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Analysis of physiological function of calcium channel β subunit in heart muscle

心肌钙通道β亚基生理功能分析

基本信息

批准号：
14370015
负责人：
TOHSE Noritsugu
金额：
$ 8.9万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370015/
关键词：
L-type Ca2+ channel cardiomyocytes patch clamp β subunit CSN-5 Jab1 カルシウムチャネル

项目摘要

In order to clarify contribution of β subunit to function of cardiac L-type Ca^<2+> channel, genes of β subunit were cloned from rat cardiomyocytes. The obtained sequence of β subunit was different from those reported previously. The obtained β subunit was named as β _<2C> subunit. After introduction of β _<2C> subunit, Ca^<2+> current through the reconstituted L-type Ca^<2+> channels in cultured cell lines was observed in use of the patch clamp method. Characteristics of the reconstituted Ca^<2+> current were similar to those of the native Ca^<2+> current in cardiomyocytes. In single channel recordings, the single channel activity of the reconstituted Ca^<2+> channel exhibited similar behaviors to that of the native Ca^<2+> channel. These findings indicate that β _<2C> subunit is a functional splice variant of L-type Ca^<2+> channel in native cardiomyocytes. In addition, we have explored a possible protein molecule that can access and modulate L-type Ca^<2+> channel. In use of the yeast-two-hybrid method, CSN-5/Jab1, a transcription factor, directly bound to the II-III linker of α subunit of L-type Ca^<2+> channel. The finding was confirmed by the coimmunoprecipitation method. CSN-5/Jab1 colocalized with α subunit in sarcolemmal membrane of rat cardiomyocytes under the double immunofluorescence staining. The reconstituted Ca^<2+> current was increased in the cultured cell in which CSN-5/Jab1 was abolished by siRNA method. Therefore, CSN-5/Jab1 may produce an inhibitory regulation of L-type Ca^<2+> channel function.

为了阐明β亚基在心肌L型钙通道功能中的作用，从大鼠心肌细胞中克隆了β亚基基因。获得的β亚基序列与以往报道的序列不同。将获得的β亚基命名为β_&lt；2C&gt；亚基。在膜片钳方法中，引入β_lt；2C&gt；亚单位后，用膜片钳方法观察到细胞内钙电流通过重组的L型钙通道。重组钙电流的特性与心肌细胞天然钙电流的特征相似。在单通道记录中，重组钙通道的单通道活动表现出与天然钙通道相似的行为。这些发现表明，β_&lt；2C&gt；亚基是天然心肌细胞中L型钙通道的功能剪接变体。此外，我们还探索了一种可能的蛋白质分子，它可以进入并调节L钙通道。采用酵母双杂交的方法，将转录因子Csn-5/Jab1直接结合到L钙通道α亚基的II-III连接子上。免疫共沉淀法证实了这一发现。免疫荧光双标记法显示csn-5/jBA1与α亚基共存于大鼠心肌细胞的肌膜。用siRNA方法去除CSN-5/Jab1后，细胞内重组钙电流增加。因此，Csn-5/Jab1可能对L型钙通道功能产生抑制调节作用。