Propafenone blocks human cardiac Kir2.x channels by decreasing the negative electrostatic charge in the cytoplasmic pore
Propafenone blocks human cardiac Kir2.x channels by decreasing the negative electrostatic charge in the cytoplasmic pore
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DOI：
10.1016/j.bcp.2013.04.023
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发表时间：
2013-07-15
影响因子：
5.8
通讯作者：
Delpon, Eva
中科院分区：
文献类型：
--
作者：
Amoros, Irene;Dolz-Gaiton, Pablo;Delpon, Eva
人体心脏内向整流电流 (I-K1) 由 Kir2.x 通道产生。 In 的抑制可以提供一种有效的抗心律失常策略来对抗颤动性心律失常。因此，必须阐明仍然难以捉摸的 Kir2.x 通道药理学。我们表征了抗心律失常普罗帕酮对 Kir2.x 通道 (I-Kir2.x) 产生的电流和人心房肌细胞中记录的 I-K1 产生的抑制的电生理学和分子基础。将野生型和突变的人 Kir2.x 通道瞬时转染到 CHO 和 HEK-293 细胞中。使用膜片钳技术记录宏观和单通道电流。在浓度 >1 μM 普罗帕酮抑制 I-Kir2.x 时，效力顺序为 Kir23，类似于 I-K1 > Kir2.2 > Kir2.1 通道。阻断与细胞外 K+ 浓度无关，而当细胞内 K+ 浓度显着增加时。浓度降低了。普罗帕酮降低了内向整流，因为在 K+ 平衡正电位处，普罗帕酮诱导的阻滞以电压依赖性方式降低。重要的是，普罗帕酮有利于 Kir2.x 通道中出现亚电导水平，并降低磷脂酰肌醇 4,5-二磷酸 (PIP2) 通道亲和力。盲对接和定点诱变实验表明，普罗帕酮在细胞质结构域结合 Kir2.x 通道，靠近但不在孔本身，涉及两个保守 Arg 残基（Kir2.1 中的残基 228 和 260）的结合位点。我们的结果表明，普罗帕酮并入通道的细胞质结构域，从而减少了 K+ 离子和多胺感知的净负电荷，进而促进亚电导水平的出现和通道 PIP2 亲和力的降低。 (C) 2013 Elsevier Inc. 保留所有权利。
Human cardiac inward rectifier current (I-K1) is generated by Kir2.x channels. Inhibition of In could offer a useful antiarrhythmic strategy against fibrillatory arrhythmias. Therefore, elucidation of Kir2.x channels pharmacology, which still remains elusive, is mandatory. We characterized the electrophysiological and molecular basis of the inhibition produced by the antiarrhythmic propafenone of the current generated by Kir2.x channels (I-Kir2.x) and the I-K1 recorded in human atrial myocytes. Wild type and mutated human Kir2.x channels were transiently transfected in CHO and HEK-293 cells. Macroscopic and single-channel currents were recorded using the patch-clamp technique. At concentrations >1 mu M propafenone inhibited I-Kir2.x, the order of potency being Kir23 similar to I-K1 > Kir2.2 > Kir2.1 channels. Blockade was irrespective of the extracellular K+ concentration whereas markedly increased when the intracellular K+. concentration was decreased. Propafenone decreased inward rectification since at potentials positive to the K+ equilibrium potential propafenone-induced block decreased in a voltage-dependent manner. Importantly, propafenone favored the occurrence of subconductance levels in Kir2.x channels and decreased phosphatidylinositol 4,5-bisphosphate (PIP2)-channel affinity. Blind docking and site-directed mutagenesis experiments demonstrated that propafenone bound Kir2.x channels at the cytoplasmic domain, close to, but not in the pore itself, the binding site involving two conserved Arg residues (residues 228 and 260 in Kir2.1). Our results suggested that propafenone incorporated into the cytoplasmic domain of the channel in such a way that it decreased the net negative charge sensed by K+ ions and polyamines which, in turn, promotes the appearance of subconductance levels and the decrease of PIP2 affinity of the channels. (C) 2013 Elsevier Inc. All rights reserved.