PATCH CLAMPING MOVES TO CHIPS
COMMERCIAL CHIPS ARE DESIGNED TO REPLACE THE A R T O F PAT C H C L A M P I N G W I T H T H E E A S E O F A U T O M AT I O N .
Hanns-J. Neubert
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atch clamping is one of the most predictive analytical methods in cell and drug research. For the past two decades, only highly skilled professionals have used this most sophisticated method. Now, new chip-based devices are helping it reach the high-throughput world of testing and screening in biotechnology and drug development. However, it has been an arduous route since 1976, when biophysicist Erwin Neher and medic Bert Sakmann successfully measured electrical currents through the ion channels of cell walls. The so-called patch clamp method, now routinely used by cell physiologists around the world, won them the Nobel Prize for medicine in 1991. Because ion channels are crucial for physiological cell processes (see “Crucial channels” on p 330 A) and are important targets for drugs against diseases such as epilepsy, cardiac arrhythmia, high blood pressure, and diabetes, the need for a high-throughput version of the laborious technique has long been great. And because critical ion channels
© 2004 AMERICAN CHEMICAL SOCIETY
can be adversely affected by drugs intended for unrelated targets, the U.S. Food and Drug Administration (FDA) has proposed new testing to rule out such potential side effects. This move is expected to drive demand for patch clamp assays even higher. For the few companies who have ventured to make microfabricated, high-throughput patch clamp devices, experts say this might be a multimillion-dollar business.
Early patch clamping Neher and Sakmann’s patch clamp method helped them answer a great number of basic questions about ion channels, important cellular switches that regulate the flow of ions into and out of the cell. Using a pipette that had a very small hole (~1 µm) in the tip and was filled with a conductive fluid, they trapped a cell and “clamped” the flowing current through the ion channels within the hole area. Typically, an idle potential difference of 70–90 mV exists between the inner, negatively charged membrane surface S E P T E M B E R 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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The need to screen new drugs for sary high-resistance seals. The second and the outer, positively charged mempossible side effects problem was to position a single cell on brane surface, leading to a considerable a micrometer-scale hole without a microfield strength (~107 V/m) when a on ion channels is scope and micromanipulator. Last but channel is active. not least, the complex fluidic and elec“Our first attempts to measure ion one driving force tronic procedures for taking the meachannel currents were only of limited surements had to be controlled in a fully use,” says Neher, director of the Departbehind on-chip automated process. ment of Membrane Biophysics at the Max For the past two or three years, a Planck Institute for Biophysical Chempatch clamping. small number of companies have been istry (Germany). “We were only able to solving these problems and developing make loose patches, which could not be increasingly reliable devices for highsealed against bypassing currents. Thus, throughput purposes. They use multiwe had a very strong background noise.” The breakthrough came in 1980, says Neher. “We were able well chips, where each well has a 1-µm hole in the bottom in lieu to improve the seal between cell and pipette, thus producing a of the pipette tip. The whole chip rests on a conductive fluid; the gigaseal. This paved also the path for whole-cell measurements.” wells contain microfluids with the target substances of a drug. With a resistance of >1 GΩ, the seal reduced the background Reversible electrodes plunge into the fluids. The crucial ginoise considerably, which made whole-cell measurements pos- gaseal around each cell is generated in a first step by gentle sucsible. This type of experiment is still the gold standard for as- tion, which fixes the cell to the hole. The electrical current is sessing the actions of compounds on ion channels. To measure continuously monitored; when the cell blocks the hole, the the ionic current over the cell membrane, a cell is partly sucked current suddenly rises. Then the suction is slowly released until into a glass micropipette filled with the ionic solution to form a gigaseal of >1 GΩ is built up. During this phase it is possible the tight electrical gigaseal. The vacuum ruptures the cell mem- to record the currents from one or several ion channels in the brane, and intracellular access is provided through the small, area of the 1-µm hole. If a stronger suction is applied, the cell open “patch” that results. The ion current flow between the is ruptured, exposing its contents for whole-cell measurements. This principle is the base for all manufacturers of automated inner cell and the outer cell can thus be measured over the entire cell membrane. The electrical charge is “clamped” at a constant patch clamp devices, companies such as Aviva Biosciences, Axon Instruments, Cytocentrics, Cellectricon, Molecular Devices, Nanlevel to measure the ion current through the membranes. But these experiments aren’t so easy in practice. Obtaining the ion Technologies, and Sophion Bioscience. Their solutions difnecessary seal between the glass and the cell is an art. And only fer in the choice of materials used for the wells (glass, silica, or extremely well-skilled practitioners can fabricate the electrodes polymer), the number of wells on a chip, and the method of creproperly, identify the cells through a microscope, and successfully ating a laminar flow in order to exchange different microfluids attach the electrode to the cell. Furthermore, these measurements containing distinctive target substances. The quality of the lamihave to be performed cell by cell; on a good day, an expert tech- nar flow is important because the goal of a testing project is always to evaluate a number of different solutions or different connician might clamp 15 cells—hardly high throughput. centrations of a solution. Thus, the fluids have to be exchanged without mixing. High-throughput assays Building up secure gigaseals and cell contacts is even trickier. The genomics revolution was largely enabled by a single technological advance: high-throughput sequencing. But as proteomics This is the main hurdle of the technique and accounts for a great rapidly advances, a number of key tools require increased through- proportion of the errors, because the process of making a giput. In the case of ion channels, the lack of adequate assays has gaseal is still not understood. The geometry of the well hole may been felt particularly strongly. “Other high-throughput screen- substantially differ from that of a classical patch clamp pipette, ing methods for ion channels generated insufficient informa- and the contact of the extracellular buffer solution with the inner tion,” says Owe Owar, a researcher at the Chalmers University wall of the hole may result in a blockage and a compromised seal. of Technology (Sweden) who combines biological systems with Some manufacturers, like Nanion Technologies, use special fabsolid-state devices in the development of complex sensor, sam- rication techniques to achieve sharply defined holes in their glass pling, and computation technologies. “Fluorescence measure- chips, while others, like Cytocentrics, use one channel for suction ments, for example, are indirect, [and] raw binding of target sub- and positioning and an independent channel for the final patch stances can only be carried out in a nonfunctional context.” That clamp recording. Moreover, the devices must be simple enough for a wide is, they deliver only structural and dynamic information, but not functional information that could be related to the physiological range of workers to use. One approach is to design user-friendeffect of drugs. Thus, the pharmaceutical industry sought to de- ly software for recording the currents and evaluating the results. velop high-throughput electrophysical techniques. But patch clamping turned out to be mulish against automa- Basic cell research tion. The first obstacle was the preparation of an ultraclean sur- Genomics and the study of the crystal structures of ion channels face on a suitable and affordable substrate to obtain the neces- have led to better understanding of biophysics. Even the biophys328 A
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ical interpretation of cellular excitability has greatly improved. But the discovery and improvement of ion-channel-active drugs have lagged far behind. “Patch clamping on a chip now provides an important advantage for the study of cell membrane physiology: It can be combined with other cell examination techniques, such as high-resolution fluorescence microscopy, in order to facilitate biophysical and neurobiological experiments on ion channel proteins,” says Nils Fertig, CEO of Nanion Technologies, a spin-off of the Center for NanoScience at the University of Munich (Germany). Also very promising is the combination of patch clamping with a scanning ion conductance microscope for imaging living cells. The latter technique, developed in 2001 by Andrew Shevchuk and others, reveals the true topography of cell surfaces without damaging them. With the help of this and other microscopic techniques, it is possible to study the small-scale dynamics of cell surfaces and the distribution of single active ion channels, plasma membranes, or submicrometer cellular structures—for example, microvilli, fine neuronal dendrites, and synapses. Nevertheless, experts predict that basic research labs will continue to use the classical “low-throughput” method for studying complex organized tissues, for example, brain sections, because in most of these cases cells must be selected specifically. Automation is fine for the screening of target substances on many cells of the same type, because the researchers who perform these experiments (“screeners”) usually need only one data point per cell. “Patchers”, on the other hand, try to get as much information from a single cell as possible in order to elucidate the various functions of ion channels from a vast number of different cell types. This approach requires the careful eye of the experimenter.
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The impact of hERG For the pharmaceutical industry, the most important domains of automated patch clamp devices are the screening of test compounds and the examination of genetically created ion channel mutants produced by biotech companies. High-throughput automation even opens up the possibility of testing changes in the numbers and functions of ion channels after stress or in animal models of human diseases like epilepsy, high blood pressure, or arrhythmia. Ion channels are considered desirable targets for pharmaceuticals. Because ion channels gate the movement of charged particles and mediate the flow of electrical currents over the cell membrane, they essentially determine the way cells exchange information in all tissues. Moreover, they are easily accessible for drugs because they are membrane proteins and they connect the outer world of the cell with both the inner realm and the lipid membrane compartments. But ion channels can also be adversely affected by drugs that are designed for totally unrelated targets. A recent example that received considerable attention and led to the withdrawal of several drugs was the human ether-a-go-go-related gene (hERG) channel, a protein that transports potassium in the heart muscle. If that channel is fully or partially blocked, potassium ions cannot be released from the cell, which is then unable to relax. This leads to a malfunction in cardiac excitability, clinically ex-
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In on-chip patch clamping, (a) a 1-µm hole replaces a traditional pipette tip. (b) Suction or an applied electric field helps position the cell over the hole, and (c) the pressure in the lower chamber is reduced to form a high-resistance seal. (d) The cell is ruptured for whole-cell measurements. (Adapted with permission. Nat. Rev. Drug Discov. 2004, 3, 237–278. Copyright 2004 Macmillan Magazines, Ltd.) S E P T E M B E R 1 , 2 0 0 4 / A N A LY T I C A L C H E M I S T R Y
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Crucial channels A cell’s membrane consists mainly of lipid and protein molecules that prevent the outer world from directly interacting with its interior. A double layer of lipids ~6–10 nm thick is a barrier for polar atoms and molecules. But particular protein structures built into the membrane function as “pumps” or “channels” that enable certain ions to pass through the membrane. They are the most minute functional structures in biology. The channel proteins can transport up to 100 million ions per second from one side of the membrane to the other, thus bringing about an electrical current. Their activity is modified by metabolic reactions, drugs, and poisons. The changes in the voltage of the cell membrane are responsible for the transmission of nerve signals or the stimulation of contractible cells such as heart muscle cells. Approximately 1000 different ion channels, which serve many purposes, are distributed over the membrane surface. Pain, for example, is mediated through the sodium channels, which are blocked by the drugs used in local anesthesia. Cardiovascular anti-arrhythmics block sodium, calcium, and potassium channels, and hypertension and angina drugs prevent a particular kind of calcium channel, called “L-type”, from sluicing ions. The hERG channel, which plays a crucial role in arrhythmia, can be unintentionally blocked by a wide range of pharmaceuticals, in the worst case causing sudden death.
pressed as either cardiac arrhythmia (torsades des pointes) or long QT syndrome, a ventricular fibrillation. Long QT syndrome can be induced by a wide range of drugs that unintentionally block ion channels. Because of this, several drugs have been withdrawn from the market since 1997, including Seldane, an antihistamine; sertindole, an antipsychotic; cisapride, a gastric prokinetic; and even the antibiotic grepafloxacin. In accordance with the International Conference on Harmonization’s guidelines for nonclinical toxicology and pharmacology testing and clinical trials, which were adopted in June 2004, regulatory organizations such as FDA and the European Medicines Agency require drug testing of known ion channels, especially hERG, to rule out possible side effects before clinical trials are performed.
Economic and market outlook Because of FDA’s new recommendations, a growing number of whole-cell recordings will be performed on hERG, and special assays are already on the market. This is an important breakthrough for automated high-throughput patch clamp devices. At this time, fewer than 10 companies offer such tools, most of them located in Europe; only two are in the United States. The new rules also open markets for screening service providers, who offer safety tests on ion channels, which, in turn, generates new orders for the manufacturers. The strongest competition is in the area of multichannel devices, which register 16 or more target channels. Single-channel devices, on the other hand, are mainly used for routine tests on specific ion channels. The development of new drugs targeted to ion channels is expected to be another fast-growing market for the chip-based devices. The manufacturers of the automated devices have already acquired a considerable number of orders during the past two years from big pharmaceutical companies and a number of screening service providers. The analysis of the human genome has shown that ion channels may comprise up to 25% of all potential drug targets. Currently, such drugs represent 5–30% of 330 A
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the drugs on the market. Forty of them already generate a total of at least $100 million in annual revenues, and 7 more bring in $80–100 million. However, although all big pharmaceutical companies—together with a steadily growing number of biotechnology companies—run large programs for the discovery of ion channel drugs, not one of these new drugs has received FDA approval since 1997. Also, venture capital companies, especially in the United States, have shown comparatively little interest in ion channel programs. This may change in the near future if the high-throughput patch clamp devices live up to expectations and make it possible to test many more targets, especially those that could not be evaluated in the past.
Future directions The new patch clamp tools may eventually reveal that the chemistry of ion channels is much more sophisticated than that of other drug targets. Many components tend to bind to multiple subclasses of channels, and because of this, most of the newly developed ion channel compounds failed and caused more problems than they solved. The future will show whether high-throughput discovery programs are able to deal with the subtle chemistry and the ever-changing biology of the living systems that channels regulate. And patch clamping—even in a high-throughput, automated form—may not be the answer by itself. Even Neher, the father of the method, and his research group are not putting all of their eggs in this basket. They are working on microfluorometric measurement methods for tracking calcium through ion channels, on techniques to elucidate the mechanisms of cellular exocytosis, and on refining the amplifier technology needed to make the ion current measurements more accurate. “The tools are now fully mature, and automation is waiting to be more used,” he says. “Especially the combination of patch clamping with fluorescent techniques will open a new field in the upcoming field of genetically coded ion channels.” Hanns-J. Neubert is a freelance writer based in Hamburg, Germany.
