MOLECULAR MECHANISM, PATHOLOGY AND PHARMACOLOGY OF CALCIUM CHANNELS

钙通道的分子机制、病理学和药理学

• 批准号: 6109530
• 负责人: ARNOLD SCHWARTZ
• 金额: $ 36.38万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6109530
• 关键词: Xenopus oocyte; animal tissue; calcium channel; calcium channel blockers; calcium flux; electrophysiology; genetically modified animals; heart contraction; heart pharmacology; laboratory mouse; myocardium disorder; neuromuscular transmission; protein structure function; spectrometry; tissue /cell culture; voltage gated channel

Excitation-contraction coupling in the heart confers a tight linkage between the L-type calcium channel and the intracellular environment so that Ca2+ ingress upon depolarization triggers contraction. We have been studying the complex architecture, regulation and receptor drug sites on the subunits that comprise the cardiac calcium channel. Considering the importance of this channel in maintaining normal cardiac function, it is not surprising that defects have been found in some types of human heart failure. This continuing project focuses on subunit functional interaction in vivo on the normal and falling heart. The latter is produced in the mouse by a specific increase of L-type calcium channel subunits in a transgenic mouse, which has provided us with a cardiomyopathy very close to the human type. Transgenic "remodeling" of the heart is not only a convenient method of altering subunit stoichiometry in vivo, but provides a way to approach mechanisms of heart failure in a logical manner. The long-term objective is characterization of calcium channel regulation, in terms of the pore, subunit importance, and calcium antagonist receptor functions. The added feature in this renewal application is the transgenic and knockout approach, encompassing coordinated physiological, biochemical and microanatomical methodology. We hope to provide further molecular information on the normal L-type cardiac channel and its possible role in the diseased heart. The specific aims are: 1) to over-express the human cardiac alpha1 subunit specifically and only in the myocytes of transgenic mice. Physiological function, genotype frequency, and expression levels of the transgene, as well as comprehensive cardiac pathological assessment, hopefully will yield important new information. 2) To eliminate the functional effects of the beta-subunit in mouse heart by generating a dominant negative expression system. The high affinity alpha1 interactive domain (AID) will be over- expressed in a cardiac-specific manner, and this will then act as a trap for beta subunits. Characterization is as in Aim 1. We hope to provide new information n the role of the beta-subunit in vivo. 3) To generate conditional knockout mice lacking the alpha2/delta subunit in the heart. Studying these mice, again on the molecular, cellular and whole organ level, should give us valuable information on the role of this subunit in vivo. 4) To continue studies on the pore-lining region of calcium channels. We suggest that these studies will give us specific information concerning some structural features of the pore that are involved in regulation of the channel.

心脏的兴奋-收缩偶联使L型钙通道与细胞内环境紧密相连，使去极化时钙离子内流触发收缩。我们一直在研究组成心肌钙通道的亚单位上的复杂结构、调节和受体药物位点。考虑到这一通道在维持正常心脏功能方面的重要性，在某些类型的人类心力衰竭中发现缺陷也就不足为奇了。这个继续进行的项目专注于体内正常心脏和下落心脏上的亚单位功能相互作用。后者是由转基因小鼠体内L型钙通道亚单位的特异性增加而产生的，这为我们提供了一种非常接近人类类型的心肌病。转基因心脏重塑不仅是一种在体内改变亚单位化学计量的便捷方法，而且为以合理的方式探讨心力衰竭的机制提供了一种方法。长期的目标是根据孔、亚单位重要性和钙拮抗剂受体功能来表征钙通道的调节。在这一更新应用中增加的功能是转基因和基因敲除方法，包括协调的生理、生化和显微解剖学方法。我们希望为了解正常的L型心脏通道及其在病变心脏中的可能作用提供进一步的分子信息。其具体目的是：1)仅在转基因小鼠的心肌细胞中特异性地过表达人心脏α1亚单位。转基因的生理功能、基因频率和表达水平，以及全面的心脏病理评估，有望产生重要的新信息。2)通过产生一个显性的负性表达系统来消除小鼠心脏中β-亚基的功能效应。高亲和力的α1相互作用结构域(AID)将以心脏特有的方式过度表达，这将成为β亚基的陷阱。鉴定与目标1相同。我们希望提供有关β亚基在体内的作用的新信息。3)建立心脏α2/Delta亚基缺失的条件性基因敲除小鼠。再次在分子、细胞和整个器官水平上研究这些小鼠，将为我们提供关于这个亚单位在体内的作用的有价值的信息。4)继续研究钙通道的内膜区。我们认为，这些研究将为我们提供与通道调节有关的一些毛孔结构特征的具体信息。