Ion Channel High-throughput Screening Technology and Its Application

Ion channels are involved in a variety of fundamental physiological processes, and their malfunction leads to a variety of human diseases. Thus, ion channels represent an attractive class of drug targets and an important class of off-target sites for in vitro pharmacological assays. Rapid advances in functional assays and instrument development over the past few decades have enabled high-throughput screening (HTS) activities to be performed on an ever-expanding list of channel types. Chronologically, HTS approaches for ion channels include ligand-binding assays, flux-based assays, fluorescence-based assays, and automated electrophysiological assays.

Ligand binding assays have been widely used to screen for ion channel modulators. However, these assays are not considered functional assays because they measure the binding affinity of the compound to the ion channel rather than the ability to alter channel function. Ligand binding assays require prior knowledge of target binding sites and the formation of radiolabeled ligands specific for these binding sites. The activity of the test compound is indicated by the displacement of the labeled ligand. Therefore, conventional instruments can be used, where throughput represents its main advantage.

Ion flux assays are widely used in the pharmaceutical industry for drug discovery and hERG-related drug safety screening to identify potential QT responsibilities that may lead to fatal arrhythmias. However, these assays suffer from low temporal resolution (typically from seconds to minutes), uncontrolled membrane potential, less informative compared to voltage clamp, and lower throughput compared to fluorescence-based assays. In addition, the assay produces very weak signals for certain ion channels, which requires high levels of channel expression to achieve an acceptable signal-to-noise ratio.

Fluorescence-based methods do not directly measure ionic currents. Instead, they measure membrane potential-dependent or ion concentration-dependent changes in the fluorescent signal caused by ion flux. Because fluorescence-based methods yield robust and uniform measurements of cell populations, these assays are similar to those for other protein classes. As a result, greater instrument selection and expertise are available. Therefore, these assays are relatively easy to implement and optimize for higher throughput.

Patch clamp is widely regarded as the gold standard for direct recording of ion channel activity. This technique provides high-quality and physiologically relevant ion channel function data at the single-cell or single-channel (within a small patch of membrane) level. For pharmacological testing of compounds, it provides a standard for measuring the potency of compound-channel interactions. While conventional patch clamping provides a direct, informative, and real-time method for studying channel function, it is low-throughput and labor-intensive, requiring highly skilled and trained personnel. In the past decade, a breakthrough has been made in the development of automated planar patch clamping. Many automated electrophysiology platforms have been developed and are commercially available.

High-throughput electrophysiology has many theoretical advantages and holds great promise. The continued development of existing and new platforms for automated ion channel screening will keep pace with the need for ion channel safety analysis and ion channel-targeted drug discovery.

Before choosing the ideal screening method, it is important to determine what to look for when comparing techniques and their applications. Eight parameters commonly considered include sensitivity, specificity, throughput, temporal resolution, robustness, flexibility, cost, and physiological relevance. Of all analytical formats for ion channels, automated patch-clamp analysis is without a doubt the best option for providing high-quality data and allowing for higher throughput. Currently, automated electrophysiology testing remains expensive and not every laboratory can afford it.

Therefore, as a compromise, the combination of fluorescence-based screening techniques and automated patch clamping has become the most commonly used approach in ion channel-targeted drug discovery. As costs decrease and technology improves, automated electrophysiology will become the dominant form of assay for most ion channel subtypes. For different ion channel subclasses, high-throughput screening methods vary due to considerations of ion selectivity, channel activation kinetics, and the need for ligands.

Overall, advances and improvements in ion channel HTS technology have accelerated ion channel drug discovery. Detection of ion flux signals can be achieved using fluorescent indicator dyes and a fluorescent plate reader, such as a fluorescence imaging plate reader or FDSS (Hamamatsu Photonics). These assays have relatively low temporal resolution and information content, but are robust and low-cost. Electrophysiological methods have the most direct methods to measure ion channel activity and also allow flexibility for assay optimization for each channel type. The combination of non-electrophysiological and electrophysiological HTS methods provides an integrated and cost-effective approach to ion channel drug discovery and ensures high-quality data generation.