Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Ion channels act as gateways from one side of a cell membrane to another. When open, channels allow ions to travel through them like tunnels, thereby making it possible for charged particles to traverse the otherwise impermeable cell membrane. But channels open only when they receive a regulatory signal, such as the binding of a ligand, phosphorylation or a change in electrical membrane potential.
Ion-channel activity also can be regulated by the actions of pharmaceutical compounds, and given ion channels’ importance in neural activity, these molecules represent key drug targets for a range of psychological and neurological conditions. But testing drugs on ion channels is complicated by multiple factors, including the fact that neurons contain numerous channels capable of responding to multiple signals. One way scientists approach this problem is to simplify it by expressing individual ion channels in cultured, non-neuronal cell lines. Though far from physiological, this approach is convenient for pharmacological testing because the cells generally are easier to culture than neurons and yet can be studied using both electrophysiology and fluorescence-imaging approaches. Here are some of your options.
Off-the-shelf cell lines
Many vendors offer portfolios of cell lines stably expressing specific ion channels for ion-channel screening. Often the cell lines are validated: They show ion-channel activity that is dependable and predictable in a particular system. For example, cell lines might be designated as validated by or optimized for high-throughput, automated patch clamping using commercially available systems such as Cytocentrics’ CytoPatch™2; Molecular Devices’ PatchXpress, IonFlux and IonWorks; Nanion Technologies’ SynchoPatch®; and Biolin Scientific’s QPatch and Qube systems. They also may have been validated using fluorescence imaging or conventional patch clamping.
Although the species of the transfected ion-channel gene is important for screening (most commercially available choices are from humans, and sometimes other mammals), the genetic background of the host cell line is usually irrelevant, says Keith Cannon, executive vice president at Aviva Biosciences. The exceptions include channels (such as those activated by G-proteins) that depend on a second messenger system, where using the same species of host cell line is important. Cannon also notes that even though most ion-channel targets are human, many in vivo studies are performed in rats. Because of this, it may be best to perform screens in both species, and compare the effects on the same ion channel.
Aurora Biomed
Aurora Biomed offers cell lines that stably express a number of ion channels that are targets in drug discovery, including the hERG and Kv1.3 potassium channels and Nav1.5 sodium channels. The cell lines are validated by electrophysiology and fluorescence imaging and can be used in high-throughput screening. Aurora Biomed’s Ion Channel Reader instruments (which measure ion flux in cell-based assays for ion-channel screening) and its optimized reagents are designed for ion-channel screening assays.
Aviva Biosciences
Aviva Biosciences' cell lines express channels that include hERG potassium, transient receptor potential (TRP), acid-sensing (ASIC), sodium, calcium and chloride channels. The cell lines are optimized for high-throughput patch clamping and validated using the PatchXpress 7000 platform from Molecular Devices. Cell types include HEK-293 and CHO-K1 cells.
Charles River Laboratories
Charles River Laboratories’ associate director of pharmacology, Glenn Kirsch, says the company offers cell lines expressing 96 different ion channels. The cells may be validated by conventional and high-throughput patch-clamp assays as well as by fluorescence-imaging assays. “We provide functional, cell-based screening, profiling and safety services and make the cells available as reagents for sale to customers that prefer to use them for in-house research,” says Kirsch.
Cytocentrics
Cytocentrics offers cell lines expressing sodium, potassium, TRP and nicotinic acetylcholine receptor channels for ion-channel screening. The expressed channels are verified using electrophysiology/patch-clamp methods.
Revvity
Revvity’s ValiScreen Ion Channel Cell Lines include potassium and TRP channels. Available in HEK-293, CHO and IMR32 cells, these cell lines have been validated by electrophysiology and by imaging, using the AequoScreen/Aequorin and PhotoScreen/Photina® systems.
SB Ion Channels
SB Ion Channels (a division of SB Drug Discovery) offers cell lines expressing several types of sodium, potassium, TRP, purinergic and other channels. Most cell lines are based on HEK-293 cells. Validation methods include fluorescence imaging, conventional patch clamping and high-throughput patch clamping using QPatch and IonWorks.
Custom cell lines
Although many cell lines for ion-channel screening are available, there may be times when you want a cell line with particular characteristics, and it is unavailable. For example, something about the cells themselves may be complicating your experiment. “Endogenous expression of ion channels or receptors can interfere with the detection of signals from the exogenous target of interest,” says Kirsch. “We control for this by testing untransfected, parental cells as part of cell-line validation.”
Sometimes interference from endogenous proteins can be avoided by expressing your ion channel in a different cell type. Although HEK-293 and CHO cells are often used because they are relatively easy to maintain in culture, other cell types are available. “Depending on the channel that is being assayed, it is often important to select a host cell that exhibits low expression levels for endogenous channels or that expresses desired levels of accessory proteins,” says Meg Duskin, marketing communications manager at MaxCyte. MaxCyte does not offer cell lines, but rather technology for transiently transfecting ion-channel constructs into a variety of cell types with the MaxCyte STX® Scalable and VLX® Large Scale Transfection Systems.
Trouble with patch-clamping assays may lead some researchers to choose a different cell type for their ion channels. “Physical characteristics of the cell, such as size and membrane composition, can influence seal formation in automated patch-clamp assays,” says Duskin. In patch clamping, a tight seal between the cell membrane and the recording pipette is crucial for obtaining high-quality data, whether from custom or off-the-shelf cell lines.
Indeed, according to Cannon, obtaining a tight (“gigaohm”) seal between the instrument and the cell membrane is crucial for ion-channel screening by patch-clamp assays. Gigaohm seals are needed to effectively voltage clamp the cell and obtain quality data. “Many cell lines produce cells that either have membranes with many fenestrations or invaginations or with many processes, such that they cannot be voltage clamped or will quickly escape voltage clamp,” he says. “Or they adhere to the culture surface so strongly that the process of harvesting the cells produces too much debris in the harvest media, which interferes with the sealing process required for voltage clamp.” Cannon recommends first asking cell-line providers about success rates in voltage clamping with gigaohm seals.
If you need a stably transfected ion-channel cell line that isn’t already available for purchase, look for custom cell-line services. Several companies offer custom stable cell-line development for ion-channel screening; these include Aviva Biosciences, Charles River and SB Ion Channels. Aviva Biosciences, for example, assists in a variety of ways. “With any of our cell lines, we can develop and optimize the customers’ assays, then provide a complete package including the optimized assay, or even help to provide overflow screening services with our cell lines or with the customers’ own lines,” says Cannon.
Expression level and quality
Unfortunately, not all cell lines, stably transfected or otherwise, are created equal. Kirsch says one of his customers’ primary concerns is the quality of cell-line expression, “which can be quantified in terms of sensitivity to reference-standard compounds—such as agonists, antagonists and modulators—cell-to-cell uniformity, signal strength and stability in culture.” Charles River recently acquired the company ChanTest to help maintain quality control. “ChanTest continuously monitors and documents the characteristics of our cell lines and provides thorough technical support to help our customers achieve their research goals,” Kirsch says.
The level of expression of ion channels is also an important consideration, because it directly affects the signal strength measured in experiments. Charles River’s cell lines are optimized to give appropriate signal windows for their respective ion channels—this is the difference between the unstimulated control signal and the fully activated maximal signal. “The signal window and the variability in signal amplitudes are essential characteristics of a robust assay for screening and profiling,” says Kirsch. If the ion-channel expression level is too high, a cell might “escape” the voltage clamp in patch-clamp assays, rendering the data useless. To maintain a good voltage clamp, Cannon says ion-channel cell lines ideally should give whole-cell currents of about 1 to 5 nanoampere (nA), if the ion channel itself is not affected by the ions it conducts. “If the ion channel is affected by the ion it conducts, such as with feedback inhibition by calcium in calcium channels, then the ideal current should be reduced,” for example, to 0.5 to 1.5 nA for calcium-channel cell lines.
With high-throughput and high-content screening becoming increasingly valuable as tools in drug discovery, more cell lines expressing different types of ion channels are becoming available. They don’t come cheap, though. The hERG potassium channel cell line (for testing cardiac safety) runs about $30,000, and the Nav1.7 cell line (a drug-discovery target for pain) about $50,000. Whether you’re in the market for cell lines for ion-channel screening or already use them, check regularly on the latest cell-line offerings—there are likely to be some useful innovations ahead.