Molecular basis for the regulation of the cardiac delayed rectifier K^+ channel by membrane PIP_2

本课题采用全细胞膜片钳法，研究了膜磷脂磷脂酰肌醇4,5-二磷酸（PIP_2）调控延迟整流器K^+电流慢活化组分（I_<ks>）的分子基础。α_1-肾上腺素能激动剂苯肾上腺素（30μM）对I_<ks>的刺激作用被蛋白激酶C抑制剂双吲哚聚马来酰亚胺I（BIS-I）部分（60%）抑制，通过贴片电极在细胞内添加PIP_2 （100μM）几乎完全消除。p2Y激动剂ATP （50 μM）对I_<ks>的增强作用几乎不受BIS-I的影响，但在细胞内应用PIP_2可很大程度上阻断I_<ks>。暴露于70%低渗外液（200 mOsm）对I_<ks>的刺激显著减弱了通过贴片电极在细胞内应用抗pip_2单克隆抗体（1:40稀释）。这些结果为PIP_2代谢参与i_k_bb_0的增强提供了实验证据，i_k_bb_0可通过刺激gq -磷脂酶C （PLC）偶联受体（如α_1和p2y受体）或低渗细胞肿胀引起。在瞬时转染KCNQ1、KCNE1和M_1-R的CHO细胞中，乙酰胆碱（1 nM和10 nM）对m_1 -毒碱受体（M_1-R）的刺激使通过KCNQ1/KCNE1通道（心脏I_<ks>的分子成分）的膜电流增加20%，支持膜PIP_2在I_<ks>调控中的功能作用。这些观察结果也可能表明，膜PIP_2通过不同的gq - plc偶联受体或不同的细胞刺激，在I_<ks>的增强中起着不同的作用。

In this research project, we investigated the molecular basis for the regulation of the slowly activating component of delayed rectifier K^+ current (I_<ks>) by membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP_2), using the whole-cell patch-clamp method. The stimulatory action of α_1-adrenergic agonist phenylephrine (30μM) on I_<ks>, was partially (by 60%) inhibited by the protein kinase C inhibitor bisindolylymaleimide I(BIS-I) and was almost totally abolished by intracellular addition of PIP_2 (100μM) through a patch electrode. The potentiation of I_<ks>by the p2Y agonist ATP (50 μM) was scarcely affected by BIS-I but was largely blocked by intracellular application of PIP_2. The stimulation of I_<ks> by exposure to 70% hyposmotic external solution (200 mOsm) was significantly attenuated intracellular application of anti-PIP_2 monoclonal antibody(1:40 dilution) via a patch electrode. These results provide the experimental evidence for the involvement of PIP_2 metabolism in the potentiation of I_<ks>, evoked by stimulation a Gq-phospholipase C (PLC)-coupled receptors (e.g., α_1-and P2Y-receptors) or by hyposmotic cell swelling. The stimulation of M_1-muscarinic receptors (M_1-R) by acetylcholine (1 and 10 nM) in CHO cells transiently trasnfected with KCNQ1, KCNE1 and M_1-R increased the membrane current through the KCNQ1/KCNE1 channels, molecular constituents of cardiac I_<ks>, by 20%, supporting a functional role for membrane PIP_2 in the regulation of I_<ks>. These observation may also indicate the possibility that membrane PIP_2 plays a differential role in the potentiation of I_<ks> through the stimulation of distinct Gq-PLC-coupled receptors or various cellular stimuli.