Ion channels are increasingly common targets for drug discovery, but traditional plate-based screening methods, such as FRET and calcium flux assays, provide only indirect measurements of ion conductance and may not correlate closely with in vivo electrophysiology. While manual patch-clamp techniques remain the gold standard for screening ion channels, automated electrophysiology has not yet achieved sufficiently high throughput for use in large-scale screening and typically requires secondary assays to confirm hits and eliminate artifacts.
There remains a “gap between manual patch clamp and high throughput,” noted Juna Kammonen, senior scientist at Pfizer. A shift to plate-based screening will require assays that can be performed in smaller numbers of primary cells. Kammonen described his group’s adoption of two new strategies that have enabled higher-throughput whole cell electrophysiology assays.
They are using the FluxOR™ thallium flux assay from Invitrogen (a Life Technologies) to measure ion flow across an ion channel, together with Genedata’s Screener® Kinetics Analyzer software to enable multiparametric and time-series data analysis to support decision making on which compounds to take forward.
Pfizer is also working to achieve higher-throughput implementation of the IonWorks Quattro automated patch-clamp system (Molecular Devices) and improved correlation with PatchXpress® to yield greater confidence in the data generated. Furthermore, the company is using Port-a-Patch (Nanion Technologies) technology in automated plate-based applications for biophysical characterization of new ion-channel cell lines.
Experimentation with the Fluxion Biosciences IonFlux automated patch-clamp system is yielding promising results, and is an example of how technology is “starting to bridge the gap between plate-based assays and electrophysiology.” Kammonen reported that the instrument’s microfluidics technology provides fast, precise perfusion. A single instrument can perform a dose-response assay on 32 compounds in about 15 minutes, and multiple units can be stacked on a robotic platform to increase throughput.
He noted, in particular, the design of the system, with 12 interconnected wells per experimental zone: eight compound wells, two cell groups, and two cell suspension wells that provide built-in redundancy. This 12-well pattern is repeated in 32 experimental zones on a 384-well plate; screening of eight compounds with two recordings generated for each yields 512 data points/plate. The system’s fast liquid exchange rate allows for detection of rapid responses and screening of fast-gated channels such as GABA.