Membrane Potential-Sensitive Fluorescent Dyes

Baxter DF, Kirk M, Garcia AF et al. (2002) A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels. J Biomol Screen 7(1) 79—85 Epps DE, Wolfe ML, Groppi V (1994) Characterization of the steady-state and dynamic fluorescence properties of the potential-sensitive dye bis-(l,3-dibutylbarbituric acid)trimethine oxonol (BiBAC4(3)) in model systems and cells. Chem Phys Lipids 69(2) 137—150 Gonzalez JE, Maher MP (2002) Cellular fluorescent indicators and voltage/ion probe reader (VIPR(TM)) tools for ion channel and receptor drug discovery. Recept Channels 8(5—6) 283—295... 

The assays are based on membrane potential sensitive fluorescent dyes, which are relocated from the inside of the cells (or vice versa) and cause an alteration in the fluorescence intensity or flip between the inner and the outer sides of the membrane bilayer and transfer fluorescence resonance energy to another dye located outside of the membrane [25, 26]. In this assay, cells, transfected with hERG potassium channel, contribute to the resting membrane potential and the inhibition of this channel by test compounds results in depolarization of the cell membrane, entry of fluorescent dye in... 

Note 3 The membrane potential sensitive fluorescence dye-based assays do not reflect the behavior of the ion channels and are prone to fluorescence quenching. The assays can be used in early phase of drug discovery for lead identification. 

See, for example, Baxter, D.F. et al. A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels. J. Biomol. Screen 2002, 7, 79-85. 

Several methods are available today to test candidate ion channel active drugs (ICADs) electrophysiology (patch clamp), binding assays, radioactive flux assays, membrane potential-sensitive fluorescent dyes, ion-sensitive dyes, and voltage sensing based on fluorescence resonant energy transfer (FRET). 

Baxter, D.F., Kirk, M., Garcia, A.F., Raimondi, A., Holmqvist, M.H., Flint, K. K., Bojanic, D., Distefono, P.S., Curtis, R., and Xie, Y. (2002) A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels. Journal of Biomolecular Screening, 7 (1), 79-85. 

Schummer, U., Schiefer, H. G. and Gerhardt, U. (1979). Mycoplasma membrane-potentials determined by potential-sensitive fluorescent dyes. Curr. Microbiol. 2, 191-194. 

Mohr G.J., Wolfbeis O.S., Application of Potential-Sensitive Fluorescent Dyes in Anion-Sensitive and Cation-Sensitive Polymer Membranes, G Sensor Actuat B-Chem. 1997 37 103. 

Experimentally it is the transmembrane potential difference that is observed by the use of potential-sensitive fluorescent dyes. Two of the components of the transmembrane potential difference are the intra- and extracellular potential differences (Fig. 17.2). If (f)x is the potential in the interior of the cell and 0O the exterior potential, then the transmembrane potential, Em, is given, in the case of a symmetric membrane where D for the two sides cancels, by... 

The effect of clioquinol-metal chelates has been tested on neural crest-derived melanoma cells (6). The effect of clioquinol chelates on cells was further studied by electron microscopy and by a mitochondrial potential-sensitive fluorescent dye. Of the ions tested, only clioquinol-zinc chelate was cytotoxic. This cytotoxicity was extremely rapid, suggesting that its primary effect was on the mitochondria, and electron microscopic analysis showed that the chelate caused mitochondrial damage. This was further confirmed by the observation that the chelate reduced the mitochondrial membrane potential. The phenomenon of cUoquinol-mediated toxicity appeared to be specific to zinc and was not seen with other metals tested. Since clioquinol causes increased systemic absorption of zinc, it is likely that clioquinol-zinc chelate was present in appreciable concentrations in patients with SMON and may have been the causative toxin. 

The fourth type of mediator-based cation optical sensing is using potential sensitive dye and a cation selective ionophore doped in polymer membrane. Strong fluorophores, e.g. Rhodamine-B C-18 ester exhibits differences in fluorescence intensity because of the concentration redistribution in membranes. PVC membranes doped with a potassium ionophore, can selectively extract potassium into the membrane, and therefore produce a potential at the membrane/solu-tion interface. This potential will cause the fluorescent dye to redistribute within the membrane and therefore changes its fluorescence intensity. Here, the ionophore and the fluorescence have no interaction, therefore it can be applied to develop other cation sensors with a selective neutral ionophore. 

Fluorescent, voltage-sensitive dyes can be employed to investigate the activity of ion channels by reflecting the cellular membrane potential (Epps et al. 1994 Plasek and Sigler 1996). This approach is suitable for use in conjunction with different measurement systems including Fluorometric imaging plate readers (FLIPR), Voltage/Ion probe readers (VIPR) or conventional fluorescence readers. 

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