, play a key role in intracellular Ca2+ release. There are three isoforms of RyRs, of which type 1 RyR (RyR1) is dominantly expressed in the skeletal muscle. Mutations in the RyR1 gene (RyR1) cause severe muscle diseases, such as malignant hyperthermia (MH), which is a disorder of Ca2+-induced Ca2+ release via the RyR1 in skeletal muscle. So far, more than 300 mutations have been reported in RyR1 of patients with MH, and most of those mutations have been found in three “hot spot” regions of RyR1. However, due to lack of comprehensive analysis of the structure-function relationship of mutant RyR1 Ca2+ release channels, the mechanism remains largely unknown. Here, we combined functional studies and molecular dynamics (MD) simulation of RyR1 channels carrying disease-associated mutations at the N-terminal region. When expressed in HEK293 cells, those mutant RyR1 Ca2+ release channels caused abnormalities in Ca2+ homeostasis. MD simulation of the mutant RyR1 revealed that hydrogen bonds/salt bridges between subdomains strongly correlate with the channel function of RyR1. Especially, the mutations of Arg402, which plays key role in connecting three subdomains (A, B, C) of the N-terminal region, exhibit interesting result. Indeed, B and C subdomains together rotate clockwise with respect to A subdomain in the mutants. This movement may increase the open probability of the channel, and this increase may be the main cause of MH in the Arg402 mutants.