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 .

Public Health Relevance

When undergoing surgery, some patients can have a life-threatening condition known as `Malignant Hyperthermia ?. It arises in people who have mutations in a gene known as ?RyR1?, and the general anesthetic used for the surgery is usually what triggers MH. In this proposal, we aim to find out how general anesthetics interact with the RyR1 protein and how this interaction and the mutations affect its 3D structure and function, in order to enable the development of more effect therapies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM135633-01
Application #
9863486
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Justinova, Zuzana
Project Start
2020-01-10
Project End
2023-12-31
Budget Start
2020-01-10
Budget End
2020-12-31
Support Year
1
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104