Membrane potential assays

Cronk D (2001) Cell-based high-throughput screening of ion channels using FLIPR and VIPR membrane potential assay technology. Presented at 7th annual conference of Society for Biomolecular Screening, Baltimore, MD, 10-13 September 2001. SBS, Danbury, Abstract no 119... 

Limitations of Fluorescent-Based Flux or Membrane Potential Assays. .. 99... 

Fluorescent-based flux or membrane potential assays provide superior throughput compared to traditional electrophysiology-based screening technologies. For fluorescent-based membrane potential assays, however, the response to increasing channel activity can be non-linear due to the inherent non-linear relationship between channel activity and membrane potential (Fig. 4A). This prevents an accurate readout of efficacy and reduces the reso-... 

Jensen, A. A. Kristiansen, U. Functional characterisation of the human otl glycine receptor in a fluorescence-based membrane potential assay. Biochem. Pharmacol, 2004, 67 1789-1799. 

Aurora-FRET-based membrane-potential assay VIPR 96-well (20000) Toxin to activate channel or delay inactivation High-through-put functional readout, rapid reporter of changes in membrane potential, amenable to automation, Ratio-metiic Proprietary technology available through corporate alliance... 

Ordonez, J. V. Wehman, N. M. Amphotericin B susceptibility of Candida species assessed by rapid flow cytometric membrane potential assay. Cytometry 1995, 22, 154-157. 

Azas, N. Di Giorgio, C. Delmas, E Gasquet, M. Timon-David, P. Assessment of amphotericin B susceptibility in Leishmania infantum promastigotes by flow cytometric membrane potential assay. Cytometry 1997,28,165-169. 

Fluorescence-based methods do not directly measure ionic current but, rather, measure either membrane-potential-dependent or ion-concentration-dependent changes of fluorescence signals (from fluorescent dyes loaded into the cytosol or cell membrane) as a result of ionic flux. Because fluorescence-based methods give robust and homogeneous cell population measurement, these assays are relatively easy to set up and achieve high throughput. 

Zhu, C. Jiang, L. Chen, T.-M. Hwang, K.-K., A comparative study of artificial membrane permeability assay for high-throughput profiling of drug absorption potential, Eur. J. Med. Chem. 37, 399 107 (2002). 

Fig. 21.3. Two-micropipette current-clamp recording and effect of maintained application of 30 pM levamisole, which produces a 15 mV depolarization (upward movement of trace). The downward transients are the result of injected current pulses used to measure membrane conductance. The trace gets narrower as the input conductance increases from 2.35 pS to 4.35 pS as the levamisole ion channels open. The peak amplitude of the membrane potential response and change in input conductance are used as an assay of the number and activity of the levamisole ion channel receptors present in the muscle cell membrane. The response was fully reversible on washing (not shown).

A series of 4,4-disubstituted quinazolin-2-ones derived from HTV nonnucleoside reverse transcriptase inhibitor leads have shown good in vitro potency and in vivo efficacy [28]. Using FLIPR assays on cell lines with different resting membrane potentials, TTA-Q3 (10) and TTA-Q6 (11)... 

Intracellular Enzymes Stem Cell Isolation Cellular Membrane Potential Mitochondrial Membrane Potential Gene Reporter Assay Gene Silencing (siRNA)... 

Bagley, D.M., Dong, B.M. and De Salva, S.J. (1989). Assessing the eye irritation potential of surfactant-based materials using the chorioallantoic membrane vascular assay (CAMVA). In Alternative Methods in Toxicology, Vol. 7 (Goldberg, A.M., ed.) Mary Arm Liebert, New York, pp. 265-272. 

Dye oxidation (e.g., tetrazolium reductase activity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, MTT 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt, WST-1 3- (4,5 -carboxymethoxyphenyl) -2-(4-sulfophenyl)-2 H-tetra-zolium, MTS 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt, XTT 2,2 -di-p-nitrophenyl-5,5 -diphenyl-3,3 -(3,3 -dimethoxy-4,4 -diphe-nylenej-ditetrazolium chloride, NET), Alamar blue assays, ATP concentration (e.g., luciferase assay), oxygen consumption (e.g., oxygen electrodes, phosphorescent oxygen-sensitive dyes), mitochondrial protein and nucleic acid synthesis mitochondrial mass (e.g., mitotracker dyes) mitochondrial membrane potential (e.g., tetramethylrho-damine methyl ester, TMRM tetramethylrhodamine ethyl ester, TMRE)... 

A next-level assay is usually an isolated heart/cardiac tissue preparation. The canine Purkinje fiber assay (GLP) measures several action potential parameters, like resting membrane potential, upstroke velocity, action potential duration and shape, but also if a drug acts reverse-use dependently [72]. Based on changes of the action potential shape it is possible to conclude which ion channels are modulated (e.g., L-type calcium channel block would abolish the plateau phase). The papillary muscle assay (e.g., guinea pigs) determines similar parameters [73]. 

---