FluxOR™ Potassium Ion Channel Assay
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The FluxOR™ Potassium Ion Channel Assay kit enables high-throughput screening (HTS) of potassium ion channel and transporter activities in a fluorescence-based functional assay. This assay takes advantage of the permeability of potassium channels and transporters to thallium ions coupled with the highly sensitive fluorogenic indicator, FluxOR™ dye. With more than 80 distinct potassium ion channel isoforms, the fluorescent signal reported in this assay serves as a surrogate readout of the activity of any ion channel or transporter that is permeant to thallium, including hERG, Kv1.3, Kir2.1, KATP, and many other of >80 distinct potassium ion channel gene products. The FluxOR™ assay has been validated in cells expressing potassium channels either stably or transiently, in 96-, 384- and 1,536 well plate formats.
Assay Principle
To run the assay, cells are loaded with non-fluorescent, thallium specific FluxOR™ dye (Figure 1). Drugs to be screened are pre-incubated with the cells and the microplates are loaded into the reader, where they are injected with a stimulus buffer containing a low level of thallium ions. The thallium ions freely flow through open potassium channels, acting as a surrogate for K+. When the potassium channel is stimulated, thallium flows into the cell and binds the FluxOR™ dye, generating a fluorescent signal, proportional to channel activity—in physiological saline conditions. Multiple potassium ion channels including Kv1.3 (Figure 2), Kv7.2/Kv7.3 (Figure 3), Kv11.1 (hERG) (Figure 4), Kir1.1, Kir2.1, Kv1.1, Kv2.1 (data not shown) have been demonstrated to work with the FluxOR™ assay for reliable results.
Figure 1. Principle of the FluxOR™ assay. Basal fluorescence from cells loaded with the FluxOR™ dye is low, as shown in the left panel, until potassium channels are stimulated. When thallium is added to the assay with the stimulus, the thallium flows down its concentration gradient into the cells, activating the dye as shown in the right panel.
Figure 3. FluxOR™ potassium ion channel assay signatures of Kv7.2 and Kv 7.3, expressed alone (left panel) or together (right panel). Cells were transduced with 5% vol/vol of each construct alone or together and subjected to the FluxOR™ assay. A control virus (null BacMam) was used to show the background level of activity in the assay (lowest trace). The stimulus was injected 1:5 vol/vol to yield a final added potassium concentration of 10 mM, and a final added thallium concentration of 2 mM. Kv7.2 and Kv7.3 must be co-expressed and properly fold into a tetramer in order to obtain potassium ion flux detectable with the FluxOR™ assay.
) | Figure 4 FluxOR™ assays performed on fresh and frozen U-2 OS cells transduced with BacMam-hERG. (Click to enlarge) Figure 4a shows raw data obtained in the FluxOR™ assay determination of thallium flux in U-2 OS cells transduced with BacMam-hERG and kept in frozen storage until the day of use. U-2 OS cells were thawed and plated onto Poly-D-Lysine coated microplates four hours in advance of the assay, which was run on a FlexStation II 384 (Molecular Devices, Sunnyvale CA). The arrow indicates the addition of the thallium/potassium stimulus, and upper and lower traces indicate data taken from the minimum and maximum doses of cisapride used in the determination of the dose-response curves shown in 2D. Raw pre-stimulus peak and baseline values were boxcar averaged and normalized to generate the data shown in Figure 4b, which indicates the fold increase in fluorescence over time. Figure 4c shows data generated in a dose-response determination of cisapride block on BacMam hERG expressed in U-2 OS cells freshly prepared from overnight expression after viral transduction, and Figure 4d shows parallel data obtained from cells transduced with BacMam-hERG, stored for two weeks in liquid nitrogen, thawed, and plated four hours prior to running the assay. Error bars indicate standard deviation, n=4 per determination. |
Assay ready Ion Channel Reagents
The BacMam system (modified baculovirus mediated gene delivery) has been used to create K+ and Na+ ion channel reagents (shown in Figures 2-4) which can be transduced into mammalian cells with ease. This portability allows for faster assay development in pharmacologically relevant cell types. Combining the BacMam-potassium ion channel gene reagents with the FluxOR™ potassium channel assay kit allows for a convenient and reliable integrated screening solution.
View our collection of BacMam Ion Channel Reagents
View our collection of BacMam Ion Channel Reagents
Technical Resources
Posters presented at scientific meetings:
- FluxOR™, a Universal Potassium Channel Assay. Screening BacMam Delivered and Stably Expressed Targets.
- Primary Cell Assays using BacMam Delivery: Combining Targets and Tools in a Relevant Physiological Setting.
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