Flux assay

In combination with methods such as fluorescence and flux assays, the new automated patch clamp electrophysiology systems have revolutionized the way in which ion channels are now screened. However, the future is not without major challenges, and important limitations of these technologies remain. 

A medium throughput approach to evaluating sodium channel activity is the measurement of sodium flux across cell membranes [103]. In these experiments, a tracer that permeates the channel and is easily quantifiable is used to analyze sodium influx. Traditionally, radioactive tracers such as 22Na+ or [14C]guanidinium have been used. Alternatively, Li+ can be used as a tracer and analyzed by atomic absorption spectrometry. Sodium flux assays can be used to test approximately 105 compounds per year. They offer a robust readout of channel activity, but lack voltage control and temporal resolution. To examine sodium channel blockade by measuring sodium flux,... 

I. Thallium Flux Assay for KCNQ Channel Openers... 

Key words Cardiotoxicity, In vitro assays for cardiotoxicity, Cardiac safety, Functional flux assays, Fluorescence-based assays, hERG, Patch clamp, Alternative tests, Microelectrode array, hESC-derived cardiomyocytes, FLIPER, Q-T interval, HEK 293, CHO, Embryonic stem cells, Differentiation, hiPSCs, Drug interaction, Safety pharmacology, ICH guidelines, Arrhythmia... 

H] Dofetilide hERG-transfected pKi (-log Ki) for [3H1 [3H] dofetilide TT Flux assay APPC [11]... 

Schmalhofer WA, Swensen AM, Thomas BS et al (2010) A pharmacologically validated, high-capacity, functional thallium flux assay for the human Ether-a-go-go related gene potassium channel. Assay Drug Dev Technol 8(6) 714-726... 

Li QY, Rottlander M, Xu MK et al (2011) Identification of novel KCNQ4 openers by a high-throughput fluorescence-based thallium flux assay. Anal Biochem 418(l) 66-72... 

Bridal TR, Margulis M, Wang X, Donio M, Sorota S (2010) Comparison of hitman Ether-a-go-go related gene screening assays based on IonWorks Quattro and thallium flux. Assay Drug Dev Technol 8(6) 755-765... 

Gill S, GiU R, Sen Lee S et al (2003) Flux assays in high throughput screening of ion channels in drug discovery. Assay Drug Dev Technol 1(5) 709-717... 

Chloride flux assay. Synaptic vesicles were prepared from brains of male ICR gice (20-30 g Blue Spruce Farms, Altamont, NY) (15) Assays of °C1 uptake involved preincubation (10-20 min) of vesicles with carrier solvent (ethanol or acetone, 0.5-1 yl) or insecticide, followed by incubation (4 sec) with bCl with or without GABA or added insecticides and isolation of labelled vesicles by rapid vacuum filtration. Detailed descriptions of the assay are published elsewhere ( 15,1 7) Abamectin stock solutions were prepared in absolute ethanol in silanized glass vials Abamectin in ethanol (1 yl) was added to give concentrations of 3 nM-3 yM in a final volume of 200 yl of vesicle suspension during preincubation or 3 yM in a final volume of 200 yl of bCl uptake medium. 

Recent studies have grouped lindane with the cyclodienes on the basisLjaf its potent and stereospecific inhibition of [3H]DHPTX (.2,3) and [3DS]TBPS (4 J>) binding. However, the quantitative correlations between potency in the chloride flux assay, potency in the [3 >S]TBPS binding assay, and acute toxicity that were observed for the cyclodienes do not extend to lindane. Lindane was a very weak inhibitor of chloride uptake (I 1 mM J7). Inclusion of the data... 

Figure 3. Correlation by least squares linear regression of inhibitory potencies in [ 5S]TBPS binding assays (4) with inhibitory potencies in chloride flux assays (1 5, 17) for seven cyclodienes. The data point for lindane is also shown but not included in the calculated regression. Redrawn from Ref. 17 ...

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