Relative Affinities of General Anesthetics for Experimentally Identified Binding Sites in Ryanodine Receptors (RyR1)

Malignant hyperthermia (MH) is a pharmacogenetic disorder that manifests as a hypermetabolic response to certain anesthetic agents (halothane, isoflurane, sevoflurane, desflurane, succinylcholine). A defective or disordered Calcium ion channel, ryanodine receptor 1 (RyR1), located in the sarcoplasmic reticulum (SR) membrane, underlies MH susceptibility. RyR1, the largest known ion channel, is responsible for the rapid release of Ca2+ ions from SR into the cytoplasm, a key event that triggers skeletal muscle contraction. It has a homotetrameric organization and approximately 5000 residues in each protomer. There are a large number (>100) of known mutations in RyR1 or its binding partners which are associated with MH sensitivity. In the presence of a triggering agent and an MH-causative mutation, the channel may be biased open, allowing uncontrolled Ca2+ release, generating an extreme metabolic load. In order to understand how MH is triggered mechanistically by anesthetics, we use molecular dynamics simulations to study experimentally-identified binding sites in specific domains of the channel and alchemical free energy perturbation calculations to evaluate binding configurations and affinities. We computationally refine residue-level binding sites identified by photoaffinity labeling for selected anesthetic ligands on RyR1. We further evaluate MH-causative mutations that may precipitate an increase or decrease in the binding affinity of anesthetic molecules to RyR1. Evaluating and comparing binding sites of MH-causative anesthetics, in wild-type and mutated RyR1, helps us understand combinations of an RYR1 mutation and a bound anesthetic configuration that bias an open conformation of the channel and trigger MH.