Mechanisms of RyR1 Modulation by General Anesthetics

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Description: The skeletal muscle ryanodine receptor (RyR1) is a critical regulator of cellular calcium to facilitate excitation contraction coupling. Mutations in this receptor underlie a number of serious disorders, one of which is called malignant hyperthermia (MH). MH typically occurs unexpectedly during a general anesthetic, and can be fatal. The intramolecular mechanism by which anesthetics modulate this receptor, or even if they interact directly with RyR1, is completely unknown. For both wild type and MH mutant (R615C) porcine RyR1, we will use photolabeling technology to determine whether and where two volatile anesthetics, isoflurane and sevoflurane, bind (Aim 1); then reconcile these sites with the structure and structural changes using cryo-electron microscopy (Aim 2). We will determine their single-channel effects on ion transit using planar lipid bilayer electrophysiology (Aim 3). Finally, we will gauge relative binding affinity among the expected multiple sites and which of these are most likely to be linked to functional effects, using sophisticated computational approaches (equilibrium molecular dynamics and alchemical free energy perturbation, Aim 4). We will test the effect of site-directed and natural mutations on binding affinity in individual sites, which will then be iteratively tested by experiment in Aims 1 and 3. The included preliminary data lends confidence for a successful outcome in each aim, and also has yielded surprising results in the case of propofol, an injectable general anesthetic. The RyR1 is a challenging target due to its size and complexity, but the technologies (photolabeling, mass spectrometry, cryo-electron microscopy, planar bilayer electrophysiology, molecular dynamics) have matured to a point where this multidisciplinary campaign has a very high probability of revealing the intramolecular mechanism of anesthetic modulation .