Studies on the molecular mechanism for calcium signaling

以往的研究表明，心肌和骨骼肌钙通道激活速度的差异可以归因于四个重复的同源单位的第一个重复的氨基酸序列的不同。为了确定Repeat I中负责不同激活动力学的区域，我们系统地构建了一系列‘Repeat I嵌合体’DNA结构，并在培养的肌肉发育不全小鼠骨骼肌细胞中表达了它们。对激活速度的分析表明，第三个可能的跨膜区(S3)和S3和S4之间的连接区是决定激活速度的关键。结合S4作为电压传感器的概念，我们推测，膜电位的变化触发了S4的运动，S4通过影响S3及其邻近部分而传递，最终导致通道开放。电压门控钙通道包含更小的亚基。当主打S…较多的亚基(α1亚基)与较小的亚基共表达，功能活性明显增强。为了了解小亚基功能的分子机制，我们单独或与小亚基一起表达了心肌钙通道α1亚基。根据Northern杂交、蛋白质凝胶电泳、二氢吡啶结合实验和电生理测试的结果，我们认为这些小亚基不影响主要亚基的代谢，它们是正常功能构象所必需的。从兔脑的cDNA库中，我们克隆了编码一种新的钙通道(命名为BIII)的基因。通过测序，我们已经阐明了BIII钙通道的一级结构。该钙通道与其他电压依赖性钙通道具有同源性。特别是BIII与BI和BII脑钙通道有较高的相似性。这三种钙通道形成不同于二氢吡啶敏感的L型钙通道的亚群。将DNA注入肌肉发育不全小鼠骨骼肌细胞中表达BIII通道时，BIII通道表现出omega-conooxoxin敏感的钙通道活性。其动力学和电压依赖性与已报道的N型钙通道相似。由于N型钙通道对神经递质的释放很重要，克隆N型钙通道BIII将有助于未来脑功能的研究。较少

Previous studies demonstrated that the difference in speed of activation between the cardiac and skeletal muscle calcium channels can be attributed to the difference in amino acid sequence of the first repeat of the four repeated units of homology. To identify the region in repeat I responsible for the different activation kinetics, we have made series of 'repeat I chimera' DNA constructs systematically, and expressed them in cultured skeletal muscle cells derived from mice with muscular dysgenesis. Analyses of speed of activation have revealed that the third putative transmembrane region (S3) and the linker region between S3 and S4 is critical to determine the speed of activation. Together with the notion that S4 functions as a voltage sensor, we speculate that a change in membrane potential triggers movement of S4, which is transmitted by affecting S3 and its adjacent portion, finally leading to channel openings.Voltage-gated calcium channels contain smaller subunits. When the main s … More ubunit (alpha1 subunit) is coexpressed with smaller subunits, functional activity is markedly increased. To understand the molecular mechanism of small subunit function, we have expressed cardiac calcium channel alpha1 subunit alone or together with small subunits. From the results of Northern blots, protein gel electrophoresis, dihydropyridine binding assay, and electrophysiological measurements, we concluded that the small subunits do not affect metabolism of the main subunit, and that they are necessary for proper functional conformation.From cDNA libraries of rabbit brain, we have cloned cDNA encoding a new calcium channel (named BIII). By sequencing the cDNA, we have elucidated the primary structure of BIII calcium channel. This calcium channel showed homology to other voltage-dependent calcium channel. Especially, BIII showed higher similarity to BI and BII brain calcium channel. These three types of calcium channel form a subgroup distinct from dihydropyridine-sensitive L-type calcium channel. When BIII channel is expressed by injecting DNA into cultured skeletal muscle cells derived from mice with muscular dysgenesis, it showed omega-conotoxin sensitive calcium channel activity. Its kinetics and voltage-dependence were similar to those reported for N-type calcium channels. Since the N-type calcium channel is important for neurotransmitter release, cloning of an N-type calcium channel BIII will contribute to future studies of brain function. Less