Inhibition of hERG K channels by verapamil at physiological temperature: Implications for the CiPA initiative

Abstract

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative reassesses using the inhibition of hERG potassium channels by drugs as the major determinant for the potential to cause drug-induced Torsades de Pointes (TdP) cardiac arrhythmias. Here we report our findings on the next phase of CiPA: Determination of hERG inhibitory properties using the standard CiPA-defined data acquisition protocol, here called the standard protocol, at physiological temperature (37 degrees Celsius). To do this, we measured inhibition of hERG1a potassium channels stably expressed in HEK293 cells by the small molecule verapamil, using manual whole-cell patch-clamp electrophysiology recordings with the standard protocol, which is characterized, in part, by a series of 10 s duration voltage steps to 0 mV, ultimately leading to a cumulative recording time of approximately 30 min. Using the standard protocol, we measured an IC₅₀ for verapamil of 225 nM, a Hill coefficient of 1, and time constant of inhibition at 0 mV of 0.64 s. But, using the standard protocol resulted in a very low (5 %) experimental success rate per cell, which had low practicality for future experiments. To address the 5 % success rate, we generated a revised protocol characterized, in part, by a series of 3 s duration voltage steps to 0 mV, leading to a cumulative recording time of approximately 10 min. Using the revised protocol, we found an IC₅₀ for verapamil of 252 nM, a Hill coefficient of 0.8, and time constant of inhibition at 0 mV of 0.67 s. The values measured with the revised protocol were similar to those measured using the standard protocol and, furthermore, our success rate using the revised protocol rose to 25 %, an increase of 5-fold over the standard protocol, and more in line with the success rate for biophysical studies. In summary, we captured key pharmacological data for subsequent analysis in CiPA using a revised protocol with an increased success rate and an overall enhanced feasibility and practicality. We propose that the revised protocol may be more pragmatic for generation of some hERG channel drug inhibition data for CiPA and other regulatory sciences.