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

本研究采用全细胞膜片钳技术，研究了膜磷脂磷脂酰肌醇4，5-二磷酸(PIP_2)调节延迟整流钾电流慢激活成分(I_&lt；ks&gt；)的分子基础。α_1肾上腺素能激动剂苯肾上腺素(30μM)对Ks和Gt的兴奋作用可被蛋白激酶C抑制剂双吲哚马来酰亚胺I(BIS-I)部分抑制(60%)，并可被经膜片电极细胞内加入PIP_2(100μM)几乎完全阻断。P2Y激动剂三磷酸腺苷(50μM)对Ks和Gt；的增强作用几乎不受Bis-I的影响，但可被PIP_2细胞内应用所阻断。70%低渗外液(200mOsm)刺激Ks和Gt；可通过膜电极显著减弱抗PIP_2单抗(1：40稀释度)的细胞内应用。这些结果为PIP_2代谢参与刺激GQ-磷脂酶C偶联受体(如α_1和P_2Y-受体)或低渗细胞肿胀引起的K_s和GT的增强提供了实验证据。在KCNQ1、KCNE1和M_1-R瞬时转导的CHO细胞上，乙酰胆碱(1和10 nM)对M_1-M受体(M_1-R)的刺激使通过KCNQ1/KCNE_1通道的膜电流增加20%，从而支持膜PIP_2在调节心肌细胞膜电流中的作用。这些观察也可能表明，膜上的PIP_2可能通过刺激不同的Gq-PLC偶联受体或各种细胞刺激而在增强I_lt；ks&gt；中起不同的作用。

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.