Recording ten-fold larger IKr conductances with automated patch clamping using equimolar Cs+ solutions

Abstract

Background: The rapid delayed rectifier potassium current (I_Kr) is important for cardiac repolarization and is most often involved in drug-induced arrhythmias. However, accurately measuring this current can be challenging in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes because of its small current density. Interestingly, the ion channel conducting I_Kr, hERG channel, is not only permeable to K⁺ ions but also to Cs⁺ ions when present in equimolar concentrations inside and outside of the cell. Methods: In this study, I_hERG was measured from Chinese hamster ovary (CHO)-hERG cells and hiPSC-CM using either Cs⁺ or K⁺ as the charge carrier. Equimolar Cs⁺ has been used in the literature in manual patch-clamp experiments, and here, we apply this approach using automated patch-clamp systems. Four different (pre)clinical drugs were tested to compare their effects on Cs⁺- and K⁺-based currents. Results: Using equimolar Cs⁺ solutions gave rise to approximately ten-fold larger hERG conductances. Comparison of Cs⁺- and K⁺-mediated currents upon application of dofetilide, desipramine, moxifloxacin, or LUF7244 revealed many similarities in inhibition or activation properties of the drugs studied. Using equimolar Cs⁺ solutions gave rise to approximately ten-fold larger hERG conductances. In hiPSC-CM, the Cs⁺-based conductance is larger compared to the known K⁺-based conductance, and the Cs⁺ hERG conductance can be inhibited similarly to the K⁺-based conductance. Conclusion: Using equimolar Cs⁺ instead of K⁺ for I_hERG measurements in an automated patch-clamp system gives rise to a new method by which, for example, quick scans can be performed on effects of drugs on hERG currents. This application is specifically relevant when such experiments are performed using cells which express small I_Kr current densities in combination with small membrane capacitances.

Keywords: Cs+; automated patch clamp; cardiac electrophysiology; conductance; drugs; hERG; hiPSC-CM; ion channel.

FIGURE 1

hERG Current traces and conductance for K⁺ and Cs⁺ solutions with different concentrations of desipramine, dofetilide, LUF7244, and moxifloxacin treatment in CHO-hERG cells. Data are shown as mean ± SEM.

FIGURE 2

Concentration response curves for different drugs with either Cs⁺ or K⁺ solutions of the fully activated hERG current at + 40 mV.

FIGURE 3

I_Kr measurements in hiPSC-CM with K⁺ solutions (n = 36) and Cs⁺ solutions (n = 40). The left panels show a current trace for baseline (light blue) and after acute treatment with the hERG inhibitor E4031 (10 µM, dark blue) in K⁺ solutions (upper) and Cs⁺ solutions (lower). The right panels show the relation between test potential and conductance of the E4031-sensitive component of the tail current in K⁺ solutions (upper) and Cs⁺ solutions (lower).