Unlike other ion channels that interact only with ligands of specific structural classes, the hERG potassium ion channel can be blocked or modulated by a broad spectrum of structurally diverse compounds. Thus, the ligand binding assay has clear limitations on the assessment of compound activity at hERG channels because competition with a labeled ligand is limited to structurally similar compounds. This means that binding/displacement assays that pick up compounds occupying the same binding site as the labeled ligand may show false-negative results for hERG inhibitors with different binding sites and that negative results in a binding assay cannot exclude the possibility of an allosteric or modulatory hERG inhibitory effect of the compounds. Therefore, a functional assay of hERG channels is the ultimate methodology for examining the hERG activity of compounds. We found that the activities of hERG channel inhibitors in this thallium flux assay are well correlated with those obtained in automated patch clamp experiments. Thus, this thallium flux assay was validated as an effective and alternative method for large-scale compound screens to assess compound activity on the hERG channel in vitro.
The whole cell patch clamp experiment is traditionally used as a gold standard method for the functional screen assay for voltage-gated ion channels, including the hERG channel. However, the screening throughput using manual instrumentation and devices is very low and not practically approachable for compound screens. The automated patch clamp instruments developed during recent years have significantly increased compound screening throughput and become more routinely used in the compound follow-up stage for drug development. However, the costs of supplies and the instrument both are still high, and the throughput is limited to small-scale screens, with extensive optimization needed for each new cell type and ion channel target class. The other functional assays for potassium channels available include membrane potential dye and ion flux assay using radioisotope or atomic absorbance measurement with rubidium. However, these methods suffer from nonhomogeneous format and/or poor correlation of the IC50 values with the patch clamp data. The concentrations of ions such as Na+, K+, Ca2+, and Cl− in cytosol are markedly different from those in the extracellular medium, and ion concentration gradients are important for maintaining membrane potential, cell function, and cell viability. In the resting state the concentration of cytosolic Ca2+ is 0.0002 mM inside cell compared with 1.8 mM outside cell, whereas in the stimulated state the cytosolic Ca2+ increases dramatically to 0.1 mM. The low concentration in the resting state and huge increase in the stimulated state of cytosol Ca2+ enables the measurement of the change in cytosol Ca2+ using fluorescent dyes preloaded inside cells. Based on this mechanism, Fluo-4-based Ca2+ assays have become important compound screening methods for GPCRs and calcium channels. However, the difference in K+ concentrations, 139 mM inside cell and 4 mM outside cell, does not allow the use of a K+ fluorescence dye to detect K+ flux through the potassium channels. Thus, thallium, a surrogate ion that is not present in cells physiologically, is used in this ion flux assay to measure potassium channel function with a fluorescence dye.
Thallium ions are permeable to potassium channels and have been used as a research tool for many years [39
]. Fluorescence dye-based thallium assays have been reported for KCNQ2
, GIRKs, and calcium-activated potassium channels [34
]. These assays used a fluorescent dye, BTC–AM (benzothiazole coumarin acetoxymethyl ester, Invitrogen), to detect thallium flux into cells. However, the wash steps, high thallium concentrations, limited signal window, and Cl−
-free buffer requirement limited the application of the assay to a 1536-well format. Here we optimized a new fluorescent thallium dye, FluxOR, with better sensitivity and physiological medium/buffer than was used previously. In addition, we applied a quenching agent, enabling a homogeneous assay format without a cell wash step. The thallium flux assay is an equilibrium assay that reports the relative activity of ion channel populations as they cycle among open, closed, and inactivated states over a time course in the range of tens of seconds. Lacking the kinetic information and exquisite voltage control allowed in patch clamp electrophysiology, the flux-based ion assay relies on the steady accumulation of a surrogate ion following the change in state that accompanies the modest depolarization in the stimulus. Despite these differences, our results indicated that the IC50
values of 10 known hERG channel inhibitors from thallium assay were in good correlation with those obtained from patch clamp measurements. Thus, this thallium assay can be used as an alternative screening method for large-scale compound profiling or as a library screen to which the patch clamp assay could not be applied.
Mammalian transduction of the hERG baculovirus is straightforward and quite tolerant of changes in protocol parameters. For example, we recently transduced hERG–BacMam by adding virus directly to adherent cells in complete medium and incubating overnight at 37 °C. There was no appreciable difference in signal-to-basal ratio or EC50
between 4 h room temperature transduction in PBS and overnight transduction in complete medium at 37 °C (data not shown). In addition, the manufacturer’s protocol recommends trying BacMam enhancer to see whether it helps with expression of the gene of interest. In the case of U-2 OS cells expressing hERG, enhancer had no benefit on expression (data not shown). Recently there have been reports that expression of hERG and clinically relevant mutant constructs of hERG exhibit increased expression (via translocation to the plasma membrane) when grown at decreased temperatures [35
]. We have not observed increased expression when cells were transduced and grown overnight at 32 °C (data not shown). A limitation in this study was that two different cell types were used in the thallium flux assay and patch clamp experiment. Although a good IC50
correlation of the 10 known hERG inhibitors was observed, it would be of interest to test these compounds using the same cell lines in the thallium flux assay and patch clamp experiment.
The application of fluorescence quenching agents has been reported for reducing the fluorescence background in other cell-based assays. Crystal violet and trypan blue were originally used as quenching dyes to reduce the extracellular fluorescence background while measuring the intracellular internalized vesicles [41
]. This quenching method was also used in flow cytometry studies to reduce the cellular autofluorescence background [43
]. Recently, the quenching method was used in conjunction with fluorescent calcium dye assays to mask the extracellular fluorescence while the intracellular fluorescence signal is recorded [44
]. We applied Red 40 as a quenching agent in the FluxOR dye-based thallium flux assay to suppress the extracellular fluorescence while the intracellular fluorescence signal is measured for hERG channel activity. We found that Red 40 has broad absorbance spectra for green and blue fluorescence and can completely inhibit the fluorescence intensity emitted from extracellular FluxOR dye. Thus, the addition of the fluorescence quenching agent Red 40 allows the homogeneous measurement of the thallium flux into cells without a cell wash step. This new homogeneous thallium flux assay is robust and has been miniaturized to a 1536-well plate format for HTS.
In summary, we have developed a homogeneous thallium flux assay in a 1536-well plate format for assessment of compound activity on the hERG channel based on the commercially available FluxOR assay and the addition of a fluorescence quenching dye, Red 40, in assay buffer. The IC50 values of ten known hERG inhibitors from this assay correlated well with those determined in the patch clamp assay. Therefore, this thallium flux assay can be used as a valid functional assay to profile the hERG channel activity of large compound collections. In addition, this assay can be used for other potassium channels in the HTS campaign to identify the lead compounds and generate structure–activity relationship profiles for compounds in safety screens.