STRUCTURE/FUNCTION STUDIES OF CALCIUM CHANNEL RYR3

脑蓝嘌呤受体/钙通道异构体RyR3在哺乳动物脑和骨骼肌中被检测到。然而，人们对RyR3的生理作用和调控知之甚少。它的低丰度和它经常与其他RyR亚型共表达的事实推迟了它的功能表征。本项目的目的是研究人类骨骼肌中RyR3在1B5成肌细胞系中表达的分子生理特性。这种转基因细胞系统允许；1-一个类似于肌肉生理状况的环境；2-它将允许表达高水平的RyR3，而不受其他RyR污染。使用钙荧光染料，并通过评价表达的RyR3的钙通道活性来评估转染细胞的功能表征，以评估经典RyR激动剂的钙释放特性。通过免疫沉淀实验研究共表达RyR的分子相互作用类型，以评估DHPR的四聚体组成（与其他RyR异构体的相互作用）或四聚体排列。RyR3 cDNA序列中涉及RyR激动剂敏感性（咖啡因和钙）的特定区域的点突变也将被评估。

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.