Mechanisms of 'run-down' of calcium channels

利用电生理和生化方法研究了一种激活l型Ca^<2+>通道的细胞质蛋白。首先，我们试图建立一种使用脂质囊的纯化程序，其中重建了牛心脏Ca^<2+>通道。Ca^<2+>通道激活蛋白（CCAP）从凝胶过滤柱中洗脱出来，其表观分子量（M′_r）为200-300 x 10^3。DEAE-sepharose色谱法，以100-150 mM KCl洗脱CCAP。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。其次，CCAP可以恢复心肌细胞内外斑块Ca^<2+>通道的活性。CCAP对通道数量和单通道电导均无影响，但可显著提高通道的开状态概率。第三，我们研究了CCAP的组织分布。将大脑、心脏、骨骼肌、肝脏或肾脏匀浆的上清部分应用于心脏Ca^<2+>通道，这些通道以前会导致内而外斑块“衰竭”。从大脑、心脏、骨骼肌和肝脏提取的组织，但没有从肾脏提取的组织，可以从衰竭中恢复通道。这些结果表明，l型Ca^<2+>通道的活性是由胞质蛋白CCAP维持的。这很重要。在未来的研究中进一步表征该蛋白并阐明CCAP可能参与的调节机制。

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.