New strategies in ion channel screening for drug discovery

Medicilon provides a wide range of ion channel assays to quickly and accurately provide results for drug discovery and development research worldwide. We offer ion channel screening services on a variety of different high quality electrophysiology platforms. Email:marketing@medicilon.com.cn web:www.medicilon.com

1. Introduction

Ion channels are essential for every breath, step or heartbeat, and occur in each cell of the human body. Therefore, they play an important role in pathophysiology, and consequently also as drug targets. The gold standard for direct, real-time measurements of ion channel activity is the patch clamp technique, celebrating its 33rd birthday this year. Patch clamp is an extraordinary technique when you think about it: a glass tube is pulled to a tip diameter of micrometer dimensions and after filling with buffer, it is carefully pressed onto a cell’s membrane. A tight interaction forms between the glass and the cellular membrane, the ‘gigaseal’, with giga referring to the Giga-Ohm electrical resistance between the interior of the patch clamp pipette and the cell bath. A brief suction pulse ruptures the membrane patch covering the pipette tip, offering full electrical contact and control over the cellular membrane. In this way, the ion channel activity, measured as transmembrane currents flowing over the open channels, and response to voltage pulses or added compounds can be monitored with ultra-high resolution (µs, pA). 2. Patch clamp -- the gold standard Patch clamp yields detailed information not only about the ion channel itself, but also about the interaction between ion channel and compound, and is therefore suitable for a broad range of pharmacological experiments: kinetics, permeability, dose--response relationship, and state- and usedependence of compound interactions. However, because of its tedious nature, manual patch clamp is light years from being adaptable for ion channel drug screening, where the throughput of 104 -- 105 data points are required per day, compared with a daily throughput of roughly 10 data points for manual patch clamp. Consequently, screeners had to rely on other techniques for ion channel screening, often indirect and with drastically lower sensitivity and information content than patch clamp, and with limited or no means to investigate state- and use-dependence of ion channels and compounds, due to poor or no control of cell membrane voltage. Only far down the discovery path, promising drug candidates were analyzed using patch clamp. This gap between throughput, functionality and sensitivity spurred the development of machines doing the patch clamping. The pipettes were replaced by micro-machined plates containing micron-sized apertures, where a cell was captured from solution using suction. In this way, the manual handling was reduced to preparing cells and solutions and adding them to the recording substrate or machine, described already in 2002. The first commercial products followed shortly; the PatchXpress from Axon Instruments (acquired by Molecular Devices in 2005) the Ionworks from Molecular devices, and the Port-a-Patch from Nanion Technologies. The Port-a-Patch, launched in 2003, records from only one cell at a time, and was early adopted by academic and industrial laboratories replacing conventional patch clamp setups. Until now, about a dozen of different platforms have entered the market, with different technical solutions and standpoints regarding data quality, for excellent reviews on automated patch clamp (APC) platforms see Dunlop and Comley. PatchXpress was the first system supporting gigaseal recordings from 16 cells in parallel, followed by the high data quality systems QPatch 16 (2004, Sophion Biosciences) and the Patchliner (2006, Nanion Technologies). The Ionworks platforms (HT, Quattro, and Barracuda from Molecular Devices) do not rely on gigaseal recordings and have in comparison with other platforms limited perfusion capabilities. The Ionworks Barracuda has proven to work excellently for diverse ion channel targets, but can be a limitation when it comes to more challenging targets, for example, ligandgated ion channels exhibiting rapid desensitization kinetics, or investigations of drug use-dependency of voltage-gated ion channels. For studies on drug use-dependence, precise control of the membrane potential is essential for accurate results, where a high-quality seal in combination with a low series resistance allow for a good voltage-clamp of the membrane. Relying on a low seal resistance, ‘loose patch,’ in combination with high series resistance conferred by perforated patch recording, makes a good voltage control of the membrane difficult, if not impossible. Ionworks HT and Barracuda were the first platforms supporting recordings from cellular populations, that is, substrates with multiple apertures instead of only one, in this case 64, and 384 parallel recordings, respectively, and have provided significant input on screening broad range of ion channels. Population patch clamp has proven very helpful with cell lines exhibiting a highly variable protein expression rate or if the ion channel has a low conductance. A screening assay is never better than the cell line, meaning that care needs to be taken to find a reasonable protein expression level, and to optimize parameters affecting gigaseal formation and stability. There is a fairly great variation between existing APC platforms in how much assay development is required to get acceptable success rates for a given cell line. Putting it simple, some platforms are more forgiving than others. 3. Development of APC platforms Over the past decade, the increases in obtainable throughput in patch clamping or the numbers of parallel recording channels of the instruments have steadily scaled upward. This somewhat reminds of the scaling in semiconductor technology, nicely described by the famous law of the co-founder of Intel, Gordon E. Moore: Moore’s law is the observation that, over the history of computing hardware, the number of transistors on integrated circuits doubles approximately every 2 years. However, for our APC devices the number of parallel recording channels increased with one order of magnitude every 4 years (Figure 1). We started with one recording channel in 2003 with the Port-a-Patch, and in 2013 we introduced the SyncroPatch 384/768PE platform capable of 768 parallel whole-cell, gigaseal recordings. In 2014, Sophion introduced the Qube, also capable of 384 parallel gigaseal recordings. At this point, a throughput has been reached that fulfills the requirements of high-throughput screening (HTS) with a machine setup and design allowing full integration into HTS-environments and processes. For example, the SyncroPatch 384PE employs automated liquid handling using a 384 channel pipettor head, a 384 channel parallel amplifier and offers a daily throughput of 20,000 data points per day, while still maintaining high-quality, gigaseal recordings and fast fluidics suitable for both voltage- and ligandgated targets. One unique property of this system is the open design, allowing for full integration into automated drug screening environments, both regarding hardware and software. It remains to be seen how far this will push the boundaries of APC in HTS.