Ion channels are a very important membrane protein family involved in a variety of fundamental physiological processes. Their malfunction causes a variety of human diseases. Therefore, ion channels represent a class of attractive drug targets and a class of important off-targets for in vitro pharmacological profiling. In the past decades, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. This review will summarize the current technologies and commonly used screening methods for different ion channel classes.

Introduction of Ion Channel Screening

Traditionally, patch clamp electrophysiology is the gold standard for ion channel studies. However, the method is labor-intensive with a low throughput and requires highly trained staff to perform the experiments. Ion channels are difficult targets to be investigated using high throughput approaches, which hinders the use of ion channels compared with other targets. Recently, the rapid progress in developing functional assays and instrumentation has enabled high throughput screening (HTS) campaigns on an expanding list of channel types. Consequently, HTS was designed to identify active compounds for ion channel targets, which are of great interest to academic and industrial researchers.

High Throughput Screening Technologies

In the past, HTS methods for ion channels have been extensively developed and applied to most ion channels. In chronological order, the approaches include: the ligand binding assay, flux-based assay, fluorescence-based assay and automated electrophysiological assay.

• Ligand binding assays

Ligand binding assays have been widely used to screen for ion channel modulators. However, these assays are not considered as functional assays because they detect the binding affinity of a compound to an ion channel rather than the ability of altering channel function. Ligand binding assays require a previous knowledge of the target binding sites and of the formation of a radio-labeled ligand which is specific to those binding sites. Activity of the test compound is indicated by the displacement of the labeled ligand. Consequently, conventional instrumentation may be used, in which throughput represents its major strength. Thus far, the assay format has been rarely used for general screening but is still good for identifying modulators specific to some given ligands.

• Flux-based assays

Ion flux assay has been successfully applied to directly access a functional change of ion channel activity. Radioactive isotopes have been used to trace the cellular influx or efflux of specific ions, such as Na, Ca²⁺ and Rb, for the studies of Na, Ca²⁺ and K channels, respectively. A commonly used assay format is the Rb efflux for K channels or non-selective cation channels. In this format, the cells that express the ion channel of interest are incubated with a buffer that contains Rb for several hours before they are washed and stimulated with an agonist to allow for Rb efflux. Then the cells and supernatant are collected for radioactive counting. However, these assays have the disadvantages of low temporal resolution (typically from seconds to minutes), uncontrolled membrane potential, less information content compared with voltage-clamping and lower throughput compared with fluorescence-based assays. Furthermore, this assay generates a very weak signal for some ion channels, which requires a high level of channel expression to achieve an acceptable signal-to-noise ratio.

• Fluorescence-based assays

Fluorescence-based methods do not directly measure ionic current. Rather, they measure either the membrane-potential-dependent or ion-concentration-dependent changes of fluorescence signals as a result of ionic flux. Because fluorescence-based methods produce a robust and homogeneous cell population measurement, these assays are similar to those for other protein classes. Therefore, more instrument choices and expertise are available. Consequently, these assays are relatively easy to implement and to optimize to achieve a higher throughput.

• Automated electrophysiological assays

Patch-clamp has been widely considered as the gold standard to directly record ion channel activity. This technology provides high quality and physiologically relevant data of ion channel function 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.

Fig. 1 Pipeline of high-throughput screening targeting ion channels. (Yu, H. et al., 2016)

Prevalent HTS Methods for The Specific Ion Channel Families

Before choosing the ideal screening method(s), it is important to determine what to look for when comparing technologies and their applications. Eight parameters commonly considered include sensitivity, specificity, throughput, temporal resolution, robustness, flexibility, cost, and physiological relevance. Because there are many ion channel families and subclasses available, the most commonly used screening methods will be discussed based on ion selectivity or permeability.

• Potassium-selective channels

Potassium-selective channels are the largest and most diverse group among the ion channel families. The classes of channels include voltage-gated (K_v), inward-rectifying (K_IR), two-pore (K_2P) and Ca²⁺-activated (K_Ca) potassium channels. Multiple assay formats have been applied to this large family, including the ligand binding assay, Rb flux assay, voltage-sensitive dye-assay and Tl flux assay. Among them, the Tl flux assay is most often used to identify potassium channel modulators⁺⁺⁺.

• Ca²⁺-involved ion channels

An intracellular calcium ion (Ca²⁺) is a universal second messenger that controls both physiological and pathological processes. Depending on the application, calcium dyes are available in a range of affinities to calcium ions, excitation and emission spectra, and chemical forms (membrane permeable or not). They show different temporal resolution (from milliseconds, such as Fluo-3 and Fluo-4, to tens of seconds, such as Fura-2), and different degrees of accuracy for each range of calcium concentrations. For the high-throughput screening of Ca²⁺-involved ion channels, commercial kits, such as the Fluo-4 calcium assay kits, are available. They are extensively applied in Ca²⁺-involved ion channel assays, such as voltage-gated calcium channels, TRP channels, and NMDA receptors. Furthermore, calcium channels exist among closed, open and inactivated states. To distinguish the state-dependent inhibitors, usually an inward rectifier potassium channel gene (eg, Kir2.3) is co-expressed with calcium channels. Thus, the membrane potential can be adjusted by altering the external K concentrations. This approach has successfully been used to identify state-dependent inhibitors and to characterize the molecular selectivity, even offering some advantages over electrophysiology.

• Voltage-gated sodium channels

Voltage-gated sodium channels are important targets for treating excitable diseases, such as epilepsy and neuropathic pain. It is known that voltage-gated sodium channels exist in closed, open and inactivated states.

The fluorescence-based membrane potential dye assay (eg, DiBAC and FRET dyes) is often used for sodium channel screenings. For the assay itself, the signal change is primarily affected by the membrane potential. Recently, a thallium flux (Tl flux)-based method, a valuable technique for potassium channels, has been successfully developed as a functional assay for Nav1.7 sodium channels. Tl flux methods produce dramatically larger signals, which are superior to the state-of-the-art Na-sensitive dyes and are amenable for HTS of sodium channels.

• Chloride-selective channels

For chloride channels, the yellow fluorescent protein (YFP) quenching assay has been developed. The YFP assay is a noninvasive technique that measures fast responses. The assay has been widely developed for chloride selective channels and receptors, including CFTR, calcium-activated Cl-channels (CaCC) (TMEM16A), Glycine receptors and GABA receptors. Additionally, the voltage-sensitive dye method can be used to assay the chloride channels but is rarely used for this class of channels.

Fig. 2 Diagram of high-throughput screening methods. (Yu, H. et al., 2016)

Conclusion and Perspective

Overall, progress and improvements in ion channel HTS technologies have sped up ion channel drug discovery. The development of ion channel screening technologies has met most needs for drug discovery. Significant instrumentation development efforts continue to improve the capabilities of automated electrophysiological instruments, which are being used for more ion channel classes and cell types. Emerging trends focus on the exploration of reagents and the development of strategies that may be applied to the screening process of ion channels, including the highly expressed ion channel stable cell lines, sensitive and specific indicators and optimized screening strategies.