Mechanisms of 'run-down' of calcium channels

用电生理和生化方法研究了一种激活L型钙离子通道的细胞质蛋白。首先，我们试图建立一种利用脂泡纯化牛心肌钙离子通道的方法。钙通道激活蛋白(CCAP)由凝胶过滤柱层析出，表观分子量(M‘r)为200-300×10^3。在DEAE-Sepharose层析柱上，CCAP在100-150 mM KCl处洗脱。经SDS-PAGE分析，部分纯化的CCAP的M‘r约为100×10~(-3)。这些生化特性明显不同于cAMP依赖的蛋白激酶或钙通道的任何亚单位。第二，CCAP可恢复心肌细胞内向外膜片钙通道的活动。通道数和单通道电导均不受CCAP的影响，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。来自大脑、心脏、骨骼肌、肝脏或肾脏的匀浆的上清液部分被应用于心脏钙离子通道，而这些通道以前是由内向外的补丁。脑、心脏、骨骼肌和肝脏的组织提取液，而不是肾脏的组织提取物，使通道从衰败中恢复。这些结果表明，L型钙通道的活性是由细胞质蛋白CCAP维持的。这将是非常重要的。在未来的研究中，进一步确定该蛋白的特征，并阐明CCAP参与的可能调控机制。

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.