Characterization of Potassium Channel Modulators with QPatch^™ Automated Patch-Clamp Technology: System Characteristics and Performance

Abstract

Planar silicon chips with 1-2-microm etched holes (average resistance: 2.04 +/- 0.02 MOmega in physiological buffer, n = 274) have been developed for patch-clamp recordings of whole-cell currents from cells in suspension. An automated 16-channel parallel screening system, QPatch 16, has been developed using this technology. A single-channel prototype of the QPatch system was used for validation of the patch-clamp chip technology. We present here data on the quality of patch-clamp recordings and from actual drug screening studies of human potassium channels expressed in cultured cell lines. Using Chinese hamster ovary (CHO) and human embryonic kidney cells (HEK), gigaseals of 4.1 +/- 0.4 GOmega (n = 146) and high-quality whole-cell current recordings were obtained from hERG and KCNQ4 potassium channels. Success rates for gigaseal recordings varied from 40 to 95%, and 67% of the whole-cell configurations lasted for >20 min. Cells were maintained in suspension up to 4 h in a cell storage facility that is integrated in the QPatch 16. No decline in patchability was observed during this time course. A series of screens was conducted with known inhibitors of the hERG and KCNQ4 potassium channels. Dose-response relationship characterizations of verapamil and rBeKm-1 blockage of hERG currents provided IC(50) values similar to values reported in the literature.

Preclinical Drug Safety and Cardiac Ion Channel Screening

“…A good pharmacological correlation with conventional MPC data was observed in terms of rank-order of compounds with hERG activity although the concentration-response curves were shifted to the right (within 3-to 5-fold). PatchXpress, QPatch, and Patchliner allow stable recordings of high-quality hERG current from giga-ohm seals for 20-30 min, allow the complete concentration-response experiment to be performed on a single cell, and exhibit good pharmacological data correlation with conventional MPC experiments, although potency shift has also been reported for one of the automated patch-clamp systems [25][26][27][28][29]. In comparison to the MPC technique, the most significant advantage of the APC technique is high throughput, which facilitates analysis of structure-activity relationships across compounds.…”

CHAPTER 4. Venoms-Based Drug Discovery: Bioassays, Electrophysiology, High-Throughput Screens and Target Identification

“…109 In addition, a glass-coated microuidics channel has been incorporated into the Qube, reducing the inadvertent loss of compounds, and IC 50 values obtained using this technology are generally in good agreement with literature values obtained using conventional electrophysiology. 121,[125][126][127] The Flyscreen 8500 (FlyIon) uses 'ip-the-tip' technology, an adaption of traditional electrophysiology in which suction pulses applied to a cell inside a glass pipette result in true whole-cell conguration, gigaohm seals and resultant high-quality electrophysiology data. 105,128 This platform is fully automated and requires comparatively low volumes of compound.…”

Beat-to-Beat Variability in Field Potential Duration in Human Embryonic Stem Cell-Derived Cardiomyocyte Clusters for Assessment of Arrhythmogenic Risk, and a Case Study of Its Application

“…Its basic protocol has been described previously [21]. The external solution consisted of (in mM) 145 NaCl, 4 KCl, 2 CaCl 2 , 1 MgCl 2 , 10 glucose, and 10 HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) (pH adjusted to 7.4 with 1 M NaOH, and osmolarity to 305 mOsm with 1 M sucrose), and the internal solution consisted of (in mM) 120 KCl, 5.374 CaCl 2 , 1.75 MgCl 2 , 10 EGTA (ethylene glycol-bis(2-aminoethylether)-N, N, N', N'-tetraacetic acid), and 10 HEPES (pH adjusted to 7.2 with 1 M KOH, and osmolarity to 300 mOsm with 1 M sucrose).…”