心肌细胞的向内整流钾电流(IK1)是静息电位的主要决定因素。离子通道Kir2.1,Kir2.2和Kir2.3形成四聚体,并且是宏观IK1电流的分子相关性。维拉帕米是一种抗心律失常药物,用于抑制心房和心室心律失常。其主要作用机制是通过阻断钙通道。此外,它已被证明阻断IK1电流和Kir2.1亚基。迄今为止,尚未研究其对有助于IK1电流的其他亚基的影响。因此,我们分析了维拉帕米对非洲爪猴卵母细胞表达系统中Kir通道2.1、2.2和2.3的作用。Kir2.1、Kir2.2和Kir2.3通道在非洲爪的卵母细胞中异源表达。用电压钳技术测量相应的电流,并测量维拉帕米对电流的影响。在300µM的浓度下,维拉帕米抑制Kir2.1通道的初始电流的41.36%±2.7,Kir2.2通道下降16.51±3.6%,Kir2.3下降69.98±4.2%。因为维拉帕米对kir2.3的作用是一个以前未知的发现,我们进一步分析了这种影响。在用300μM维拉帕米清洗时,在输注后20分钟内观察到最大效果。用对照溶液冲洗后,只有部分电流恢复。在-120mV(73.2±3.7%)时,维拉帕米的电流降低相同,-40mV(85.5±6.5%),和0mV(61.5±10.6%)暗示块没有电压依赖性。在推定的结合位点使用定点突变,我们证明了孔突变体E291A的作用降低,而对D251A则没有维拉帕米的作用。用突变体I214L,这显示了对PIP2结合更强的亲和力,我们观察到归一化电流降低到对照电流的61.9±0.06%,与野生型通道相比,明显不那么明显。维拉帕米阻断Kir2.1、Kir2.2和Kir2.3亚基。在Kir2.3中,阻断依赖于位点E291和D251,并干扰经由PIP2的通道激活。对于其他Kir通道阻断药物,也已经描述了对这些位点和PIP2结合的干扰。由于Kir2.3优先在心房中表达,选择性Kir2.3阻断剂将构成一个有趣的抗心律失常概念.
The inwardly rectifying potassium current of the cardiomyocyte (IK1) is the main determinant of the resting potential. Ion channels Kir2.1, Kir2.2, and Kir2.3 form tetramers and are the molecular correlate of macroscopic IK1 current. Verapamil is an antiarrhythmic drug used to suppress atrial and ventricular arrhythmias. Its primary mechanism of action is via blocking calcium channels. In addition, it has been demonstrated to block IK1 current and the Kir2.1 subunit. Its effect on other subunits that contribute to IK1 current has not been studied to date. We therefore analyzed the effect of verapamil on the Kir channels 2.1, 2.2, and 2.3 in the Xenopus oocyte expression system. Kir2.1, Kir2.2, and Kir2.3 channels were heterologously expressed in Xenopus oocytes. Respective currents were measured with the voltage clamp technique and the effect of verapamil on the current was measured. At a concentration of 300 µM, verapamil inhibited Kir2.1 channels by 41.36% ± 2.7 of the initial current, Kir2.2 channels by 16.51 ± 3.6%, and Kir2.3 by 69.98 ± 4.2%. As a verapamil effect on kir2.3 was a previously unknown finding, we analyzed this effect further. At wash in with 300 µM verapamil, the maximal effect was seen within 20 min of the infusion. After washing out with control solution, there was only a partial current recovery. The current reduction from verapamil was the same at - 120 mV (73.2 ± 3.7%), - 40 mV (85.5 ± 6.5%), and 0 mV (61.5 ± 10.6%) implying no voltage dependency of the block. Using site directed mutations in putative binding sites, we demonstrated a decrease of effect with pore mutant E291A and absence of verapamil effect for D251A. With mutant I214L, which shows a stronger affinity for PIP2 binding, we observed a normalized current reduction to 61.9 ± 0.06% of the control current, which was significantly less pronounced compared to wild type channels. Verapamil blocks Kir2.1, Kir2.2, and Kir2.3 subunits. In Kir2.3, blockade is dependent on sites E291 and D251 and interferes with activation of the channel via PIP2. Interference with these sites and with PIP2 binding has also been described for other Kir channels blocking drugs. As Kir2.3 is preferentially expressed in atrium, a selective Kir2.3 blocking agent would constitute an interesting antiarrhythmic concept.