CALCIUM CHANNELS IN NORMAL AND DISEASED TISSUE

正常和病变组织中的钙通道

• 批准号: 3161547
• 负责人: RICHARD A STEINHARDT
• 金额: $ 20.94万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3161547
• 关键词: G protein; Xenopus oocyte; calcium channel; disease /disorder model; dystrophin; fluorescent dye /probe; gene expression; gene therapy; homeostasis; human tissue; laboratory mouse; laboratory rat; muscular dystrophy; myoblasts; myotubes; protein kinase; sarcolemma; sex linked trait; voltage /patch clamp

Our findings in the DMD human and the mdx mouse indicate that the absence of dystrophin in Duchenne-type muscular dystrophy results in elevated intracellular free calcium. Therefore, we propose to characterize the calcium leak (calcium background) channels in normal human and mouse skeletal myotubes in culture and compare them to the calcium leak channels in dystrophic DMD human and mdx mouse myotubes. In parallel experiments we will examine the degree of regulation of intracellular free calcium in response to levels of external calcium as the myotubes differentiate from populations of the respective myoblasts. In addition to the characterizations of channels and calcium regulation after myotube formation we will follow both channel properties and calcium regulation as the myoblasts undergo fusion, differentiate and develop as myotubes, over a period of 2-3 weeks in culture. By using heterocaryons formed by fusion of mdx mouse and normal rat myoblasts we will observe degree of rescue of normal channel properties and correlate degree of rescue with number of and proximity to fused normal nuclei. We will also study the restoration of normal regulation of channel properties by the expression of a full-length dystrophin cDNA in dystrophic mouse myotube cell lines. This would be a critical step in developing gene therapy for muscular dystrophy. We will extend these studies to cardiac myocytes since that tissue also expresses dystrophin. Unlike skeletal muscle, cardiac muscle preparations of RNA have been successfully used to express functional calcium channels in Xenopus oocytes, therefore we will use total RNA from cardiac tissue to develop an assay in Xenopus oocytes for the expression of calcium leak channels.

我们在DMD人和mdx小鼠中的发现表明， 杜氏型肌营养不良症中的抗肌萎缩蛋白的水平升高 细胞内游离钙 因此，我们建议将 正常人和小鼠的钙漏（钙背景）通道 骨骼肌管在文化和比较他们的钙泄漏 在营养不良的DMD人和mdx小鼠肌管中的通道。 并行 实验中，我们将检查细胞内的调节程度， 游离钙对外界钙水平的反应， 与相应的成肌细胞群区分。 此外 肌管化后的通道特性和钙调节 形成我们将遵循通道特性和钙调节 当成肌细胞经历融合、分化并发育为肌管时， 培养2-3周。 通过使用由以下物质形成的异核体 mdx小鼠和正常大鼠成肌细胞的融合，我们将观察 恢复正常通道特性，并将恢复程度与 融合的正常核的数量和接近度。 我们亦会研究 通过表达恢复通道特性的正常调节 的全长肌营养不良蛋白cDNA在营养不良小鼠肌管细胞系。 这将是开发肌肉萎缩症基因疗法的关键一步。 营养不良 我们将把这些研究扩展到心肌细胞，因为 组织也表达肌营养不良蛋白。 与骨骼肌不同， RNA的制备已经成功地用于表达功能性的 钙通道，因此我们将使用总RNA从 在非洲爪蟾卵母细胞中开发用于表达 钙渗漏通道。