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STRUCTURE/FUNCTION STUDIES OF CALCIUM CHANNEL RYR3

钙通道 RYR3 的结构/功能研究

基本信息

批准号：
6194465
负责人：
Claudio F Perez
金额：
$ 2.67万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-05-01 至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/6194465
关键词：
animal genetic material tag caffeine calcium channel complementary DNA electron microscopy human genetic material tag immunoprecipitation lipid bilayer membrane point mutation protein isoforms protein structure function ryanodine striated muscles tissue /cell culture transfection /expression vector

项目摘要

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.

脑兰尼碱受体/钙通道亚型RyR 3已在哺乳动物脑和骨骼肌中检测到。然而，关于RyR 3的生理作用和调节知之甚少。它的低丰度和它经常与其他RyR同种型共表达的事实延迟了其功能表征。本课题的目的是研究人骨骼肌RyR 3在1B 5肌原细胞系中表达的分子生理学特征。这种转基因细胞系统允许：1-类似于肌肉生理条件的环境，和2-它将允许高水平的RyR 3表达而没有其他RyR污染。将使用钙荧光染料并通过评价表达的RyR 3的钙通道活性，评价转染细胞的经典RyR激动剂的钙释放性质的功能表征。将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亚型的相互作用）或四聚体排列。还将评价涉及RyR激动剂敏感性（咖啡因和钙）的RyR 3 cDNA序列特定区域的点突变。