Speedy patch clamping of primary blood cells

Schematic of high-throughput patch clamping of primary cells. The difference between the current (a) before and (b) after selective blocking of the target ion channel gives the activity of the channel of interest.
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Patch clamping may sound like a medieval torture method, but in reality, it’s a technique that has led to a wealth of information about the electrophysiology of cells. Manual patch clamping involves positioning a cell onto the tip of a fine glass pipette by suction. The tight seal forces ions to flow through ion channels contained in a target patch of the membrane, and this current can be measured by an ultrasensitive electronic amplifier attached to the pipette. Alternatively, this technique can be modified to measure ion channels in the entire area of the plasma membrane of a cell.

More recently, a planar patch clamp technique has been developed; in this method, cells are positioned onto a hole in a membrane. Instruments can process tens to hundreds of cells at a time by using planar patch clamping, but to date, these instruments have been limited to large cultured cells that are overexpressing the ion channel of interest. Now, in work published in AC (2008, 80, 3728–3735), Michael Mayer and colleagues at the University of Michigan have developed an automated, high-throughput method to study the electrophysiology of relatively small primary blood cells.

The idea grew out of a would-be student’s academic frustration. The student, who had initially been rejected from the University of Michigan’s biomedical engineering department, contacted Mayer (then an incoming professor) with an idea. The student had been working at a company that had developed a high-throughput electrophysiology instrument to study overexpressed ion channels, and he suggested that the instrument could also be used to study native ion channels in primary cells. Mayer was intrigued: “I actually overall was quite impressed with the student, so I did accept him into my group, and he was then accepted into the department,” he says.

The researchers chose to work with lymphocytes because in patients with several immunological diseases, some subsets of these cells have up-regulated activity of the Kv1.3 K⁺ ion channel. The first order of business for the group was to modify the experimental protocol to work with the smaller blood cells. The cultured cell lines used in previous experiments are ~15–20 µm in diameter, whereas lymphocytes are only ~10 µm in diameter. “One important thing was to find out we needed specific plates that had the small pore sizes,” says Mayer.

The team also had to distinguish the activity of the Kv1.3 channel from leak currents or other ion channels. It took a while to figure out how to apply the instrument to primary cells that do not overexpress the ion channels, says Mayer. “You have the additional challenge that you must play some tricks to be sure that you’re measuring only the one channel of interest.”

The researchers ultimately built on manual patch clamping studies that had been previously carried out in the groups of George Chandy and Michael Cahalan at the University of California Irvine. To accomplish high-throughput recordings, Mayer’s group developed a method that measures the activity of the Kv1.3 ion channel before and after a complete and selective blockage of the channel; the difference between the two measurements gives the conductivity of the channel of interest.

Armed with their new method, the scientists could look at populations of lymphocytes from blood samples. For example, they conducted experiments in which they stimulated the blood cells from several human subjects with an antibody and watched Kv1.3 ion channel activity rise. Most people reached peak stimulation after 48–72 hours—but not everyone. “We had one example of one group member who . . . when we added this antibody, he had a decrease in the ion channel activity,” says Mayer. “It makes you wonder: how can that person still be completely healthy?” He postulates that this individual may display different mechanisms of T cell activation and that measuring Kv1.3 activity may enable such differences to be explained.

Mayer says that he hopes other people will see his group’s work as an enabling technology for high-throughput ion channel profiling in primary cells. “Of course, more work needs to be done to really see how big the potential is here,” he says. “One has to see: now, what can you do with it?” His group is hard at work to answer this question.

—Jennifer Griffiths