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: 6335048
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6335048
• 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.

心脏中的兴奋-收缩偶联赋予L-型钙通道和细胞内环境之间的紧密联系，使得去极化后的Ca 2+进入触发收缩。我们一直在研究组成心脏钙通道的亚单位的复杂结构、调节和受体药物位点。考虑到该通道在维持正常心脏功能中的重要性，在某些类型的人类心力衰竭中发现缺陷并不奇怪。这个持续的项目集中在亚基功能的相互作用，在体内的正常和下降的心脏。后者是通过转基因小鼠中L型钙通道亚单位的特异性增加而在小鼠中产生的，这为我们提供了非常接近人类类型的心肌病。心脏的转基因“重塑”不仅是一种改变体内亚基化学计量的方便方法，而且提供了一种以逻辑方式接近心力衰竭机制的方法。长期的目标是表征钙通道调节，在孔，亚基的重要性，和钙拮抗剂受体功能。 该更新申请的新增功能是转基因和敲除方法，包括协调的生理、生化和显微解剖学方法。我们希望提供进一步的分子信息，正常的L-型心脏通道及其可能的作用，在患病的心脏。具体目的是：1）特异性地且仅在转基因小鼠的心肌细胞中过表达人心脏α 1亚基。转基因的生理功能、基因型频率和表达水平，以及全面的心脏病理评估，有望产生重要的新信息。2)建立显性负性表达系统，消除小鼠心脏β亚基的功能效应。高亲和力α 1相互作用结构域（AID）将以心脏特异性方式过表达，并且这将随后充当β亚基的陷阱。特征描述与目标1相同。我们希望为β亚基在体内的作用提供新的信息。3)产生心脏中缺乏α 2/δ亚基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，应该会给我们关于这种亚基在体内作用的有价值的信息。4)继续研究钙通道的孔衬区。我们认为，这些研究将为我们提供具体的信息，涉及在调节通道的孔的一些结构特征。