Chloride channels are involved in a variety of important physiological functions that include transepithelial ion transport, cellular electrical excitability and ion homeostasis. Defects in chloride channel function underlying a number of chronic disease states and they therefore represent valuable drug targets. To facilitate measurements of chloride channel activity Invitrogen has developed Premo™ Halide Sensor, a fluorescent protein based biosensor.
The Premo™ Halide Sensor is a pharmacologically relevant sensor for functional studies of ligand- and voltage-gated chloride channels. It is based on a halide-sensitive form of Venus, the brightest available YFP molecule, affording an excellent signal window. Packaged in BacMam format, the transient expression is efficient in a range of cell types, including human primary cells. The combination of BacMam delivery and the bright fluorescence of Venus provide a highly sensitive, robust and easy-to-use tool for efficient screening of halide ion channels and transporter modulators in a range of mammalian cellular models. The spectral properties of the Premo™ Halide Sensor are compatible with all standard HTS platforms.
Chloride channel–specific—functional assay for voltage- and ligand-gated chloride channels and transporters
Fast—measure chloride flux in high-throughput mode with highly reproducible results
Robust—reliably high expression, no-wash format and excellent signal window
Convenient—study chloride channel activity in your cellular model by efficient and non-cytopathic delivery of Premo™ Halide Sensor by BacMam technology
Pharmacologically relevant—known modulators show dose-dependent quenching and BacMam delivery enables assays in primary cells
Premo™ Halide Sensor Assay
The Premo™ Halide Sensor is based on the Venus variant of
Aequorea victoria GFP (Figure 1). Additional mutations were made within the Venus sequence to increase its sensitivity to changes in local halide concentration. Chloride channels are permeable to iodide and the Premo™ Halide Sensor exploits the ability of this surrogate ion to effectively quench Venus fluorescence; when a chloride channel opens, iodide enters the cell due to the large gradient across the plasma membrane. This channel opening is detected by a decrease (quench) of the Premo™ Halide Sensor fluorescence. The amount of quench is directly proportional to the ion flux (chloride channel or transporter activity).
To uncouple cell maintenance and preparation from screening, simply freeze transduced cells and bring out as little as 4 hours before the screen. Both stable cell lines and human primary cells can be prepared frozen and “assay-ready”.
Principle of the Premo™ Halide Sensor
The Premo™ Halide Sensor is provided as a ready-to-use solution of virus for robust and reproducible assays of chloride channel activity using high throughput screening fluorescence plate readers or fluorescent microscopes. BacMam-mediated delivery ensures highly efficient and reproducibly transient expression in a wide range of mammalian cells, including human primary cells.. The kit contains all components required for cellular delivery and expression. Screening can be conducted in complete medium and without any wash steps. The Premo™ Halide Sensor has excitation and emission maxima (515nm/530nm) similar to YFP and can therefore be detected using standard GFP/FITC or YFP filter sets; the assay is therefore easily performed on most standard HTS microplate readers, or fluorescent microscopes.
The combination of genetically encoded biosensors with BacMam delivery technology yields robust and easy-to-use cell-based assays. BacMam (baculoviral) particles carrying the Venus gene (yellow) are taken up by endocytosis. The DNA traffics to the nucleus where only the CMV promoter-driven Venus gene is transcribed; baculovirus promoters are not recognized by the mammalian transcriptional machinery—hence no virus replication and no toxicity. Following transcription, the Venus mRNA is expressed in the cytosol and cells are ready to assay. This process begins within 4-6 hours after transduction and in many cell types is completed after an overnight period. In the absence of halide ions, the YFP halide sensor fluoresces brightly; on influx of halide ions (particularly the iodide surrogate ion) fluorescence is quenched (Figure 2).
Premo™ Halide Sensor Workflow
Using the Premo™ Halide Sensor is simple.
1. Add virus to cells for 2 to 4 hours.
2. Treat with an enhancer for 2 hours.
3. Incubate overnight and plate or freeze for future use.
Figure 3. Premo™ Halide Sensor Workflow
In particular U-2 OS cells are efficiently transduced (near 100%) and are recommended for initial assay development work. However, BacMam has been shown to efficiently transduce multiple mammalian cell types, including human primary and stem cells. Transduced cells can be divided into aliquots for a homogenous cell population and stored frozen. Assay-ready frozen cells can be plated as little as 4 hours prior to the assay.