STRUCTURE/FUNCTION STUDIES OF CALCIUM CHANNEL RYR3

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Brain ryanodine receptor/calcium channel isoform, RyR3, has been detected in mammalian brain and skeletal muscle. However, little is known about the physiological role and the regulation of RyR3. Its low abundance and the fact that it is frequently co-expressed with other RyR isoforms has delayed its functional characterization. The goal of this project is to study the molecular-physiological characteristic of the RyR3 from human skeletal muscle expressed in the 1B5 myogenic cell line. This trangenic cell system allows; 1- An environment that resembles the muscle physiological condition and 2- It will allow expression of a high level of the RyR3 without other RyR contamination. Functional characterization of the transfected cell will be evaluated for the calcium releasing properties of classical RyR agonist, using calcium fluorescence dye, and by evaluation of the calcium channel activity of RyR3 expressed. The type of molecular interaction of co-expressed RyR will be studied through immunoprecipitation experiments to evaluate tetrameric composition (interaction with other RyR isoform) or tetradic arrangement of the DHPR. Point mutations over the specific region of RyR3 cDNA sequence involved in RyR agonist sensitivity (caffeine and calcium) will be evaluated as well.