Grant: CIHR Fellowship

Some individuals have a single mutation within their skeletal muscle ryanodine receptor (RYR1) resulting in Malignant Hyperthermia (MH), a life-threatening complication associated with general anesthesia. The RYR1 is a channel responsible for the rapid, but controlled, release of calcium from internal stores within the cells of our skeletal muscles that triggers muscle contraction. These individuals, only when exposed to general anesthetics, show uncontrolled contraction of their muscles and body temperatures that reach over 40.6 degrees Celsius. While considerable advances have emerged regarding our understanding of RYR1, there is still much to learn about the relationship between the protein's structure and how small molecules like anesthetics can significantly alter their normal function. Our objective is to use techniques from chemistry and biology as well as a novel combined strategy to identify the binding sites for anesthetics within normal and MH mutant RYR1 in order to determine and compare the consequence of binding on RYR1 structure. To accomplish this we will employ photoactive analogs of the anesthetics isoflurane and sevoflurane to 'photolabel' their binding sites within both RYR1s. We will determine the affect of anesthetic binding on the structure of the ion channel using a novel strategy that couples cryo-electron microscopy (cryo-EM) with the photoactive analogs in order to obtain a snapshot of the anesthetic interactions with the channel. Preliminary studies have indicated a potential anesthetic binding site within a unique region of RYR1 that would have long-range effects on the structure within other channel regions. Ultimately this investigation will provide 1) a strategy for the advancement of scientific methods and 2) define the transitions that occur within RYR1 and the impact of anesthetics on the channel's function.