Because ion channels are so fundamental to cellular physiology, diseases resulting from their malfunction spur pursuit of drug targets and other flurries of research. Ion channel function can be studied directly using electrophysiology, and indirectly using biochemistry, cell biology, and genomics, among others. Electrophysiology methods such as patch clamping, in which the currents flowing through ion channels are studied using an electrode tightly sealed to the cell membrane, are the most direct way to study ion channel function. This article looks at methods used today in ion channel screening.
Manual vs. automated patch clamp
Manual patch clamping performed by a skilled scientist provides high-quality data; by conventional wisdom, it returns the best possible data when expertly done. It is ideal for obtaining answers to very focused experimental questions about the nature of ion channel physiology, such as testing the effects of a few compounds on ion channel properties. Academic labs mainly use manual patch clamping, while larger pharma labs usually use both manual and automated patch clamping.
Manual patch clamp’s low-throughput, training-intensive nature and high time expenditure make it less efficient for screening large numbers of compounds. Typically, pharma labs will use automated patch clamp for high-throughput screening with a large library of compounds (say, on the order of tens of thousands), for instance, “to find which candidates might block the channel,” says Ariel Louwrier, president and CEO of Stressmarq Biosciences. “Then once they have a few candidates, they’ll switch to manual patch clamp to drill down and start looking at details of how the ion channel is blocked.”
High-throughput screening
SB Drug Discovery operates “both manual patch clamp systems and the latest technology in high-throughput automated patch clamp electrophysiology to study ion channel function and modulation,” says Davide Pau, ion channel group leader at SB Drug Discovery. They use electrophysiology and fluorescence-based assays to study ion channels over-expressed in recombinant cell lines. “High-throughput automated patch clamp has significantly enhanced the ability to study ion channels and identify potent modulators of function,” he says.
SB Drug Discovery uses the SyncroPatch 384i Automated Patch Clamp (APC) systems from Nanion Technologies. Nanion’s SyncroPatch 384i APC system can measure electrophysiological data from up to 384 wells simultaneously, and is integrated with Beckman Coulter’s Biomek i5 liquid handling robot. Pau says that automated electrophysiology systems like the SyncroPatch generate high-quality data on a par with manual patch clamp electrophysiology, but with a significant enhancement in throughput. Because of that, “these [instruments] are the ‘workhorses’ in our organization, and have the flexibility to be used for a broad range of applications, such as high-throughput screening, lead optimization, and biophysical characterization,” he says.
Charles River Laboratories also relies on both automated and manual patch clamp, along with fluorescence-based assays such as calcium-flux, thallium-flux, and membrane potential dye. They use the Sophion Qube automated patch clamp platform, which can measure ion channels directly, as opposed to using fluorescent dyes as surrogate indicators of ion channel activity. “The platform also allows for complex multiparametric analysis of a variety of different ion channel metrics, which can speed up the drug discovery process by eliminating the need for counterscreen assays that are often required if using more traditional plate-based methods,” says Juha Kammonen, ion channel group leader at Charles River Discovery. Disadvantages can sometimes include a higher cost per data point and lower throughput than fluorescence-based assays.
Fluorescence-based assays, such as the FLIPR platform used by Charles River, are a cost-effective and high-throughput method for studying the effects of compounds on ion channels. “As this method has no means of voltage-control, it is better suited toward ligand-gated ion channels rather than voltage-gated ion channels,” Kammonen says. “However, an automated patch clamp assay can still be required to validate the hits due to potential false positives or false negatives, possibly increasing the overall time required for the drug discovery process.” Cost can present an obstacle to widespread automated patch clamp use for high-throughput screening, especially compared to traditional fluorescence-based assays.
Screening tools
StressMarq Biosciences offers research tools for studying ion channels in the form of monoclonal and polyclonal antibodies, antibody conjugates, small molecule activators and inhibitors, immunoassays, and proteins. While antibody-based tools are often used for ion channel immunostaining, they can also be used to study functional ion channels in patch clamp experiments. “Researchers using patch clamp are looking specifically at the physiology of the channel, studying its interactions, studying how it depolarizes, and other electrochemical processes,” says Louwrier. As such, their antibody tools can reveal pertinent ion channel information. “Small molecules interacting with ion channels can show measurable properties, such as whether the antibody binds to or blocks the channel, and how quickly it falls off,” he says. Such properties of ion channels are of interest to pharma labs studying how drugs affect ion channels important for brain functions, for example.
Ion channel activity can also be measured by Aurora Biomed’s Ion Channel Reader (ICR) line of instruments using atomic absorption spectroscopy and flux assays. “Ion Channel Reader combines Aurora’s advanced atomic absorption spectroscopy with a patented microsampling technology to accurately measure ion movement in a cell-based assay format,” says Fekre Mulugeta, product manager for ion channel technologies and microarray systems at Aurora Biomed.
An advantage of this method is that it can measure the activities of voltage- and ligand-gated ion channels, co-transporters, and pumps, including non-electrogenic transporters that aren’t easily studied with traditional patch-clamp methods. “This technology works in combination with our high-throughput screening flux assay, which uses a tracer ion to measure the direct ion flux,” says Mulugeta, such as Aurora Biomed’s Nonradioactive Rubidium Efflux Assay. Non-radioactive flux assays are a high-throughput method to study membrane proteins without patch-clamping. The disadvantage of this method is that it makes endpoint measurements, and cannot gather data on ion channel kinetics.
Going forward, Kammonen sees new disease targets emerging in the ion channel field, such as lysosomal and mitochondrial channels. “At the same time, the field is moving toward more physiologically relevant cell models like iPSC-derived neuronal cells measured using multi-electrode array platforms,” he says. These advances may soon give scientists a significant advantage in pursuing ion channels as therapeutic targets.