High-throughput electrophysiological assays for voltage gated ion channels using SyncroPatch 768PE

Abstract

Ion channels regulate a variety of physiological processes and represent an important class of drug target. Among the many methods of studying ion channel function, patch clamp electrophysiology is considered the gold standard by providing the ultimate precision and flexibility. However, its utility in ion channel drug discovery is impeded by low throughput. Additionally, characterization of endogenous ion channels in primary cells remains technical challenging. In recent years, many automated patch clamp (APC) platforms have been developed to overcome these challenges, albeit with varying throughput, data quality and success rate. In this study, we utilized SyncroPatch 768PE, one of the latest generation APC platforms which conducts parallel recording from two-384 modules with giga-seal data quality, to push these 2 boundaries. By optimizing various cell patching parameters and a two-step voltage protocol, we developed a high throughput APC assay for the voltage-gated sodium channel Nav1.7. By testing a group of Nav1.7 reference compounds’ IC50, this assay was proved to be highly consistent with manual patch clamp (R > 0.9). In a pilot screening of 10,000 compounds, the success rate, defined by > 500 MΩ seal resistance and >500 pA peak current, was 79%. The assay was robust with daily throughput ~ 6,000 data points and Z’ factor 0.72. Using the same platform, we also successfully recorded endogenous voltage-gated potassium channel Kv1.3 in primary T cells. Together, our data suggest that SyncroPatch 768PE provides a powerful platform for ion channel research and drug discovery.

Mechanism-specific assay design facilitates the discovery of Nav1.7-selective inhibitors

“…To validate the N1742K-based membrane potential assay, we characterized the 9,975 screening hits and 384 randomly selected compounds by using SyncroPatch 768 electrophysiology ( 18 ). All compounds were tested at a concentration of 6.6 µM on WT Nav1.7 channels.…”

“…To identify subtype-selective chemical scaffolds, large libraries of compounds, sometimes exceeding millions of individual compounds, are screened against the targets of interest (e.g., Nav1.7), followed by selectivity tests on other isoforms (e.g., Nav1.5). The effects of compounds are determined using in vitro assays such as electrophysiology ( 17 – 19 ) or fluorescence-based membrane potential assays ( 20 – 22 ). The major considerations for assay design have been signal robustness, throughput, and cost, whereas mechanisms of action, such as drug binding sites and molecular basis for selectivity, are often not taken into consideration.…”

Deep Mutational Scan of a cardiac sodium channel voltage sensor

“…Therefore, in terms of scale and cost-effectiveness, the Deep Mutational Scan approach compares favorably with both conventional manual patch-clamp as well as emerging methods such as automated patch-clamp or optical screening in multiwell plates. 39 A challenge of assaying Na V 1.5 function is that the channels' default state is closed, and they open for only ~5 ms in response to a rapid increase in the cell's membrane potential before inactivating. In this study, we used two sodium channel agonists to bias the cell towards the open state and promote sodium influx, which we made more toxic to the cell by treatment with an inhibitor of the Na+/K+ exchanger.…”

Sensing Senses: Optical Biosensors to Study Gustation

“…In addition, optical tissue clearing protocols start also to be applied to 3D cultures, which could largely improve expression analysis in three dimensions [340]. For enhanced throughput of electrical recordings, new devices such as multielectrode arrays or automatic patch-clamp platforms allow to clamp hundreds of cells simultaneously [341].…”