Multiple Compound Addition Assay for hERG Ion Channel on IonWorks Barracuda

It is important to screen drugs candidates for hERG (human ether-à-go-go) channel activity very early in the drug discovery process, due to the potentially fatal effects of hERG inhibition. Conventional patch clamp, the current gold standard for studying ion channel activity and pharmacology, is impractical for screening large libraries due to high costs, low throughput and the need for highly skilled operators. Even with the development of automated patch clamp systems in the past decade, the ability to screen hERG ion channel has been limited due to high cost and low to medium throughput

The IonWorks Barracuda® Automated Patch Clamp System offers a real solution to the pharmaceutical and biotechnology industries, with the capability to screen thousands of compounds against hERG ion channel quickly and cost-effectively. Its ability to record from 384 sites in parallel using proven, affordable substrate makes it the ideal system for screening mid to large sized libraries.

The multiple-compound addition assay has the added benefit of generating cumulative concentration-response data similar to those generated in conventional patch clamp assays. This method is broadly applicable to many ion channel assays, particularly those which study compound block of voltage-gated ion channels.


  • Throughput capable of screening large compound libraries
    • Increased throughput of > 6,000 data points per hour
    • Record from 384 sites in parallel
  • Ultra low running costs
    • Direct electrophysiological screening of ion channels at costs approaching indirect high throughput screening assays
    • Based on proven consumables
  • Within-well method generates cumulative concentration-response data, similar to those generated in conventional patch clamp assays
  • Consistent, reproducible data
    • Patented Population Patch Clamp technology results in highly reproducible recordings

Purpose of study

The use of multiple compound addition assays can increase throughput and decrease the cost of screening compounds against ion channels. This study was set up to evaluate the use of within-well, multiple compound addition method for hERG compound screening on the IonWorks Barracuda System, and to compare the result with the previously validated, lower throughput cross-plate method, also on the IonWorks Barracuda System.

IonWorks Barracuda System supports the addition of up to eight compounds per protocol and unlimited protocols per experiment, resulting in virtually unlimited compound additions per experiment, and the ability to apply multiple experimental treatments to each recording site. Accumulating concentrations of a compound can be applied while measuring its pharmacology, all within one well on a plate. This within-well method offers:

  • increased throughput
  • decreased cost
  • alignment with traditional methods for testing compound effects on ion channels, in which one cell or recording site receives multiple concentrations of test compound and cumulative activity is measured.

A within-well experiment with 8 compound additions takes approximately 30 minutes to run and can produce >3000 data points per experiment, or >6000 data points per hour.

The IonWorks Barracuda System has been validated for screening hERG compounds based on a crossplate method, in which each well receives only one compound concentration or vehicle control. Using this method 384 data points are collected in an approximately 20-minute assay, with a throughput of >1100 data points per hour (Cook et al., 2011).

To effectively compare pharmacology data from the within-well versus the cross-plate method, this study used the same set of reference hERG blockers as was originally used to validate the cross-plate method, but tested them using a six-compound addition assay.


The within-well, multiple compound assay developed increased throughput more than four-fold while maintaining comparable pharmacology to the cross-plate assay.

Seven reference compounds were applied to hERG channel at six incremental concentrations per compound, using the withinwell method. The pharmacology data are comparable between the two assay methods on IonWorks Barracuda System; cross-plate (1 compound addition) and within-well (multiplecompound addition). Throughput was increased to 2300 data points per experiment and 4600 data points per hour.

Summary of the average IC50 values for each compound, obtained from within-well versus cross-plate methods. As the fold difference values demonstrate, IC50 values obtained using the two methods were in good agreement; the two values for each compound were within one half-log of each other, with the exception of Astemizole, for which the difference was slightly greater than one half-log. There is no trend toward either method producing more or less potent pharmacology values.

Materials and methods

Chinese hamster ovary (CHO) cells stably transfected with human KV11.1 channel were provided by ChanTest Corporation (Cleveland, OH). Reference hERG blockers were obtained from Sigma-Aldrich (St. Louis, MO).

Development of a multiple compound addition assay for hERG ion channel

Within-well assays subject cells to multiple fluid exchanges and prolonged electrical stimulations that present the technical challenge of minimizing channel run down. To achieve stability of recording over extended experiment time, a single-pulse voltage protocol was used instead of the fivepulse protocol employed in the cross-plate study.

Compounds were added at 3X final assay concentration in 1% DMSO and mixed with buffer in the well to achieve a 1X final compound concentration in 0.33% DMSO. Compounds were incubated for one minute, after which a voltage protocol was applied with a one second stimulation at +40 mV, followed by a one second step to -50 mV for measurement of peak tail current (Figure 1). Six compound plates were prepared per experiment, with each plate containing only one concentration of test compound, for a six compound addition assay. Once optimal assay conditions were determined, seven experiments were run, each containing the same seven reference compounds used in the cross-plate study (Cook et al., 2011).

Representative recording of control hERG current (Figure 1)

Cells were held at -70 mV for 200 msec, stepped to +40 mV for 1000 msec, stepped to -50 mV for 1000 msec, and stepped back to -70 mV for 200 msec.

The stability of the optimized experimental condition is demonstrated in Figure 2. This example shows average seal resistances for all wells in one representative 384-well plate, as well as average percentage of channel activity determined by measuring peak tail current amplitudes for six control additions of 0.33% DMSO in 24 control wells.

Average seal resistances and peak tail current amplitudes (Figure 2)

Average seal resistances were measured with each current recording, corresponding to pre-compound addition and each post-compound addition. % Channel Activity for each current recording was determined by dividing the peak tail current amplitude after each compound addition by the peak tail current amplitude during the pre-compound addition recording, multiplied by 100%.

Within-well pharmacology: Generating cumulative concentration-response data similar to those generated in conventional patch clamp assays

Six compound plates were run sequentially for each of seven pharmacology experiments. Each plate contained only one concentration of each test compound, in replicates of 24, such that the first plate contained the lowest concentration to be tested, and the sixth plate contained the highest concentration to be tested.

One concentration-response curve and corresponding IC50 value were generated for each compound in each of the seven independent experiments. Figure 3 shows pharmacology curves generated in one representative experiment.

Concentration-dependent inhibition of hERG ion channel using within-well method (Figure 3)

Concentration-response curves from one representative within-well experiment. % Channel Activity for each current recording was determined by dividing the peak tail current amplitude after each compound addition by the peak tail current amplitude during the pre-compound addition recording, multiplied by 100%. Each data point represents an average of 24 wells. Sigmoidal curve fit was applied.

Testing reproducibility of the multiple compound addition assay

It is important that screening assay data are shown to be reproducible and robust. Using the optimized experimental conditions described, reproducible pharmacology data was produced across seven experiments for reference hERG blockers. The robustness of this six-compound addition assay is illustrated in Figure 4. This robustness is particularly notable considering the technically demanding nature of producing stable recordings from the cells before and after the addition of six compounds with mixing and the corresponding voltage stimulations.

One of the compounds tested, Pimozide, was observed to have less reproducible results than expected. This could perhaps be explained by the compound’s high lipophilicity, which may cause its behavior to be less stable in an environment where plastic materials are necessary for many aspects of the screening workflow.

Reproducibility of the hERG multiple compound addition assay (Figure 4)

Data mapped day-to-day, experiment-to-experiment. The data spread illustrates the robustness and reproducibility of this assay.


This study demonstrates the within-well, multiple compound addition assay can significantly increase throughput and cut costs without sacrificing quality of data.

  • Throughput was increased from 1100 to 4600 data points per hour
  • Pharmacology was comparable to crossplate with average IC50 values in good agreement
  • The assay is robust, reproducible and produces stable recordings
  • Cumulative concentration-response data was generated similar to those in conventional patch clamp assays


Application protocol: “Validation of the IonWorks Barracuda System for hERG Ion Channel Assay”, by Karen Cook, M.S., James L. Costantin, Ph.D., and Xin Jiang, Ph.D., Molecular Devices, LLC (2011)

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