Automated Patch-Clamp Methods for the hERG Cardiac Potassium Channel

Abstract

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.

Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells

“…These compounds display variable effects on hERG currents 12,35 and should be characterized in the assay systems used. Surfactants such as the poly(ethylene oxide)-poly(propylene oxide) block copolymer Pluronic F-68 can be used up to 1% v/v in APC experiments on QPatch platforms without affecting hERG current amplitude 36 .…”

Profiling antimicrobial peptides from the medical maggotLucilia sericataas potential antibiotics for MDR Gram-negative bacteria

“…AMPs were tested at concentrations of 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200 and 400 μM. AMP interaction with the human Ether-à-go-go-Related Gene (hERG) potassium channel was analysed using an automated patch-clamp method as previously described, 25 with AMPs at concentrations of 0.12, 0.37, 1.1, 3.3, 10 and 30 μM.…”

Antimicrobial Peptides from Rat-Tailed Maggots of the Drone Fly Eristalis tenax Show Potent Activity against Multidrug-Resistant Gram-Negative Bacteria

“…The interaction of the peptides with the human ERG (hERG) potassium channel was analyzed using the automated patch-clamp method [13]. Chinese hamster ovary (CHO) hERG Duo cells constitutively expressing hERG (B'SYS) were diluted to 8 × 10 6 cells/mL in Ex-Cell CHO medium (Sigma-Aldrich) containing 25 mM HEPES, 100 U/mL penicillin-streptomycin, and 0.004% soybean trypsin inhibitor.…”

“…We then investigated the cardiotoxicity of the peptides by measuring the effect on hERG. This voltage gated potassium channel is important for the repolarization of the cardiac action potential, and functional disruption can cause fatal ventricular tachyarrhythmia (torsade de pointes) [13,21]. No target-specific activity was observed.…”