Study on mechanism of modulation of cardiac L-type Ca^<2+> channel by phosphorylation and Ca^<2+>

本研究的主要目的是基于单通道分析，阐明由通道磷酸化引起的心脏l型Ca^<2+>通道门控过程的变化。在研究过程中，出现了Ca^<2+>通道β -亚基参与磷酸化依赖性调节的新问题。异丙肾上腺素主要通过增加l型通道的可用性（Ps）和微开放概率（Po）来增加通道电流。在1 nM ~ 0.1 mM范围内，其浓度的增加并没有增加2型扫描的速率。在BAY K 8644存在的情况下，异丙肾上腺素显著提高了2型扫描率。尼群地平可延长宏观Ba^<2+>电流失活的时间过程，异丙肾上腺素可延长。体外培养的成年兔心室肌细胞β -亚基反肽处理可使l型电流密度降低30%，使电流上升和衰减的时间延长2倍，并显著降低异丙肾上腺素增加电流的效果。未来展望利用异源表达系统积累的分子生物学研究表明，l型Ca^<2+>通道的多亚单位组织，α -1亚基磷酸化位点和β亚基磷酸化。利用对天然心肌细胞的反义技术和单通道记录技术，我们有很好的机会揭示β亚基在Ca^<2+>通道调节中的作用。

The major aim of the present study was to clarify the changes of the gating process of the cardiac L-type Ca^<2+> channels produced by channel phoisphorylation based on single channel analysis. During the course of the study, new problem of the involvement of Ca^<2+> channel beta-subunit on the phosphorylation-dependent modulation arised.Results1.Isoproterenolincreased mainly availability (Ps) and slightly openprobability (Po) of the L-type channel to increase the channel current. The increase of its concentration between 1 nM and 0.1 mM did not increase the rate of mode-2 sweeps.2.The rate of mode-2 sweeps in the presence of BAY K 8644 was significantly increased by isoproterenol.3.The time course of inactivation of macroscopic Ba^<2+> current was enhanced by nitrendipine and this was enhanced by isoproterenol.4.Treatment by beta-subunit antisence of cultured adult rabbit ventricular myocytes decreased the L-type current density by 30%, increased the time course of rise and decay of the current by a factor of two and significantly decreased the efficacy of isoproterenol to increase the current.Future prospectAccumulating knowledges from molecular biology study using heterologous expression system indicated the multisubuinit organization of L-type Ca^<2+> channels, phosphorylation sites in alpha-1 subunit and phosphorylation of beta subunit. Using antisence technology towards native cardiac myocytes and applying single channel recording technique we have a good chance to disclose the role of beta subunit in Ca^<2+> channel regulation.