Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.
Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay’s performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.
1. Generation of Stable Polyclonal Cell Lines
2. Generation of Stable Monoclonal Cell Lines
3. General Tl+ Flux Assay Procedure
4. Determination of Optimal Tl+ Concentration
5. Determination of Assay Sensitivity to DMSO
6. Determination of Assay Sensitivity to Known Pharmacological Modulators
7. Checkerboard Analysis
8. Pilot Screen
The use of a tetracycline-inducible expression system provides a convenient internal control for distinguishing Tl+ flux through endogenous pathways and the Kir channel of interest. Figure 1 shows some examples of cell plating maps used in different types of experiments. The positions of wells containing uninduced or tetracycline-induced cells are indicated with different colors. Figure 2A shows the source plate map used to determine the optimal Tl+ concentration for assay development and compound screening. The color gradient represents the 3-fold dilution series ranging from 100% to 0.002% Tl+. A representative fluorescence intensity map is shown in Figure 2B, with cool-to-hot colors indicating low-to-high Tl+ flux values, respectively. The cell plating map shown in Figure 1C was used for this experiment. A fit of a 4-parameter logistic function to the Tl+ CRC (Figure 2C) is used to determine an EC80 value of 15% Tl+. Figure 3A shows the source plate map used to determine the DMSO tolerance of an assay. Columns 1 and 24 contain assay buffer only, whereas the color gradient indicates the 2-fold dilution series ranging from 10% to 0.01% DMSO. A representative fluorescence intensity map is shown in Figure 3B, with low Tl+ flux values indicated with darker blue. The cell plating map shown in Figure 1B was used in this experiment. The average Tl+ flux values recorded from wells containing the indicated concentrations of DMSO are summarized in Figure 3C. The data are plotted as the percentage of Tl+ flux recorded in the absence of DMSO. For this particular Kir channel, DMSO concentrations up to 2.5% had no effect on Tl+ flux and can therefore be used in experiments. Figure 4A shows the source plate map used to establish concentration-response curves for known inhibitors of a Kir channel. Each compound is indicated with a different color and is typically plated as a 3-fold dilution series. A representative fluorescence intensity map is shown in Figure 4B. The cell plating map shown in Figure 1C was used for this experiment. A representative experiment showing dose-dependent inhibition of Tl+ flux by an inhibitor is shown in Figure 4C. The robustness of an assay is determined in so-called “checkerboard” assays, which are summarized in Figure 5. In the source plate map shown in Figure 5A, a maximally effective concentration of an inhibitor or 0.1% DMSO as a vehicle control are plated in alternating wells. Figure 5B shows a representative fluorescence intensity map. A scatter plot of the peak Tl+ flux recorded from individual wells is plotted in Figure 5C. The mean fluorescence values are indicated with solid line, whereas 3 standard deviations are indicated with a dotted line. The Z prime value, a statistical measure of how well separated the two cell populations are separated, calculated for this plate is 0.75, which is well above the 0.5 threshold required for high-throughput screening.
Figure 1. Cell plating maps used for Tl+ flux assay development. (A) Cell plating map use to determine the optimal assay Tl+ concentration. The wells in column 1, rows A1-23, K1-12, F13-23, and P13-23 contain uninduced cells (-Tet). The remaining wells contain cells that were treated with tetracycline (+Tet) to induce Kir channel expression. (B) Cell plate map use to determine the assay DMSO tolerance and perform checkerboard analysis. Note that all the wells are treated with tetracycline (+Tet). (C) Cell plate map used to determine the assay sensitivity to known pharmacological modulators. The wells in column 1 and row A1-23 contain uninduced cells (-Tet). The remaining wells contain cells that were treated with tetracycline (+Tet). Click here to view larger figure.
Figure 2. Determination of optimal assay Tl+ concentration. (A) Source plate map used to determine the Tl+ concentration that evokes approximately 80% of the maximal FluoZin-2 fluorescence increase (EC80). Row A and columns 1 and 24 (yellow) contain a 5x concentration of 12 mM Tl2SO4. The remaining rows contain a 3-fold dilution series ranging from 12 mM (100%) to 0.024 mM (0.002%) Tl2SO4. The series is repeated in columns 2-12 and 13-23. (B) Fluorescence intensity map depicting Tl+ flux for each well approximately 1 min after Tl+ addition. The pseudo-color bar on the right indicates the extent of Tl+ flux, with cooler and hotter colors representing low and high flux values, respectively. Note that the cooler colors in column 1, rows A1-23, K1-12, F13-23, and P13-23 are due to low Tl+ flux in uninduced cells. The remaining wells, including those in column 24, contain cells that were treated with tetracycline to induce Kir channel expression (see cell plating map in Figure 1A). (C) Mean ± SEM CRC for Tl+-dependent changes in fluorescence (n=3). Fitting a four-parameter logistic function to the data yielded an IC80 value of 15% Tl+. Click here to view larger figure.
Figure 3. Assay tolerance to DMSO. (A) Source plate map used to determine assay tolerance to DMSO. Columns 1 and 24 contain HBSS assay buffer. The rows contain a 2x concentration of a 2-fold DMSO dilution series ranging from 10% to 0.01% v/v. (B) Representative fluorescence intensity map depicting Tl+ flux in each well recorded approximately 1 min after Tl+ addition. The pseudo-color bar on the right indicates the extent of Tl+ flux, with cooler and hotter colors representing low and high flux values, respectively. (C) Mean ± SEM (n=9) Tl+ flux normalized to that recorded in the presence of HBSS assay buffer alone. Click here to view larger figure.
Figure 4. Assay sensitivity to known pharmacologically active compounds. (A) Source plate map used to assess the activity of known pharmacologically active compounds in the Tl+ flux assay. Row A and columns 1 and 24 contain 0.1% v/v DMSO. The plate layout allows for the testing of 10 compounds in triplicate in columns 2-23. The rows contain a 2x concentration of a 3-fold serial dilution series of compounds ranging from 100 μM to 2 nM (B) Fluorescence intensity map depicting Tl+ flux for each well approximately 1 min after Tl+ addition. The pseudo-color bar on the right indicates the extent of Tl+ flux, with cooler and hotter colors representing low and high flux values, respectively. Note that wells in column 1 and row A1-23 contain uninduced cells. The remaining wells contain tetracycline-induced cells expressing the Kir channel of interest (See cell plate map in Figure 1C). (C) Representative Tl+-induced changes in FluoZin-2 fluorescence in wells pre-treated with the indicated concentration of a Kir channel inhibitor. Click here to view larger figure.
Figure 5. Determination of assay suitability for HTS. (A) Source plate map used to evaluate the well-to-well variability among wells containing 0.1% DMSO (vehicle) or a maximally effective concentration of a known inhibitor. (B) Fluorescence intensity map depicting Tl+ flux in each well approximately 1 min after Tl+ addition. Note that all wells contain tetracycline-induced cells expressing the Kir channel of interest (See cell plate map in Figure 1B). (C) Representative scatter plot of steady-state fluorescence values obtained from vehicle- or inhibitor-treated wells. The mean fluorescence amplitude of each sample population is indicated with a solid line, 3 standard deviations from the mean is shown with a dashed line, and alternating samples for vehicle (VHL) and inhibitor (INH) are graphed as individual points. Click here to view larger figure.
Data treatment: Once the data are collected, a common step in the analysis involves normalizing each well’s fluorescence response, F, to its initial value at the beginning of the experiment, F0. This is commonly referred to as the “static ratio” and symbolized “F/F0“. In cases where F0 is dominated by the indicator dye the static ratio operation will substantially correct for many factors such as disuniformities in illumination, signal collection, and cell number. In cases where the dye signal is weak or background fluorescence or reflections in the system are high, the static ratio will not be effective unless the background can be appropriately dealt with prior to calculating the static ratio. After data normalization, it is typical to reduce the fluorescence waveform to a single value that will be used to quantify activity and pick hits. Most commonly this will be done by fitting the data points in the 10 seconds following addition of Tl+ to obtain an initial slope of the evoked fluorescence increase. For hit picking, a popular approach is to assume that the vast majority of compounds in the test population are inactive (the null hypothesis is in force). A mean and standard deviation is calculated for the test population and hits are selected that are three standard deviations from the mean.
Splitting the Protocol for long compound incubations: For detecting Kir channel inhibitors, particularly those acting at intracellular binding sites, it may be valuable to incubate the cells with test compounds for an extended period of time (e.g. 20 min) prior to the addition of Tl+. If the compounds are added “offline” before the assay is introduced to the plate reader, the optical properties of test compounds (e.g. fluorescence) may not be easily recognized and can adversely affect commonly used data normalization approaches such as the “static ratio” F/F0 described above resulting in false positives and/or false negatives. To avoid this problem while simultaneously avoiding having to keep an assay plate in a reader for a long incubation, one solution is to utilize a “two read” protocol. The first read is a short (e.g. 30 sec) experiment where the compound is added after 10 seconds to allow capture of the pre-compound addition F0 and to assess the compounds’ optical effects on the system. The plate is then removed from the reader and incubated for the desired period and returned to the reader for thallium addition. After both reads are completed, the first read F0 can be used to normalize the data from the second read thus avoiding many issues associated with adding compounds “offline” while allowing the opportunity to keep the reader busy collecting data thus improving screening throughput. The two read approach is most easily implemented when using an automated screening system. It is important to note that the two read approach will not eliminate problems caused by fluorescent compounds that saturate the detector and/or cause read-out artifacts in the CCD camera.
Configuring an assay for activators: Not all Kir channels are maximally activated under resting conditions, thus it may be possible to design an assay that can detect small-molecule activators. In these cases, selecting an EC80 concentration of Tl+ may not provide enough “headroom” for the assay to reliably detect activators. Thus, one may choose to use a lower concentration of Tl+ (e.g. EC20) in activator assays. In some cases, the assay may show low enough variability as to allow the use of an EC50 concentration of Tl+ and still provide suitable resolution of both activated and inhibited channel populations. In this case, the assay may be conducted in dual activator/inhibitor mode. When available, a known activator can be used to help determine the appropriate Tl+ concentration to maximize the chances of discovering channel activators.
Limitations: There are some limitations that should be considered during the development and execution of a Tl+ flux-based high-throughput screen. For example, the assay relies on an indirect measure of Kir channel activity using a fluorescent probe whose optical properties could be directly affected by compounds in a screen. Therefore, important observations from Tl+ flux experiments must be confirmed using voltage clamp electrophysiology, which is considered the “gold-standard” method for ion channel pharmacology. Furthermore, small-molecules may have direct effects on endogenous Tl+ flux pathways expressed in HEK-293 cells, leading to the identification of false-positive hits. The tetracycline-inducible system is particularly useful for distinguishing false-positive hits from Kir channel-directed modulators because the hits can be rapidly screened in uninduced cells for effects on endogenous pathways. Finally, the low solubility of Tl+ in chloride-containing buffers limit the concentration of Tl+ that can be used in an assay, requiring the use of a non-physiological Tl+ stimulus buffer that may augment the pharmacology of the target11. The compatibility of different buffers with the target of interest should be carefully evaluated during assay development. This can be done by establishing CRCs for known modulators using different Tl+ stimulus buffers and choosing conditions in which the pharmacology most closely matches that observed using physiological buffers.
The authors have nothing to disclose.
This work was supported by funding from National Institutes of Health grants 1R21NS073097-01 and 1R01DK082884 (J.S.D.) and Foundation for the National Institutes grant PIER11VCTR.
Name of the reagent | Company | Catalog number | Comments |
pcDNA5/TO | Invitrogen | V1033-20 | Tetracycline-inducible expression vector |
T-REx-HEK293 cells | Invitrogen | R71007 | Tetracycline-inducible cell line |
Lipofectamine LTX/Plus Reagent | Invitrogen | 15338100 | Transfection reagent |
FBS | ATLANTA Biologicals | S11550 | Cell culture media |
DMEM | Invitrogen | 11965 | Cell culture media |
Hygromycin B | Invitrogen | 10687-010 | Cell culture media |
Blasticidin S | Invitrogen | R210-01 | Cell culture media |
Penicillin/Streptomycin | Invitrogen | 15140 | Cell culture media |
HBSS-divalent free | Mediatech | 21022CV | Cell washing |
Trypsin-0.25% | Mediatech | 25053CI | Cell dissociation |
Tetracycline-HCl | Sigma | T9823 | Induction reagent |
Dialyzed FBS | ATLANTA Biologicals | S12650 | Plating media |
FluoZin-2 | Invitrogen | F24189 | Fluorescent dye |
Pluronic F-127 | Invitrogen | P-3000MP | Dye loading |
HBSS | Invitrogen | 14175 | Assay buffer |
HEPES | Invitrogen | 15630 | Assay buffer |
NaHCO3 | Sigma | S6297 | Tl+ stimulus buffer |
MgSO4 | Sigma | M2643 | Tl+ stimulus buffer |
CaSO4•2H2O | Sigma | C3771 | Tl+ stimulus buffer |
D-Glucose | Sigma | G7528 | Tl+ stimulus buffer |
Thallium sulfate | Aldrich | 204625 | Tl+ stimulus buffer |
HEPES | Sigma | H4034 | Tl+ stimulus buffer |
DMSO | Sigma | D4540 | Solvent |
Eight-channel electronic pipettor | Biohit | E300 | Cell plating in 384-well plates |
BD PureCoat amine-coated 384-well plates | BD Biosciences | 356719 | Assay microplates |
Echo qualified 384-Well polypropylene microplate (384PP) | Labcyte | P-05525 | Compound source microplates |
384-well polypropylene microplates | Greiner Bio-One | 781280 | |
Multidrop Combi reagent dispenser | Thermo Scientific | 5840300 | |
ELx405 microplate washer | BioTek | ELx405HT | Automated cell washing |
Echo liquid handler | Labcyte | Labcyte Echo 550 | |
Bravo automated liquid handling platform | Agilent Technologies | Standard model | |
Hamamatsu FDSS 6000 | Hamamatsu | Kinetic imaging plate reader |
Table 1. List of Materials and Reagents.