Optimization of a Cav1.2 Cell Line for Use on QPatch and PatchXpress

Abstract

Ion channel currents comprise the cardiac action potential and are important in cardiac safety liability assessment of potential drug candidates. The gold standard for assessing ion channel activity is the voltage clamp technique, but this technique is a very low throughput process. Planar patch technology (QPatch and PatchXpress) allows for moderate throughput by providing automated, simultaneous whole cell voltage clamp recordings from cells heterologously expressing the channel of interest. Ion channels routinely screened for cardiovascular safety are hERG (Kv11.1), Nav1.5, Kir2.1 and KvLQT/minK using either PatchXpress or QPatch instruments. In this study, we highlight the validation of Cav1.2 (L-type calcium channel) on our automated electrophysiology systems for cardiovascular safety screening. The L-type calcium channel is expressed in the cardiovascular system both in smooth and cardiac muscle. Potent L-type calcium channel antagonists can lower blood pressure, reduce cardiac contractility, and potentially increase the P-R interval on the electrocardiogram (ECG). Cav1.2 was expressed in CHO cells using a tetracycline inducible vector. Because of this we needed to optimize expression level by varying the induction variables. Addition of an L-type antagonist (verapamil) also provided benefit by keeping well-expressing cells viable after induction. In order to optimize flexibility in performing experiments, we also prepared the cells as a cryo-preserved subtrate. Cav1.2 channel kinetics for both activation and inactivation were investigated, and potencies of 8 reference compounds (weak and strong antagonists) were assessed on both platforms. In conclusion, we have optimized tissue culture conditions, cell preparation and voltage clamp protocols on two automated electrophysiology platforms to provide cardiac safety evaluation of drug candidates using an inducible Cav1.2 cell line.