Houtmann Sylvie, Schombert Brigitte, Sanson Camille, Partiseti Michel, Bohme G Andrees
Integrated Drug Discovery, Sanofi R&D, 13 Quai Jules Guesde, F-94403, Vitry-sur-Seine, France.
Methods Mol Biol. 2017;1641:187-199. doi: 10.1007/978-1-4939-7172-5_10.
The human Ether-a-go-go Related Gene (hERG) product has been identified as a central ion channel underlying both familial forms of elongated QT interval on the electrocardiogram and drug-induced elongation of the same QT segment. Indeed, reduced function of this potassium channel involved in the repolarization of the cardiac action potential can produce a type of life-threatening cardiac ventricular arrhythmias called Torsades de Pointes (TdP). Therefore, hERG inhibitory activity of newly synthetized molecules is a relevant structure-activity metric for compound prioritization and optimization in medicinal chemistry phases of drug discovery. Electrophysiology remains the gold standard for the functional assessment of ion channel pharmacology. The recent years have witnessed automatization and parallelization of the manual patch-clamp technique, allowing higher throughput screening on recombinant hERG channels. However, the multi-well plate format of automatized patch-clamp does not allow visual detection of potential micro-precipitation of poorly soluble compounds. In this chapter we describe bench procedures for the culture and preparation of hERG-expressing CHO cells for recording on an automated patch-clamp workstation. We also show that the sensitivity of the assay can be improved by adding a surfactant to the extracellular medium.
人类醚 - 去极化相关基因（hERG）产物已被确定为心电图上家族性长QT间期形式以及药物诱导的相同QT段延长的核心离子通道。实际上，这种参与心脏动作电位复极化的钾通道功能降低会产生一种危及生命的室性心律失常，称为尖端扭转型室速（TdP）。因此，新合成分子的hERG抑制活性是药物发现的药物化学阶段中化合物优先级排序和优化的相关构效指标。电生理学仍然是离子通道药理学功能评估的金标准。近年来，手动膜片钳技术实现了自动化和并行化，从而能够对重组hERG通道进行更高通量的筛选。然而，自动化膜片钳的多孔板形式无法目视检测难溶性化合物的潜在微沉淀。在本章中，我们描述了用于在自动膜片钳工作站上记录的hERG表达CHO细胞的培养和制备的实验台程序。我们还表明，通过向细胞外培养基中添加表面活性剂可以提高检测的灵敏度。
