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# Patch clamp electrophysiology steps up a gear

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Posted: 20 May 2005 | | No comments yet

The advent of higher throughput patch clamp electrophysiology systems has begun to change the face of ion channel drug discovery. Systems such as IonWorksHT, PatchXpress and QPatch should allow electrophysiology to be re-positioned from an occasional reagent and compound ‘spot-check’ method, to a frontline gene expression analysis tool and drug screening workhorse. This article chronicles the evolution of these systems, their technical capabilities and impact on drug discovery.

The advent of higher throughput patch clamp electrophysiology systems has begun to change the face of ion channel drug discovery. Systems such as IonWorksHT, PatchXpress and QPatch should allow electrophysiology to be re-positioned from an occasional reagent and compound ‘spot-check’ method, to a frontline gene expression analysis tool and drug screening workhorse. This article chronicles the evolution of these systems, their technical capabilities and impact on drug discovery.

The advent of higher throughput patch clamp electrophysiology systems has begun to change the face of ion channel drug discovery. Systems such as IonWorksHT, PatchXpress and QPatch should allow electrophysiology to be re-positioned from an occasional reagent and compound ‘spot-check’ method, to a frontline gene expression analysis tool and drug screening workhorse. This article chronicles the evolution of these systems, their technical capabilities and impact on drug discovery.

In their quest to prove the existence of discrete pore-like ion channel proteins in cell membranes, Bert Sakmann and Erwin Neher developed the patch clamp electrophysiology method in 1976. This technique allows the direct recording of tiny ionic currents in either a patch of, or the entire cell membrane, via a glass microelectrode and is capable of resolving single ion channel gating events on the sub-millisecond time-frame1. Unsurprisingly, patch clamp electrophysiology rapidly became the cornerstone of ion channel biophysics and molecular neuroscience and Sakmann and Neher were later awarded the Nobel Prize for Physiology & Medicine in 1991.

In the pharmaceutical workplace, the patch clamp method soon became a stock part of the ion channel pharmacologist’s armoury. The basic component parts of a patch clamp amplifier, inverted microscope, micromanipulators, anti-vibration workstation and microcomputer can be assembled for less than £40K and used to profile the effects of compounds at either heterologously-expressed recombinant or native ion channels. Our current understanding of the detailed molecular mechanism of action of many important drugs owes much to this approach. For example, nifedipine inhibits the pore of voltage-gated L-type Ca2+ channels in vascular smooth muscle and the myocardium to produce its antihypertensive effects and benzodiazepine anxiolytics such as diazepam act as allosteric positive modulators of GABAA-gated Cl- channels in the central nervous system. To this day this method remains the ‘gold standard’ for this type of analysis.

Despite this, patch clamp electrophysiology has never truly made it as a frontline, primary drug screening tool. Because of the requirement for single cell visualisation and delicate microelectrode manipulation, even an experienced electrophysiologist can at best screen 2-3 compounds per day. This is clearly insufficient to keep pace with the speed and throughput requirements of a modern medicinal chemistry team, let alone support compound library or full diversity screening where several hundred thousand samples may require profiling. Consequently, faster but indirect assay methods such as ion flux with radioisotopes or fluorodetectors, membrane potential measurements or radioligand binding have been deployed, with occasional ‘spot checking’ of compounds by electrophysiology. Whilst this has been reasonably successful for some ion channels, the inability of these alternative approaches to provide a true temporal, linear readout of function – and in the case of voltage-gated ion channels a true gating stimulus – can often lead to false positive (or worse still false negative) outcomes and confounding structure-activity relationships. In the extreme, some mechanisms of drug action such as use-dependent block of voltage-gated Na+ channels that underlies the efficacy of many clinically used anticonvulsants, anti-arrhythmic and local anaesthetic drugs cannot be reliably quantified by any method other than patch clamp electrophysiology. These factors, coupled with increasingly widespread recognition across the pharmaceutical industry that ion channels represent an under exploited opportunity for new therapeutics, were the main drivers for the development of new higher throughput systems.

## Evolution of automated electrophysiology systems

The first commercially available automated patch clamp systems such as the Neuropatch (now Apatchi – Sophion Inc) and Autopatch (CENES, now Xention) were released between 1996 and 1998. Each reduced the requirement for operator skill by using motion controllers and feedback circuitry to automate pipette manipulation and the subsequent formation of a high resistance (>1GΩ) seal between the pipette and the cell surface – a key requirement for high quality recordings. Whilst the Neuropatch system was based around an inverted microscope as in conventional electrophysiology, the Autopatch made ‘blind’ recordings from cells in suspension by inverting the electrode and using a combination of gravity and negative pressure to locate a cell at the meniscus of a droplet of buffer (the ‘Interface’ patch). With software controlled drug application, each system proved capable of unattended operation from the start to the finish of a drug screening experiment. However, since they were based on recording from a single cell at a time neither significantly improved overall screening throughput.

An alternative approach has been to use the amphibian expression system of Xenopus Oocytes for rapid electrophysiology. These extremely large cells (1-2mm diameter) can be easily microinjected with cDNA or mRNA encoding the ion channel of interest and then voltage clamped using two microelectrodes. Electrode positioning for microinjection and recording does not require microscopy and thus more naturally lends itself to automation. Following automatic microinjection, the Roboocyte instrument (2001, MultiChannel Systems) allows sequential recordings of up to 96 oocytes on a plate. The OpusXpress platform makes eight parallel recordings (2002, Axon Instruments now Molecular Devices). Each system claims to increase drug screening throughput by 10-20-fold against conventional oocyte two electrode voltage clamps. While this may be true, these estimates rely on high success rates with microinjection and recording, which is often not the case and multiple drug applications to the same cell. In random drug screening many compounds do not readily washout from the cell and this can drastically decrease the opportunity to apply further compounds. The greatest limitation for many industrial pharmacologists is the fact that substantially lower compound potency is often observed in the oocyte expression system compared to mammalian cells and this is likely due to the large phospholipid content of the cell surface which can act as a ‘sink’ for lipophilic drugs. Nevertheless, these automated platforms have proved extremely useful for accelerating structure-function studies on ion channels by enabling numerous channel chimaeras or single point mutations to be rapidly characterised2.

## Acknowledgements

The author wishes to thank several key co-workers at GSK, notably Claire Townsend, Tim Dale and Wolfgang Jarolimek for their valuable input and debate on the subject of electrophysiology screening methods.

## References

1. Hamill, OP, Marty, A, Neher, E, Sakmann, B & Sigworth, FJ (1981). Improved patch clamp techniques for high resolution current recording from cells and cell-free membrane patches. Pflugers Arch, 391, 85-100
2. Activity of ·7-Selective Agonists at Nicotinic and Serotonin 5HT3 Receptors Expressed in Xenopus Oocytes (2004). Bioorganic & Medicinal Chemistry Letters. 14(8):1849-1853
3. Fertig N, Blick RH, Behrends, JC (2002). Whole cell patch clamp recording performed on a planar glass chip. Biophys J. 2002, 82, 3056-3062
4. Schroeder K, Neagle B, Trezise DJ, Worley, J (2003). Ionworks HT: a new high-throughput electrophysiology measurement platform. J Biomol Screen. 8, 50-64
5. Kostyuk PG, Krishtal OA, & Pidoplichko, VI (1975). Effect of internal fluoride and phosphate on membrane currents during intracellular dialysis of nerve cells. Nature 257, 691-693
6. Brown AM. (2004). Drugs, hERG and sudden death. Cell Calcium. 35, 543-547