Whole-cell patch clamp recording for cardiac potassium ion channels KV1.5, KVLQT1/minK, KV4.3 and Kir2.1 on QPatch automated patch clamp system
Cardiac ion channel activity is important to generate cardiac action potentials in appropriate timing and duration. Drug-induced impairment of those ion channels cause abnormal cardiac activity such as QT interval prolongation, ventricular arrhythmia and, in most serious cases, sudden death. These effects are one of the leading causes for drug withdrawal from the market or denied regulatory approval of new therapeutic candidates. Although ICH guideline E14/S7B Q&A published in February focused on IKr (hERG) as well as INa (NaV1.5) and ICa,L (CaV1.2), the other cardiac ion channel targets, including many potassium channels such as IKur (KV1.5), Ito1 (KV4.3), IKs (KVLQT1/minK) and IK1 (Kir2.1) are also important to understand the drug effect on the generation of action potentials in cardiomyocytes. To establish the screening assay for those cardiac potassium channels, we have established the whole cell experiment method using QPatch automated patch clamp system.
Voltage protocols customized for each ion channel were applied to measure biophysical properties and current stability. The currents elicited by the designated voltage protocols were measured in each ion channel, and the half-inactivated voltages (V1/2) were calculated using Boltzmann fitting. The average inactivation V1/2 for KV4.3 and KV1.5 was -39.5 mV and -9.5 mV, respectively. The reference compounds for KV4.3 (Flecainide IC50=11.6µM), KV1.5 (4-Aminopyridine IC50=125.1µM), and KVLQT1/minK (Chromanol 293B IC50=17.4µM, XE-991 IC50=1.7µM) were also assessed. KVLQT1/minK is known to have severe rundown of the currents. The smallest rundown condition was observed in the modified intracellular solution and extracellular solutions as well as shorten voltage protocols. The remaining currents after vehicle solution application for 6 times (approximately 500 seconds) in the best successful combination were 67% in average of groups of experiments.
The optimized assays on QPatch enable measuring the biophysical properties of those four ion channels and assessing the pharmacological effect of reference compound for each of them. QPatch allows the assessment of a novel compounds proarrhythmic risk on those cardiac ion channels other than hERG, NaV1.5 and CaV1.2.