Department of Medical Physiology, Division of Heart & Lungs, University Medical Center Utrecht, Yalelaan 50, 3584 CM Utrecht, The Netherlands.
Cardiovasc Res. 2013 Jul 1;99(1):203-14. doi: 10.1093/cvr/cvt103. Epub 2013 Apr 25.
In excitable cells, KIR2.x ion-channel-carried inward rectifier current (IK₁) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific IK1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of IK1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits IK₁ by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific IK₁ inhibitors based on the P structure.
We analysed seven pentamidine analogues (PA-1 to PA-7) for IK₁ blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC₅₀ values (12-15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG -44.1 kJ/Mol) than for P (ΔG -31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect INav1.5, ICa-L, IKv4.3, IKv11.1, and IKv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of IK₁ in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM.
PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK₁ block.
在可兴奋细胞中，KIR2.x 离子通道介导的内向整流电流（IK₁）被认为设定了负的和稳定的静息膜电位，并有助于动作电位复极化。功能丧失或获得性突变与心律失常和病理性重塑相关，而病理性重塑会影响正常的 KIR2.x 蛋白水平。目前尚无特定的 IK1 抑制剂可用于体内使用，这严重阻碍了对 IK1 在正常心脏生理学和病理生理学中精确作用的研究。二胺抗原生动物药物戊脒（P）通过堵塞通道的细胞质孔区域，急性抑制 IK₁。我们旨在基于 P 结构开发更有效和更特异的 IK₁ 抑制剂。
我们使用表达 KIR2.1 的 HEK-293 细胞的内向外膜片钳，在 200 nM 时分析了七种戊脒类似物（PA-1 至 PA-7）对 IK₁ 阻断的效力。PA-6 表现出最高的效力，并进一步进行了测试。PA-6 以低 IC₅₀ 值（12-15 nM）阻断人和鼠的 KIR2.x 电流。建模表明，PA-6 与细胞质通道孔的静电相互作用较小，但亲脂性相互作用较大，导致 PA-6 对通道的亲和力高于 P（ΔG -44.1 kJ/Mol）（ΔG -31.7 kJ/Mol）。实验证实酸性氨基酸残基 E224 和 E299 参与了药物-通道相互作用。PA-6 不影响 200 nM 时的 INav1.5、ICa-L、IKv4.3、IKv11.1 和 IKv7.1/minK 电流。PA-6 抑制分离的犬成年心室肌细胞（CM）中内向（50 nM 为 40%；100 nM 为 59%；200 nM 为 77%）和外向（50 nM 为 40%；100 nM 为 76%；200 nM 为 100%）IK₁ 成分。PA-6 分别在 50、100 和 200 nM 时将 CM 的动作电位持续时间延长了 8%（n = 9）、26%（n = 5）和 34%（n = 11）。与 P 不同，PA-6 在 0.1 至 3 μM 的浓度下对 KIR2.1 通道表达没有影响。然而，PA-6 在 10 μM 时增加了 KIR2.1 的表达水平。此外，PA-6 除在 10 μM 时外，对 hERG 的成熟没有影响。
PA-6 对 KIR2.x 介导的电流的效率和特异性均高于 P，可延长动作电位持续时间，在完全阻断 IK₁ 的相关浓度下不影响通道转运。
