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

为了明确β亚基对心脏l型Ca^<2+>通道功能的贡献，我们从大鼠心肌细胞中克隆了β亚基基因。得到的β亚基序列与先前报道的序列不同。得到的β亚基命名为β _<2C>亚基。引入β _<2C>亚基后，利用膜片钳法在培养细胞系中观察到Ca^<2+>电流通过重构的l型Ca^<2+>通道。重构Ca^<2+>电流的特征与心肌细胞中Ca^<2+>电流的特征相似。在单通道记录中，重构Ca^<2+>通道的单通道活动表现出与原生Ca^<2+>通道相似的行为。这些发现表明β _<2C>亚基是天然心肌细胞l型Ca^<2+>通道的功能性剪接变体。此外，我们还探索了一种可能进入和调节l型Ca^<2+>通道的蛋白质分子。利用酵母双杂交方法，将转录因子CSN-5/Jab1直接结合到l型Ca^<2+>通道α亚基的II-III连接子上。共免疫沉淀法证实了这一发现。双免疫荧光染色显示CSN-5/Jab1与α亚基共定位于大鼠心肌细胞肌层膜。siRNA法消除CSN-5/Jab1后，细胞中Ca^<2+>电流增加。因此，CSN-5/Jab1可能对l型Ca^<2+>通道功能产生抑制调节作用。

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.