High throughput automated patch-clamp of cardiac ion channels using physiological fluoride-free internal solution

Abstract

Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (Na_V) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40 % for GΩ seals and have also extended the approach to include the medium throughput device, the Patchliner. We demonstrate the method using hERG expressed in HEK cells, as well as Na_V1.5, and K_Ca3.1 expressed in CHO cells. Although the voltage dependence of activation of hERG was not affected by internal fluoride, the voltage-dependence of activation and inactivation of Na_V1.5 was shifted to more negative potentials when fluoride was present (V_half,Act = −25.2 ± 0.5 mV (241) in fluoride-free versus − 37.5 ± 0.4 mV (484) in standard internal; V_half,Inact = −53.6 ± 0.6 mV (165) in fluoride-free versus − 68.4 ± 0.4 mV (728) in standard internal), whereas activation of K_Ca3.1 by internal calcium was more robust when physiological internal solution was used. In preliminary recordings we could successfully use the fluoride-free approach for experiments involving stem cell-derived cardiomyocytes. Therefore, APC data are more comparable to recordings done using manual patch clamp and physiological solutions. Ultimately, this will enhance ion channel characterization and cardiac safety testing for cell lines, induced pluripotent stem cells and primary cells.