Regulation of L-type Ca channel and phosphorylation

利用蛙心室肌细胞研究了细胞内Mg^<2+>通过a激酶磷酸化调控l型Ca通道的作用。当细胞内Mg^<2+>浓度低于生理水平时，由于解除了Mg^<2+>通道的阻塞，l型Ca通道电流（I_<Ca. l >）显著增强。当细胞通过a激酶刺激磷酸化时，该通道对Mg^<2+>阻滞不敏感。结果表明，磷酸化通过对Mg^<2+>阻滞的充电敏感性调节心肌细胞的l型Ca通道。在蛙背根神经节（DRG）神经元的l型Ca通道中没有发现这种特殊类型的Mg^<2+>阻滞。Mg^<2+>阻滞缺失与青蛙DRG神经元l型Ca通道a激酶调控缺失有良好的相关性。我进一步发现，修饰半胱氨酸SH残基的膜不透性甲烷乙硫磺酸化合物（MTS）可以从细胞内显著增加I_<Ca>。当I_<Ca>预先条件地通过a激酶刺激增加到次极大水平时，没有这种增强作用。这表明MTS试剂影响了磷酸化机制的调控步骤之一。这些结果表明，如果我们将l型钙通道胞内区域的半胱氨酸或其相关蛋白作为靶点，我们可能会进一步了解磷酸化途径的机制。

The role of intracellular Mg^<2+> in the regulation of L-type Ca channel through A-kinase phosphorylation was elucidated using frog ventricular myocytes. When concentration of intracellular Mg^<2+> was lowered beyond a physiological level, it enhanced L-type Ca channel current (I_<Ca.L>) remarkably due to unblock of the channel from Mg^<2+>. The channel becomes insensitive to Mg^<2+> block when cell was phosphorylated through A-kinase stimulation. It concludes that phosphorylation regulates L-type Ca channel in cardiac myocytes through charging sensitivity to Mg^<2+> block. This particular type of Mg^<2+> block was not found in L-type Ca channel in frog dorsal root ganglion (DRG) neurons. The absence of Mg^<2+> block well correlates to the absence of the regulation through A-kinase in L-type Ca channel of frog DRG neurons. I have further found that membrane impermeable methanethiosulfonate compounds (MTS) that modify SH residues of cysteine can increase I_<Ca> dramatically from the intracellular side. This enhancing effect was not seen when I_<Ca> was pre-conditionally increased to a sub-maximal level by A-kinase stimulation. This indicates that MTS reagents affect one of the regulatory steps of phosphorylation mechanism. These results indicate that we may gain a further step to understand the mechanism of phosphorylation pathway if we target cysteines of intracellular region of L-type Ca channel or its associated proteins.