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Mechanisms of 'run-down' of calcium channels

钙通道“耗尽”的机制

基本信息

批准号：
63570044
负责人：
KAMEYAMA Masaki
金额：
$ 1.34万
依托单位：
Okazaki National Research Institutes, National Inst. for Physiol. Sci.
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

A cytoplasmic protein that activates L-type Ca^<2+> channel was investigated by using both electrophysiological and biochemical methods. First, we attempted to establish a purification procedure using lipid vesicles into which bovine cardiac Ca^<2+> channels were reconstituted. The Ca^<2+> channel activating protein (CCAP) was eluted from a gel-filtration column as a mass having an apparent molecular weight (M'_r) of 200-300 x 10^3. On DEAE-sepharose chromatography, CCAP was eluted at 100-150 mM KCl. The partially Purified CCAP had M'_r of about 100 x 10^3 on SDS-polyacrylamid gel electrophoresis. These biochemical properties were clearly different from the cAMP- dependent protein kinase or any subunit of the Ca^<2+> channel. Second, CCAP could restore the activity of Ca^<2+> channel in inside-out patches of cardiac myocytes. Neither number of channels nor single channel conductance was affected by CCAP, but open-state probability of the channel was dramatically increased by CCAP. Third, we investigated tissue distribution of CCAP. A supernatant fraction of the homogenate from brain, heart, skeletal muscle, liver or kidney was applied to cardiac Ca^<2+> channels which were previously led to 'run-down' in inside-out patches. The tissue extracts from brain, heart, skeletal muscle and liver, but not kidney, recovered the channels from the run-down.These results suggest that the activity of the L-type Ca^<2+> channel is maintained by the cyto- plasmic protein CCAP. It would be important. in future studies to characterize this protein further and to clarify possible regulatory mechanisms in which CCAP is involved.

用电生理和生化方法研究了一种激活L型Ca^<2+>通道的胞质蛋白。首先，我们试图建立一种利用脂质囊泡的纯化方法，在其中重组牛心脏Ca^<2+>通道。从凝胶过滤柱中洗脱出表观分子量（M 'r）为200-300 × 10^3的Ca^2+通道激活蛋白（CCAP）。在DEAE-Sepharose色谱上，CCAP在100-150 mM KCl下洗脱。部分纯化的CCAP在SDS-聚丙烯酰胺凝胶电泳上的M '_r约为100 × 10^3。这些生化特性明显不同于cAMP依赖性蛋白激酶或Ca^2+通道的任何亚基.第二，CCAP可恢复心肌细胞内面向外膜片上Ca^<2+>通道活性。CCAP对通道数目和单通道电导均无影响，但通道的开放概率显著增加。第三，我们研究了CCAP的组织分布。将来自脑、心脏、骨骼肌、肝脏或肾脏的匀浆的上清液部分应用于心脏Ca^2+通道，该通道先前在由内而外的贴片中导致“流失”。脑、心脏、骨骼肌和肝脏的组织提取物恢复了通道的活性，但肾脏没有恢复。这些结果表明，L型Ca^2+通道的活性是由细胞质蛋白CCAP维持的。这很重要在未来的研究中，进一步表征这种蛋白质，并阐明可能的调控机制，其中CCAP参与。