The human ether-a-go-go-related gene (hERG) encodes a potassium channel responsible for the cardiac delayed rectifier current (IKr) involved in ventricular repolarization. Drugs that block hERG have been associated with QT interval prolongation and serious, sometimes fatal, cardiac arrhythmias (including torsade de pointes). While displacement of [3H]dofetilide, a potent methanesulfonanilide hERG blocker, from cells heterologously expressing hERG has been suggested as a screening assay, questions have been raised about its predictive value.
To validate the utility of this assay as a screening tool, we performed a series of saturation and competition binding studies using [3H]dofetilide as ligand and either intact cells or membrane preparations from HEK 293 cells stably transfected with hERG K+ channels. The object of these experiments was to (1) compare binding Ki values for 22 hERG blockers using intact cells or membrane homogenates to determine whether maintaining cell integrity enhanced assay reliability; (2) evaluate the ability of different K+ concentrations (2, 5, 10, 20, and 60 mM) to modulate hERG binding; and (3) to establish the predictive value of the assay by comparing Ki values from binding studies at 5 and 60 mM [K+]o to functional IC50 values for hERG current block using 56 structurally diverse drugs.
We found (a) comparable Ki values in the intact cell and isolated membrane binding assays, although there were some differences in rank order; (b) increasing [K+]o lowered the Kd and increased the Bmax for [3H]dofetilide, particularly in the membrane assay; and (c) good correlation between binding Ki values and functional IC50 values for hERG current block.
In conclusion, increasing K+ concentrations results in an increase in both [3H]dofetilide affinity for hERG and available binding sites, particularly when using membrane homogenates. There are no meaningful differences between Ki values when comparing intact cell versus membrane assay, neither are there meaningful trends with increasing [K+]o within assays. There is good correlation between binding Ki values and functional (whole-cell patch clamp) IC50 values at both 5 and 60 mM K+ concentrations (R2 values of .824 and .863, respectively). The simplicity, predictability, and adaptability to high-throughput platforms make the [3H]dofetilide membrane binding assay a useful tool for screening and ranking compounds for their potential to block the hERG K+ channel.