What are ion channels?
Ion channels are pores in the cellular membrane that allow the passage of ions across the impermeant lipid cell membrane. The flow of calcium, potassium, and sodium is very important in many cellular processes such as muscle contraction in the heart, pancreatic insulin release, the transmission of impulses in the nervous system such as pain, and T cell activation. Unlike GPCRs, ion channels facilitate passive ion flow toward equilibrium, depending on the ion concentration differences across the membrane, as well as membrane potential (i.e., difference in interior and exterior electric potentials).
High-throughput screening of ion channels in drug discovery
Mutations in ion channel genes can alter ion flow and disrupt the electrochemical balance. Abnormalities in ion channels have been associated with various disorders including epilepsy, ataxia, diabetes mellitus, cardiac arrhythmia, and cancer. That’s why ion channels have become increasingly important in drug discovery.
Ion channels are the second largest class of membrane proteins after GPCRs, with more than 400 protein families identified in the human genome.
Drug screening solutions can help us track drug-induced ion concentration changes and ion channel permeability. More importantly, monitoring ion channel activity is necessary for assessing drug cytotoxicity, as off-target drug effects on ion channels can lead to cardiac toxicity. In fact, a number of FDA-approved drugs had to be withdrawn for that reason.
Membrane Potential
Traditionally, patch clamping—considered the gold standard—has been the method of choice for measuring changes in membrane potential. Although it is the most informative, this method is very labor-intensive and time-consuming. It does not provide the solution for high-throughput drug screening of ion channels.
Molecular Devices developed the FLIPR® Membrane Potential Assay Kit to provide a fast, simple and reliable fluorescence assay for detecting changes in membrane potential. This assay provides a level of information approaching that of patch clamping. In combination with the FLIPR system, it provides a very good system for high-throughput screening of ion channel drug targets.
Potassium Channels
Potassium channels constitute the largest and most diverse group of ion channels, and they are expressed in virtually all cell types. Potassium channels are responsible for a variety of cellular functions including the maintenance and regulation of membrane potential, secretion of salt, hormone, and neurotransmitters. Not surprisingly, the dysfunction of potassium channels has been associated with many human diseases and off-target drug effects on potassium channels have been linked to cardiac toxicity.
The FLIPR® Potassium Assay Kit measures functional activity of ligand- and voltage-gated potassium channel activities on a FLIPR System. This reagent kit provides a homogeneous, fast, simple and reliable fluorescence based high-throughput assay for potassium channel activity.
Solutions for identifying early leads against ion channel targets
We offer a variety of assay and instrument solutions to support studies of ion channel function including assay kits, cellular screening and imaging systems, and microplate readers. Below we highlight key application notes for membrane potential and potassium channel assays:
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Characterization of hERG channel blockers
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 System to investigate hERG compound activity.
Read Application Note:
Characterization of hERG channel blockers
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 System to investigate hERG compound activity.
Read Application Note:
Development of a Cav 1.3 channel assay using optogenetic methods
Cav1.3 is a L-type voltage-gated calcium channel and an important therapeutic target for drug discovery. It has been shown that a number of drugs exhibit state dependent effects on Cav1.3 channels, meaning the potency of these drugs vary in response to the membrane voltage (Vm) and the consequent change of channel states (open, close, inactivated). As this likely provides highly-desired selectivity for pathologically over-activated Cav1.3 channels, there is a growing demand for the development of high-throughput assays to evaluate channel blockers under different states.
Current screening methodologies for this channel utilize either electrophysiology or fluorometric methods using potassium challenge to modulate membrane potential, yet both approaches have significant limitations.
In this study, we demonstrate the novel utility of optogenetic tools to control Vm in a reversible and precise fashion for screening state-dependent calcium channel blockers using the FLIPR System.
Development of a Cav 1.3 channel assay using optogenetic methods on the FLIPR Tetra System
Development of a Cav 1.3 channel assay using optogenetic methods
Cav1.3 is a L-type voltage-gated calcium channel and an important therapeutic target for drug discovery. It has been shown that a number of drugs exhibit state dependent effects on Cav1.3 channels, meaning the potency of these drugs vary in response to the membrane voltage (Vm) and the consequent change of channel states (open, close, inactivated). As this likely provides highly-desired selectivity for pathologically over-activated Cav1.3 channels, there is a growing demand for the development of high-throughput assays to evaluate channel blockers under different states.
Current screening methodologies for this channel utilize either electrophysiology or fluorometric methods using potassium challenge to modulate membrane potential, yet both approaches have significant limitations.
In this study, we demonstrate the novel utility of optogenetic tools to control Vm in a reversible and precise fashion for screening state-dependent calcium channel blockers using the FLIPR System.
Development of a Cav 1.3 channel assay using optogenetic methods on the FLIPR Tetra System
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Development of a cell-based potassium-chloride transporter assay
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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Development of a cell-based potassium-chloride transporter assay using the FLIPR Potassium Assay Kit
Development of a cell-based potassium-chloride transporter assay
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.
Read application note:
Development of a cell-based potassium-chloride transporter assay using the FLIPR Potassium Assay Kit
Ligand gated ion channels (LGICs)
Ligand gated ion channels (LGICs) are a large family of membrane embedded proteins that enable the passage of ions across membranes, in response to the binding of ligands such as neurotransmitters. LGICs represent a class of highly attractive drug targets because of the pivotal role they play in many physiological functions, and their association with multiple human diseases.
Ligand gated ion channels (LGICs)
Ligand gated ion channels (LGICs) are a large family of membrane embedded proteins that enable the passage of ions across membranes, in response to the binding of ligands such as neurotransmitters. LGICs represent a class of highly attractive drug targets because of the pivotal role they play in many physiological functions, and their association with multiple human diseases.
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Measuring membrane potential
This application note provides a basic protocol for performing a membrane potential assay on the FLIPR system using the FLIPR Membrane Potential Assay Kits as well as a discussion of some of the important parameters for optimization and troubleshooting of the assay.
The kit combines the benefits of highly informative data that is comparable to patch clamping data with the benefits of high-throughput screening achievable using the FLIPR system.
Measuring membrane potential
This application note provides a basic protocol for performing a membrane potential assay on the FLIPR system using the FLIPR Membrane Potential Assay Kits as well as a discussion of some of the important parameters for optimization and troubleshooting of the assay.
The kit combines the benefits of highly informative data that is comparable to patch clamping data with the benefits of high-throughput screening achievable using the FLIPR system.
Membrane potential assays
Changes in membrane potential (voltage across a cell membrane) such as in muscle cells or heart cells can have a therapeutic or adverse effect upon the cells. Voltage sensitive dyes can be used to measure membrane potential as it changes across the cell membrane. An increase in dye intensity signals a rise in voltage and a decrease in signal reflects a drop in membrane potential. If a channel is blocked, the fluorescent signal from the dye is reduced. This is an indirect method for screening large numbers of compounds to identify potential channel openers and blockers.
Membrane Potential Application Notes
Membrane potential assays
Changes in membrane potential (voltage across a cell membrane) such as in muscle cells or heart cells can have a therapeutic or adverse effect upon the cells. Voltage sensitive dyes can be used to measure membrane potential as it changes across the cell membrane. An increase in dye intensity signals a rise in voltage and a decrease in signal reflects a drop in membrane potential. If a channel is blocked, the fluorescent signal from the dye is reduced. This is an indirect method for screening large numbers of compounds to identify potential channel openers and blockers.
Membrane Potential Application Notes
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Optimization of NaV1.5 channel assay
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.
The FLIPR® Membrane Potential Assay Kits deliver homogenous fluorescence-based formulations for observation of real-time membrane potential changes associated with ion channel activation and ion transporter proteins.
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Optimization of NaV1.5 channel assay with FLIPR Membrane Potential Assay Kit
Optimization of NaV1.5 channel assay
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.
The FLIPR® Membrane Potential Assay Kits deliver homogenous fluorescence-based formulations for observation of real-time membrane potential changes associated with ion channel activation and ion transporter proteins.
Read application note:
Optimization of NaV1.5 channel assay with FLIPR Membrane Potential Assay Kit
Potassium Assays
The majority of ion channel drug discovery solutions focus on potassium channels since they are the most prevalent type of ion channel, functioning in cardiac muscle action and hormone release. Potassium assay kits can be used to measure the effect of drug molecules on thallium transport through the potassium channel as a surrogate for potassium channel activity.
Potassium assay resources:
Potassium Assays
The majority of ion channel drug discovery solutions focus on potassium channels since they are the most prevalent type of ion channel, functioning in cardiac muscle action and hormone release. Potassium assay kits can be used to measure the effect of drug molecules on thallium transport through the potassium channel as a surrogate for potassium channel activity.
Potassium assay resources:
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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.
Download scientific poster:
A Novel Homogenous Potassium Ion Channel Assay for High-Throughput Screening
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.
Download scientific poster:
A Novel Homogenous Potassium Ion Channel Assay for High-Throughput Screening
Resources of Ion Channels
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,…
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Application Note
Measuring Membrane Potential using the FLIPR® Membrane Potential Assay Kit on Fluorometric Imaging Plate Reader (FLIPR) Systems
Measuring Membrane Potential using the FLIPR® Membrane Potential Assay Kit on Fluorometric Imaging Plate Reader (FLIPR) Systems
Molecular Devices Corporation developed the FLIPR Membrane Potential Assay Kit to provide a fast, simple and reliable fluorescence assay for detecting changes in membrane potential. This…
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Application Note
Development of a Cav 1.3 channel assay using optogenetic methods on the FLIPR Tetra System
Development of a Cav 1.3 channel assay using optogenetic methods on the FLIPR Tetra System
In this study, we demonstrate the novel utility of optogenetic tools to control membrane potential in a reversible and precise fashion for screening state-dependent calcium channel blockers…
-
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…
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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…
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 chan…
