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Ionenkanäle

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).


Flipr Potassium Assay Kit Principle

High-throughput screening of ion channel 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 In fact, a number of FDA-approved drugs had to be withdrawn for that reason.

 

Membranpotenzial

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:

 

  • Beschreibung von hERG-Kanalblockern

    Beschreibung von hERG-Kanalblockern

    Die durch Wirkstoffe verursachte Inhibition des humanen Ether-à-go-go-Related Gen (hERG)-Ionenkanals wurde mit der Anfälligkeit von Patienten für die potentiell tödliche ventrikuläre Tachyarrhythmie, der Torsade-de-Pointes-Tachykardie, in Verbindung gebracht. In den letzten Jahren wurden einige der von der FDA zugelassenen Wirkstoffe wieder vom Markt genommen, da sie einen Off-Target-Effekt auf hERG zeigten. Als Folge daraus entstand eine wachsende Notwendigkeit, bereits in früheren Stadien der Wirkstoffforschung Verbindungen zu identifizieren, die den hERG-Kanal blockieren. Here we present the utility of the FLIPR Potassium Assay Kit on the FLIPR System to investigate hERG compound activity.

    Application Note lesen:

    Beschreibung von hERG-Kanalblockern mit dem FLIPR Potassium Assay Kit auf dem FLIPR Tetra System

    Development of a Cav 1,3 channel assay using optogenetic methods

    Development of a Cav 1,3 channel

    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.

    Entwicklung eines Cav 1.3-Kanal-Assays mithilfe optogenetischer Methoden auf dem FLIPR Tetra System

  • Development of a cell-based potassium-chloride transporter assay

    Development of a cell-based potassium-chloride transporter assay

    Die funktionelle Beurteilung der Kalium-Ionenkanäle einer Zelle ist von entscheidender Bedeutung in der Wirkstoffforschung, insbesondere wenn es die Sicherheit des Herzens betrifft. The FLIPR® Potassium Assay Kit exploits the permeability of thallium ions (Tl+) through both voltage- and ligand-gated potassium (K+) channels. In diesem Assay erzeugt ein neuartiger, hochempfindlicher Tl+ Indikatorfarbstoff ein helles Fluoreszenzsignal, das proportional zur Anzahl der Kaliumkanäle im offenen Zustand ist, und liefert dadurch einen funktionellen Anhaltspunkt für die Aktivität von Kaliumkanälen.

    Application note lesen:

    Entwicklung eines zellbasierten Kaliumchlorid-Transporter-Assays mit dem FLIPR Kalium-Assay-Kit

    Ligand gated ion channels (LGICs)

    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.

  • Measuring membrane potential

    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.

    Messung des Membranpotenzials mit dem FLIPR® Membrane Potential Assay Kit auf Fluorometric Imaging Plate Reader (FLIPR®) Systemen

    Membranpotenzial-Assays

    Membranpotenzial-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

  • Optimization of NaV1.5 channel assay

    Optimization of NaV1.5 channel assay

    Spannungsabhängige Ionenkanäle befinden sich in den erregbaren Zellmembranen des Herzens, der Skelettmuskeln und der Gehirn- und Nervenzellen. Das Blockieren oder Modulieren solcher Kanäle kann therapeutische Auswirkungen haben oder die normale Zellfunktion beeinträchtigen. Daher sind Verbindungen, die Auswirkungen auf spannungsabhängige Ionenkanäle haben, wichtige Zielmoleküle in der Wirkstoffforschung.

    Die FLIPR® Membrane Potential Assay Kits liefern homogene fluoreszenzbasierte Rezepturen zur Beobachtung von Veränderungen des Membranpotenzials in Zusammenhang mit der Aktivierung von Ionenkanälen und Ionentransporterproteinen in Echtzeit.

    Application note lesen:

    Optimization of NaV1.5 channel assay with FLIPR Membrane Potential Assay Kit

    Kalium-Assays

    Kalium-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:

  • Kaliumionenkanal-Assay für das Hochdurchsatz-Screening

    Kaliumionenkanal-Assay für das Hochdurchsatz-Screening

    Kaliumkanäle sind für eine Vielfalt von zellulären Funktionen verantwortlich, einschließlich der Aufrechterhaltung und Regulation des Membranpotenzials und der Freisetzung von Salzen, Hormonen und Neurotransmittern. Die Fehlfunktion des Kaliumkanals wurde mit vielen menschlichen Erkrankungen in Verbindung gebracht. Off-Target-Effekte von Wirkstoffen, die Kaliumkanäle beeinträchtigen, wurden mit Kardiotoxizität assoziiert. Aufgrund ihrer wichtigen physiologischen Funktionen und ihres Beitrags zur Wirkstoff-induzierten Toxizität werden Kaliumkanäle intensiv von der pharmazeutischen Industrie erforscht. Weiterhin wurden zellbasierte funktionelle Assays zunehmend eingesetzt, weil sie physiologisch relevantere Ergebnisse erzielen. Herausforderungen bestehen darin, die Aktivität von K+-Ionenkanälen in einem Hochdurchsatz-Format zu messen. Eine weitverbreitet übernommene Technik ist die fluorometrische Methode, bei der die Bindung von Thallium an Thallium-sensitive Fluoreszenzfarbstoffe stellvertretend für die Aktivität von Kaliumkanälen genutzt wird.

    Download scientific poster:

    Ein neuartiger homogener Kaliumionenkanal-Assay für das High-Throughput-Screening

Resources of Ion Channels

Related Products & Services of Ion Channels