GPCRs (G protein-coupled receptors) and Ion Channels

Calcium Flux

The evaluation of calcium flux is a long accepted, tried and true measure of cellular activity. Calcium flux can be used as a measurement for a host of cellular processes including neurotransmitter release, GPCR activity, voltage or ligand gated ion channels, and cardiomyocyte beat patterns, among many others. The FLIPR Tetra System is the drug discovery tool for evaluation of calcium flux in high and ultra-high throughput due to its ease of use, sensitivity and user configurability.

• Enhance your calcium screens with FLIPR Calcium 6 Assay Kits
• Comparison of a novel calcium assay to fluorescence-based calcium flux assays

Cardiomyocyte Screening

Early prediction of drug-induced functional cardiotoxicity requires robust in vitro systems suitable for high-throughput screening. Readily-available iPSC-derived human cardiomyocytes may be used in conjunction with calcium-sensitive dyes, and beat rates are monitored as changes in intracellular calcium. These calcium peaks can be analyzed with our ScreenWorks PeakPro software which provides the investigator a suite of powerful tools for quantification of cardiomyocyte activity.

• Compound effects upon calcium transients in Cor.4U human iPSC cardiomyocytes
• High throughput cardiotoxicity assays using stem cell-derived cardiomyocytes

Characterization of hERG channel blockers using the FLIPR Potassium Assay Kit

Drug-induced inhibition of the human ether-à-go-go-related gene (hERG) ion channel has been related to the susceptibility of patients to potentially fatal ventricular tachyarrhythmia, torsade de pointes. In recent years, a number of FDA-approved drugs were withdrawn from the market due to their off-target effect on hERG. As a result, there has been an increasing need for identifying compounds that block the hERG channel at earlier stages in the drug discovery process. Here we present the utility of the FLIPR Potassium Assay Kit on the FLIPR Tetra System to investigate hERG compound activity.

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Comparison of intracellular calcium measurements

G protein-coupled receptors (GPCRs) play an important role in cell signaling. When the receptor is activated by a ligand, receptor conformation is changed, triggering G-protein activation inside the cell. An active G protein has the potential to induce various cascades of intracellular messengers including calcium. The FLIPR Tetra System performs high-throughput, functional cell-based assays and is the system of choice in drug discovery for evaluating changes in intracellular calcium detected through use of fluorescent calcium-sensitive reporter dyes. Here we provide a basic protocol for performing a calcium mobilization assay on the FLIPR Tetra System using the FLIPR® Calcium Assay Kit, a homogeneous, fast and reliable fluorescence assay.

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Homogeneous solution for GPCR assays

Cell-based assays have become an indispensable method for screening and compound profiling in the early drug discovery process. To date, such assays have proven to be some of the most reliable and reproducible methods in receptor characterization studies, primary screening campaigns and compound profiling programs. For Gq-coupled GPCR targets specifically, homogeneous fluorescent calcium flux assays with masking technology are the methodology of choice.

Combining a novel fluorophore and proven masking technology, the FLIPR Calcium 5 Assay Kit delivers reliable pharmacology, a larger signal window, and improved assay performance. With the FLIPR Calcium 5 Assay Kit and the FLIPR Tetra System, consistent screening of a variety of receptors and targets, especially those with small calcium signal responses, can be obtained in an easyto-use, homogeneous format.

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Live cell Gi - and Gs -coupled GPCR second messenger signaling

Detection of G_i and G_s -coupled GPCR second messenger signal activity has been traditionally accomplished using assays such as radioactive binding or endpoint cAMP assays that require cell lysis. Such assays measure activity at a single time point in the cellular response and do not provide kinetic information. Another option utilizes forced-coupling of G_i and G_s -coupled GPCRs to Gα16 followed by fluorescence detection of calcium flux upon agonist receptor activation. Again, this assay is sub-optimal as it does not signal through the biorelevant cAMP pathway.

Here we demonstrate endogenous receptor activity in CHO-K1 and HEK-293 cell lines stably expressing the GloSensor^™ plasmid using the FLIPR Tetra System.

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Live Cell Kinetic Assays

The ImageXpress Confocal and Micro 4 High-Content Imaging Systems offer optional fluidics and environmental control modules, enabling single-channel pipetting for compound addition and media exchange during rapid kinetics or extended time-lapse experiments. The Transfluor Cell-Based GPCR Assay Kit is a valuable tool used during high content screening (HCS) to track G-protein coupled receptor (GPCR) activation by quantifying GPCR desensitization and recycling using GFP-labeled ß-arrestin with automated image analysis.

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Membrane Potential Assays

Voltage-gated ion channels are present in the excitable cell membranes of heart, skeletal muscle, brain and nerve cells. Blocking or modulating such channels can have a therapeutic effect, or may interfere with normal cell function. As a result, compounds that affect voltage-gated ion channels are important targets in drug discovery.

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Monitoring Gq-Coupled Protein Receptor Activity

Gq protein-coupled receptor activation is commonly monitored in live cells in real time using calcium-sensitive dyes on a fluorescence plate reader. Automated liquid handling within the plate reader is generally required to deliver agonist compounds to the cells in the microplate while the detection system takes real-time readings of compound-induced changes in fluorescence intensity values. Analysis of the resulting kinetic readings yields information about the compound response profiles, including EC₅₀ and IC₅₀ values for agonists and antagonists.

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Potassium Assays

Functional evaluation of potassium ion channels in a cell is of critical importance in the drug discovery process, particularly when it involves cardiac safety. The FLIPR^® Potassium Assay Kit exploits the permeability of thallium ions (Tl⁺) through both voltage- and ligand-gated potassium (K⁺) channels. In this assay, a novel, highly-sensitive Tl⁺ indicator dye produces a bright fluorescent signal proportional to the number of potassium channels in the open state providing a functional indication of the potassium channel activities

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Potassium ion channel assay for high-throughput screening

Potassium channels are responsible for a variety of cellular functions including the maintenance and regulation of membrane potential, secretion of salts, hormones, and neurotransmitters. The dysfunction of potassium channels has been associated with many human diseases. Off-target drug effects on potassium channels have been linked to cardiac toxicity. Due to their crucial physiological functions and their implication in drug-induced toxicity, potassium channels are heavily investigated by the pharmaceutical industry. Furthermore, cell-based functional assays have increasingly been used because they yield more physiologically-relevant results. Challenges exist in measuring K+ ion channel activity in a high-throughput format. A widely adopted technique is to use the fluorometric method where the binding of thallium to thallium-sensitive fluorescent dyes is utilized as a surrogate measurement of potassium channel activity.

Here we demonstrate how the FLIPR® Potassium Assay Kit analyzes potassium ion channel activities on the FLIPR Tetra System.

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