When the Nobel Assembly at the Karolinska Institute awarded the 1991 Nobel Prize in Physiology or Medicine to Erwin Neher and Bert Sakmann, the committee announced that their work on ion channels “opened a way to develop new and more specific drugs.” But nearly two decades later, that promise remains largely unfulfilled.
The problem is that while ion channels might make for fantastic drug targets—accounting for an estimated $12 billion in pharmaceutical company revenue in 2002 (1)—they also make for lousy screens. Those drugs currently on the market are the fruits of serendipity, not high-throughput screening (HTS), says Michael Dabrowski, head of AstraZeneca's three-year Global Ion Channel Initiative (GICI). GICI is a technology development program launched in 2006 whose goal “is to enable … and to raise increased awareness about ion channel lead generation internally at AstraZeneca as well as externally.”
“Ion channels were always underserved because the throughput—the rate at which you could study how drugs affect them—was very low,” says Arthur “Buzz” Brown, president and CEO of ChanTest, an ion channel screening service provider located in Cleveland, Ohio. Thanks to technologic developments over the past decade, however, that trend is beginning to change.
The “Art” of the ScreenIn a business where speed and cost are everything, ion channels don't fit neatly into the traditional process of drug discovery. Pharmaceutical companies cannot easily apply their million-compound libraries to ion channels, because these channels don't behave like other proteins. Ion channels are membrane-spanning macromolecules that regulate the flow of charged molecules such as Na+, K+, Cl−, and Ca2+ across an otherwise impermeable barrier. They don't catalyze enzymatic reactions, and induce no secondary, amplified signal. Instead, upon activation—whether by changes in voltage, ligand binding, or mechanical force—these channels open, creating a pore in the membrane through which charged molecules can flow.
The resulting currents are small and fast—on the order of picoamperes and often occurring on a millisecond timescale. Yet they are substantial enough to kick-start a chain reaction that, depending on the context and location, results in effects as varied as pain, muscle contraction, or even fertilization.
The bottom line is that it's not enough to know whether a potential drug compound binds a particular ion channel; scientists need to actually measure its impact on that flow of ions across the cell membrane. Researchers can measure these electrophysiologic changes indirectly, by using voltage-or ion-sensitive fluorescent dyes or atomic absorption spectroscopy for instance. But to do it right—that is, to directly measure the current flow across individual ion channels—requires the technique that won Neher and Sakmann their Nobel prize: patch clamping.
In traditional patch clamping, a glass microelectrode (basically a drawn-out micrometer-wide glass pipet with buffer and an electrode inside) is coupled to a cell membrane under a microscope. Suction is then applied to form a seal between the pipet tip and the piece of membrane to which it is attached (the “patch”). In such a system, ion flow across the membrane (mediated by ion channels) produces current that can be recorded by the electrode. The goal is to hold the transmembrane voltage difference steady—that is, “clamp” it—and measure the current. That's not to say the voltage remains constant throughout the experiment: oftentimes, current is recorded while modulating the voltage to mimic, for instance, a cardiac action potential.
Data-rich, the resulting technique is nonetheless a bottleneck. The low-throughput, manual process requires a skilled operator, is done one cell at a time, and is, says David Yamane, MDS Analytical Technologies' senior director for drug discovery marketing, “more art than science.” In contrast, planar patch clamp systems may be run by technicians, and are amenable to scale-up and robotic integration.
