Sub-Project #3

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The skeletal muscle Ca2+ release channel, also known as the ryanodine receptor (RYR1), regulates the release of Ca2+ from sarcoplasmic reticulum (SR) stores and is mutated in human central core disease (CCD) and in the pharmacogenetic syndrome, malignant hyperthermia (MH). The mutations cluster in three regions of RYR1: region 1 from amino acids 35 to 614, region 2 from 2163 to 2459 and region 3 from 4647-4914. The MH mutations are located primarily in cytoplasmic regionsl and 2 while most of the CCD mutations are found in the transmembrane region 3. The working hypotheses to be tested in this project are: a. Regions 1 and 2, located in the clamp domain, interact to allosterically stabilize a closed state of the channel, b. The MH mutations, by destabilizing this interaction, enhance the response of the channel (and hence muscle) to activators and elevated temperature, c. Enhanced RyR1 activity during exercise elevates endogenous cytokines that act as pyrogens (such as IL-6) and the subsequent increase in body temperature increases the probability of an MH response. To test these hypotheses, the following specific aims are proposed: A1. Define the effects of the MH/CCD mutations on the conformational changes in the clamp domain of RyR1 A2. Evaluate the effects of the MH/CCD mutations on the structural and contractile properties of the muscle. A3. Determine if the probability of exercise-induced MH responses and/or rhabdomyolysis is increased by elevated body temperature and/or increased endogenous IL-6 concentrations. These studies will use mice with MH/CCD mutations that have been generated in Projects 1 and 3.