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

Date Published: July 6, 2017

Publisher: Public Library of Science

Author(s): Tianbo Li, Gang Lu, Eugene Y. Chiang, Tania Chernov-Rogan, Jane L. Grogan, Jun Chen, Giuseppe Pignataro.

http://doi.org/10.1371/journal.pone.0180154

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.

Partial Text

Ion channels are involved in a broad spectrum of physiological processes such as neuronal firing, muscle contraction, hormone secretion and T cell activation [1]. Many ion channels have been identified as important therapeutic targets, including the voltage-gated sodium channel Nav1.7 and the voltage gated potassium channel Kv1.3. Nav1.7 is preferentially expressed in sensory neurons and is implicated as the threshold channel for pain sensation [2,3]. In humans, loss of function mutations of Nav1.7 lead to congenital insensitivity to pain (CIP), whereas gain of function mutations of Nav1.7 cause inherited erythermalgia (IEM) and proxysmal extreme pain disorder (PEPD) syndromes [4–6], therefore Nav1.7 antagonists should have an application in pain management. Kv1.3 regulates membrane potential and Ca2+ signaling in T cells, and its expression is enhanced in CD4+ and CD8+ cells following T cell receptor activation [7–9]. The inhibition of Kv1.3 suppresses Ca2+-signaling, cytokine production, and proliferation of autoantigen-specific T cells. Therefore Kv1.3 blockers may have utility in autoimmune diseases treatment [10]. Despite the recent advances in channelopathies and protein structures, the discovery of ion channel therapeutics is still facing a major challenge from the limitation of assay technologies.

Besides SyncroPatch, another two comparable APC systems, IonWorks Barracuda and Qube, each with a single 384 module, also have the capacity of parallel recording of 384 cells. IonWorks Barracuda was launched in 2010 and has been reported for compound screening on hERG, CaV2.2 and Nav channels [16–18]. Different from the other two systems, Barracuda uses perforated patch configuration, and the averaged seal resistance was ~120 MΩ for single-hole mode and ~35 MΩ for population patch mode (64 holes). Qube was introduced in 2014 and shared many similar features with SyncroPatch, including using a 384 format design (e.g., 384 channel digital amplifier, 384 pipetting robot and 384 well borosilicate glass chips) and a programmable negative pressure system to achieve whole cell configuration, thus with potential for giga-seal quality recording. In a recently reported Nav1.7 modulator screening by using Qube system, ~80% wells achieved > 15 MΩ seal resistance (in 10-hole population patch mode), equating to 150 MΩ resistance for each single cell recording, with daily throughput ~2300 compounds [29]. SyncroPatch 768PE was also developed in 2014 using negative pressure to form high quality whole cell configuration as Qube, but was equipped with different amplifier, non-micro-fluid planar chip design, and two-384 modules on a Biomek automation workstation. In our optimized Nav1.7 inhibitor screening using SyncroPatch 768PE system, 92% recordings achieved > 200 MΩ seal resistance, and 90% recordings reached > 500 MΩ seal resistance (Fig 6B), and the daily throughput was ~6,000 compounds. Our data suggested that SyncroPatch 768PE can generate both higher-quality and higher throughput data in our optimized Nav1.7 assay.

 

Source:

http://doi.org/10.1371/journal.pone.0180154

 

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