CALCIUM CHANNELS IN NORMAL AND DISEASED TISSUE

我们在DMD人类和MDX小鼠中的发现表明， 营养不良蛋白在Duchenne型肌营养不良症中的应用 细胞内游离钙。因此，我们建议将 正常人和小鼠的钙泄漏(钙本底)通道 骨骼肌管的培养，并与钙泄漏进行比较 营养不良的DMD人和MDX小鼠肌管中的通道。并行的 实验中我们将检查细胞内的调节程度 作为肌管的外源性钙水平对游离钙的反应 区别于相应成肌细胞的群体。此外 肌管后通道特性及钙调节的研究进展 我们将同时遵循通道特性和钙调节 随着成肌细胞的融合、分化和肌管的发育， 经过2-3周的培养。通过使用由以下分子形成的杂核 Mdx小鼠和正常大鼠成肌细胞的融合 正常航道属性的抢救及其与抢险的关联度 融合的正常核的数目和接近程度。我们亦会研究 通过表达恢复通道属性的正常调节 在营养不良的小鼠肌管细胞系中获得全长抗肌营养不良蛋白基因。 这将是发展肌肉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 组织也表达抗肌营养不良蛋白。与骨骼肌不同，心肌 RNA的制备已成功地用于表达功能 非洲爪哇卵母细胞中的钙通道，因此我们将使用来自 心脏组织建立在非洲爪哇卵母细胞中表达的检测方法 钙渗漏通道。

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.