Searching responsible sites for the regulation of the L-type Ca channel through phosphorylation

【概述】本研究的最终目的是确定心脏中l型Ca通道调控的负责位点，如细胞内Mg^<2+>阻滞，或通过磷酸化加速通道活性的增强。因此，我们发现仅使用非心脏细胞的l型Ca通道来完全重建通道活性是缺乏的。特别是，Mg^<2+>反应与天然心肌细胞的行为完全不同。在重建系统中，细胞内Mg^<2+>的消耗导致通道活性的快速清除，而在天然心脏细胞中，细胞内Mg^<2+>的消耗通常会导致Ca通道电流显著增加（I_< Ca >）。这表明需要针对宿主细胞条件广泛改进重建系统[结果]1。当温度低于28℃时，豚鼠心肌细胞内Mg^<2+>降低，而I_<ca>升高受到干扰。这种温度依赖性不能用简单的热力学活动来解释。我们在青蛙心室细胞中发现了编码l型Ca通道α_1、β_<2C>和α_<2/δ>亚基的全长cdna。它们与大鼠或小鼠的同源性超过80%。α_1、β_<2C>和α_<2/δ>亚基在BHK细胞中的共表达表现出功能性的l型Ca通道活性。当使用log Mg^<2+>（低于10^<-5>M）低于10^<-5>M的补片溶液透析细胞时，BHK细胞中重构的Ca通道迅速下降。这与在天然心肌细胞中发现的数据相反。我们需要寻找未知的蛋白质来适应缺乏重要的通道调节。

[Overview]The ultimate aim of this study is to identify responsible sites for the regulations of L-type Ca channels in the heart, such as intracellular Mg^<2+> block, or enhancement of channel activity accelerated by phsphorylation. As a result, we found that something is lacking to fully reconstruct channel activity only using L-type Ca channels with non-cardiac cells. Especcially, Mg^<2+> response was totally different from the behavior found in native cardiac cells. Depletion of intracellular Mg^<2+> led to the rapid wash out of channel activity in reconstructed systems, while in native cardiac cells, depletion of intracellular Mg^<2+> normally causes prominent increase in Ca channel current (I_<ca>). This indicates the requirement of extensive improvement of reconstruction systems regarding host cell conditions[Results]1. Increase in I_<ca> responding to reduction in intracellular Mg^<2+> was disturbed in guinea-pig cardiac cells, when temperature was below 28℃. This temperature dependency was not explained by simple thermodynamic activities.2. We identified whole length of cDNAs encoding α_1, β_<2C> and α_<2/δ> subunits of the L-type Ca channel in frog ventricular cells. They exhibited more than 80 % homology of those reported in rat or mouse. Co-expression of all subunits of α_1, β_<2C> and α_<2/δ> in BHK cells exhibited functional L-type Ca channel activity.3. Reconstructed Ca channels in BHK cells rapidly ran down, when cells were dialysed with log Mg^<2+> (below 10^<-5>M) patch solutions below 10^<-5>M. This is in contrast to data found in native cardiac cells. We need to search unkown proteins to accommodate the lacking of important regulations of the channel.