Molecular Devices IonWorks Barracuda Automated Patch Clamp System

Introduction

Defects in ion channel function have been implicated in several therapeutic areas such as central nervous systems diseases, cardiovascular diseases, immune and inflammatory diseases, cancer, alzhemimer’s disease, epilepsy, hypertension and many others. The conventional patch-clamp assay is the traditional method to study ion channels. However, it is too slow, costly, and labor intensive to keep pace with a modern medicinal chemistry team. Automated electrophysiology platforms have been introduced to address the limitations of conventional electrophysiology. However, their limited throughput and high cost per data point does not support compound library or full diversity screens. As a result, faster and cheaper indirect assays are used for high throughput screening of ion channel targets. While these assays have sufficient throughput and are low cost, they lack the precision, temporal resolution and voltage control of the patch-clamp assay which results in a high number of false positives requiring follow-up. Because of the limitations of these methods, Molecular Devices, Inc. developed the IonWorks Barracuda™ Automated Patch Clamp System. The IonWorks Barracuda System features:

• Simultaneous compound addition and data acquisition allows the measurement of both voltage- and rapidly desensitizing ligand-gated ion channels
• Unique flow through recording chamber design enables rapid solution exchange
• 384 discrete amplifiers and a 384-tip pipettor head for parallel acquisition of an entire 384- well patch plate in 20 minutes
• Highest available throughput of 1,100 to 6000 data points per hour
• Low-cost IonWorks PatchPlate™ consumable to keep running costs down
• Population Patch Clamp™ (PPC) recording for robust and reproducible recordings
• Flexible software with two modes of operation; Assay Development and Screening Mode streamlines assay optimization and simplifies ion channel screening
• Ultrasonic pipette tip washer with two wash solution inputs eliminates the potential of cross contamination

The IonWorks Barracuda System is the ideal platform for ion channel screening. It’s ability to assay both voltage-gated and rapidly desensitizing ligand-gated ion channels combined with it capability to assay >1,100 data points per hour enables the IonWorks Barracuda System to meet the challenges of ion channels screening in today’s drug discovery environment.

Configured to Meet Your Throughput Needs

The IonWorks Barracuda System is capable of performing a typical ion channel assay in as little as twenty minutes resulting in a throughput of 1,100–6,000 data points per hour depending on the number of compound additions per well. Up to eight compound additions per well can be performed increasing throughput and reducing the cost per data point.

The assay deck of the IonWorks Barracuda System is easily accessible for the loading of consumables and compound plates (Figure 1). The IonWorks Barracuda System features 384 discrete amplifiers and a 384-channel pipettor head which allows the recording of all 384-wells of the PatchPlate consumable in parallel. The system comes with an automated pipettor for the transfer of cell suspension to a cell boat designed to minimize cell usage (< 5 mL/run). To load cells, the user simply provides a tube of cell suspension to a position on the assay deck. To eliminate compound carryover, the system features an ultrasonic tip washer with two solution inputs.

Rapid Solution Exchange to Assay Rapidly Desensitizing LGICs

The opening and closing of LGICs can be characterized by a fast activation phase followed by a desensitization phase. Activation and desensitization are commonly reported in the range of milliseconds to seconds. Therefore, a rapid solution exchange rate is required to record rapidly desensitizing LGICs.

The unique flow through design (Figure 2) of the IonWorks Barracuda PatchPlate well positions the fluidic dispensing pipette immediately above the cells. As ligand is delivered, the solution at the cell membrane is rapidly exchanged with the solution containing the ligand to modulate the LGIC. The electrode plate array (Figure 3) has 384-electrodes and 384-holes for the ligand dispensing pipettes to fit through. The electrode plate sits on top of the PatchPlate and facilitates the ability to add ligand or compound while recording currents.

Ligand-gated ion channel (LGICs) assays on the IonWorks Barracuda System

The Acid-Sensing Ion Channel (ASIC), was used to validate the use of the IonWorks Barracuda System to study LCICs. ASICs are cation-permeable non-selective LGICs activated by extracellular protons. ASICs have been suggested to play an important role in the physiology and pathology of sensory transmission, behavioral memory, retinal function, epileptogenesis and ischemia. An example of ASIC currents is shown in Figure 4. Slower (GABA) and faster LGICs have also been measured on the IonWorks Barracuda System. Figure 5 shows recordings from nicotinic acetecholine receptors.

Voltage-gated ion channel (VGICs) assays on the IonWorks Barracuda System

IonWorks Quattro Systems have been adopted by many laboratories around the world and have resulted in a number of publications on VGICs.(1,2,3) These assays can be readily transferred to the IonWorks Barracuda System. Typical current from hERG and NaV1.5 channels are shown in Figures 6 and 7 respectively. The kinetics and pharmacology of all VGICs tested to date on IonWorks Barracuda Systems are comparable to those measured on the IonWorks Quattro System.

Flexible Software Designed to Work with You

The software is designed to run in two distinct modes; Assay Development or Screening Mode. In Screening Mode, the user simply loads a saved protocol from the library and starts the run for walk away operation. Assay Development Mode allows the user to alter experimental conditions during the course of an experiment. Applied voltage protocols can be changed multiple times during the course of an experiment for as long as the cells remain viable. During the course of the experiment, voltage protocols can be newly constructed, or the user can load and execute a series of previously saved protocols. For instance a user may want to run a series of current-voltage relationships with different voltage intervals or different voltage endpoints. In this way, assays can be quickly optimized in Assay Development Mode and the protocol saved to be executed in Screening Mode.

IonWorks Barracuda System Meets All Your Ion Channel Assay Needs

The IonWorks Barracuda System performs assays on both LGICs and VGICs utilizing the affordable PatchPlate consumable. The high throughput and low running costs of the system make it well suited to perform primary screens of large directed compound libraries and for hit-to-lead applications. The IonWorks Barracuda System is also well-suited for compound profiling and safety assessment assays; up to eight compound additions per well may be performed to further reduce running costs and increase throughput. The software is designed to simplify assay optimization when operated in Assay Development Mode and is simple to use in Screening Mode. The next generation of the family of IonWorks Systems, the IonWorks Barracuda System meets all your ion channel assay needs for both VGIC and LGIC targets. 

References

1. Castle N, et al., Sodium channel inhibitor drug discovery using automated high throughput electrophysiology platforms. Comb Chem High Throughput Screen 12: 107-122, 2009
2. Bridgland-Taylor MH, et al., Optimisation and validation of a medium-throughput electrophysiologybased hERG assay using IonWorks HT. J Pharmacol Toxicol Methods 54: 189-199, 2006.
3. Dale TJ, et al., Population patch clamp electrophysiology: a breakthrough technology for ion channel screening. Mol Biosyst 3: 714-722, 2007.
4. Priest BT, et al., Automated Electrophysiology Assays. Assay Guidance Manual - Eli Lilly & Company and the National Center for Advancing Translational Sciences Bethesda (MD): 491-539, 2017