GPCRs and Ion Channels
GPCRs (G protein-coupled receptors) are the largest protein family, with between 600 and 1000 members, and have been linked to many normal biological as well as pathological conditions. They are also known as seven transmembrane (7-TM) receptors, and about 45% of modern medicinal drugs affect this target class. The function of GPCRs is highly diverse, recognizing a wide range of ligands, including photons, small molecules, and proteins.
Ion channels are pores in the cellular membrane that allow ions to pass in and out of the cell. There are over 400 genes for ion channels in the human genome. Many of them have been targeted by drugs that are now blockbusters. Direct measurement of ion channel activity is measured using traditional electrophysiology equipment for patch-clamping. However, the throughput is very low. Ion channel activity can also be measured indirectly with much higher throughput by using fluophores sensitive to changes in membrane potential, calcium flux, and potassium flux.
Solutions for identifying early leads against GPCRs and ion channel targets
We offer a variety of assay and instrument solutions to support studies of GPCR and ion channel function including assay kits, cellular screening and imaging systems, and microplate readers.
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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.
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.
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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.
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.
Live cell Gi - and Gs -coupled GPCR second messenger signaling
Detection of Gi and Gs -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 Gi and Gs -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.
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.
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.
Resources of GPCRs and Ion Channels
eBook
Identify Targets Like a Pro
Identify Targets Like a Pro
Our FLIPR Tetra® High-Throughput Cellular Screening System is fast, reliable, and remarkably easy to configure. The system is optimized for use with both fluorescence and luminescence, an…
Application Note
Enhance your calcium screens with FLIPR Calcium 6 Assay Kits
Enhance your calcium screens with FLIPR Calcium 6 Assay Kits
FLIPR Calcium Assay Kits from Molecular Devices employ sensitive calcium indicators and proprietary masking dyes to enable researchers to conduct highly sensitive fluorescent screens of G…
Application Note
Monitoring Gq protein-coupled receptor activation on the SpectraMax i3x reader with injector module
Monitoring Gq protein-coupled receptor activation on the SpectraMax i3x reader with injector module
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…
Scientific Poster
Cell Based Assays on the FLIPR® Tetra System : Comparison of a Novel FLIPR® Calcium Assay to Other Fluorescence Based Calcium Flux Assays
Cell Based Assays on the FLIPR® Tetra System : Comparison of a Novel FLIPR® Calcium Assay to Other Fluorescence Based Calcium Flux Assays
Read this poster to see how FLIPR Calcium 6 kit is optimized and how it compares to other fluorescent calcium flux assays.
Scientific Poster
Validation of TRPV1 and ASIC1 Ligand-Gated Ion Channels using Automated Patch Clamp and FLIPR with novel Ca2+ detection dye
Validation of TRPV1 and ASIC1 Ligand-Gated Ion Channels using Automated Patch Clamp and FLIPR with novel Ca2+ detection dye
In one series of experiments, we constructed and validated cell based assays in which the FLIPR Calcium 6 assay kit containing this novel calcium fluorophore was used to examine the activ…
Scientific Poster
Fura-2 QBT Ratiometric Calcium Kit, a Novel Homogeneous Assay for Measuring Calcium Flux Assays on the FLIPR Tetra System and the FlexStation 3 Microplate Reader
Fura-2 QBT Ratiometric Calcium Kit, a Novel Homogeneous Assay for Measuring Calcium Flux Assays on the FLIPR Tetra System and the FlexStation 3 Microplate Reader
This PDF introduces Fura-2 QBT ratiometric calcium kit which saves time and buffer costs & offers less well to well variability, and a larger signal window.
Application Note
Characterization of hERG channel blockers using the FLIPR Potassium Assay Kit on the FLIPR Tetra System
Characterization of hERG channel blockers using the FLIPR Potassium Assay Kit on the FLIPR Tetra System
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,…
Scientific Poster
Optimizing a Transient Receptor Potential (TRP) Channel Assay on the FLIPRTETRA® Instrument using hTRPV1-HEK293 Cells and the FLIPR® Calcium 5 Assay Kit
Optimizing a Transient Receptor Potential (TRP) Channel Assay on the FLIPRTETRA® Instrument using hTRPV1-HEK293 Cells and the FLIPR® Calcium 5 Assay Kit
This PDF describes optimization of the TRP (transient receptor potential) channel assay on the FLIPR TETRA instrument using hTRPV1-HEK293 cells and the FLIPR calcium 5 assay kit.
Scientific Poster
Functional Characterisation of Recombinant Muscarinic M3 Receptors in Assay-Ready Frozen Cells using a Novel Calcium Assay Kit on the FLIPRTETRA® Instrument
Functional Characterisation of Recombinant Muscarinic M3 Receptors in Assay-Ready Frozen Cells using a Novel Calcium Assay Kit on the FLIPRTETRA® Instrument
This poster compares the performance of cryopreserved "frozen" cells vs. cells in culture when used for screening GPRC drug candidates on the FLIPR Tetra instrument.
Scientific Poster
Cell Based Assays on the FLIPR TETRA® System : Comparison of the FLIPR Calcium 5 Assay Kit to Other Fluorescence Based Calcium Flux Assays
Cell Based Assays on the FLIPR TETRA® System : Comparison of the FLIPR Calcium 5 Assay Kit to Other Fluorescence Based Calcium Flux Assays
This poster explores FLIPR Calcium 5 Assay Kit containing a superior performance calcium-sensitive indicator that does not require media removal & utilizes same proven quench technolo…
Application Note
Fura-2 QBT Calcium Kit: A homogenous Fura-2 calcium assay
Fura-2 QBT Calcium Kit: A homogenous Fura-2 calcium assay
The Fura-2 QBT Calcium Kit from Molecular Devices incorporates proven quench based technology with a ratiometric Fura-2 calcium indicator to provide a homogenous assay to minimize cell…
Application Note
Calcium signaling with FLIPR Calcium 6 and 6-QF Assay Kits on the FlexStation 3 reader
Calcium signaling with FLIPR Calcium 6 and 6-QF Assay Kits on the FlexStation 3 reader
FLIPR® Calcium Assay Kits from Molecular Devices employ sensitive calcium indicators and proprietary masking dyes to enable researchers to conduct highly sensitive fluorescent screens of G-…
Application Note
Live cell Gi- and Gs -coupled GPCR second messenger signaling on the FLIPR Tetra System
Live cell Gi- and Gs -coupled GPCR second messenger signaling on the FLIPR Tetra System
In this application protocol we demonstrate the use of the modified luminescent firefly luciferase-based Promega GloSensor™ cAMP Assay on the FLIPR Tetra® High Throughput Cellular Screening…
Application Note
Homogeneous solution for GPCR assays with FLIPR Calcium 5 Assay Kit
Homogeneous solution for GPCR assays with FLIPR Calcium 5 Assay Kit
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…
Application Note
High throughput cardiotoxicity assays using stem cell-derived cardiomyocytes
High throughput cardiotoxicity assays using stem cell-derived cardiomyocytes
Eliminate cardiotoxic compounds and identify potential cardiac drug candidates earlier in the drug discovery process with the FLIPR Tetra System, together with ScreenWorks Peak Pro Software…
Application Note
Comparison of intracellular calcium measurements with FLIPR Calcium Assay Kit
Comparison of intracellular calcium measurements with FLIPR Calcium Assay Kit
The FLIPR Tetra System delivers a screening platform to address real-time kinetic cell-based assays in 96-, 384- and 1536-well format. Regardless of the FLIPR Tetra System microplate…
Application Note
Development of a cell-based potassium-chloride transporter assay using the FLIPR Potassium Assay Kit
Development of a cell-based potassium-chloride transporter assay using the FLIPR Potassium Assay Kit
The FLIPR Potassium Assay Kit can be used to measure the functional activity of the hKCC2 cation-chloride cotransporter using a homogeneous, no-wash protocol. The assay kit displays a large…
Application Note
Compound effects upon calcium transients in beating Axiogenesis Cor.4U human iPS cell-derived cardiomyocytes
Compound effects upon calcium transients in beating Axiogenesis Cor.4U human iPS cell-derived cardiomyocytes
The assessment of cardiotoxicity is important in the early stages of drug discovery to enable elimination of potentially toxic compounds from further development. This is critical to reduce…
Application Note
Optimization of NaV1.5 channel assay with FLIPR Membrane Potential Assay Kits
Optimization of NaV1.5 channel assay with FLIPR Membrane Potential Assay Kits
FLIPR® Membrane Potential (FMP) Assay Kits provide a rapid and reliable fluorescence-based method to detect changes in membrane potential brought about by compounds that modulate or block…
Application Note
Live cell kinetics assay utilizing the ImageXpress Micro System
Live cell kinetics assay utilizing the ImageXpress Micro System
The ImageXpress® Micro Confocal/ XLS Widefield High-Content Imaging System from Molecular Devices includes optional Fluidics and Environmental Control modules, enabling single-channel…
Scientific Poster
Calcium Release-Activated Channel (CRAC) Assays on the FLIPR® Tetra System: Evaluation of a Novel Fluorescent Calcium Reagent
Calcium Release-Activated Channel (CRAC) Assays on the FLIPR® Tetra System: Evaluation of a Novel Fluorescent Calcium Reagent
Get detailed information about Calcium release-activated calcium channels by exploring this PDF. Learn how CRAC channels are playing an important role in intracellular Ca2+ homeostasis.…
Scientific Poster
A Novel Homogenous Potassium Ion Channel Assay for High-Throughput Screening
A Novel Homogenous Potassium Ion Channel Assay for High-Throughput Screening
Ion channels are a class of membrane proteins that mediate the movement of charged ions across the cell membrane. Potassium channels constitute the largest and most diverse group of ion c…