Many high-throughput ion channel assays require the use of voltage-sensitive dyes to detect channel activity in the presence of test compounds. Dye systems employing Förster resonance energy transfer (FRET) between 2 membrane-bound dyes are advantageous in combining high sensitivity, relatively fast response, and ratiometric output. The most widely used FRET voltage dye system employs a coumarin fluorescence donor whose excitation spectrum is pH dependent. The authors have validated a new class of voltage-sensitive FRET donors based on a pyrene moiety. These dyes are significantly brighter than CC2-DMPE and are not pH sensitive in the physiological range. With the new dye system, the authors demonstrate a new high-throughput assay for the acid-sensing ion channel (ASIC) family. They also introduce a novel method for absolute calibration of voltage-sensitive dyes, simultaneously determining the resting membrane potential of a cell. (Journal of Biomolecular Screening 2007:656-667)
Mattheakis LC , Savchenko A.: Assay technologies for screening ion channel targets. Curr Opin Drug Discov Devel2001;4:124-134.
2.
Wood C., Williams C., Waldron GJ: Patch clamping by numbers. Drug Discov Today2004;9:434-441.
3.
Cox B., Denyer JC, Binnie A., Donnelly MC, Evans B., Green DV, et al: Application of high-throughput screening techniques to drug discovery. Prog Med Chem2000;37:83-133.
4.
Wolff C., Fuks B., Chatelain P.: Comparative study of membrane potential-sensitive fluorescent probes and their use in ion channel screening assays. J Biomol Screen2003;8:533-543.
Plasek J., Sigler K.: Slow fluorescent indicators of membrane potential: a survey of different approaches to probe response analysis. J Photochem Photobiol B1996;33:101-124.
7.
Förster T.: Delocalized excitation and excitation transfer. In Sinanoglu O (ed): Modern Quantum Chemistry . New York: Academic Press, 1965:93-137.
8.
González JE, Tsien RY: Improved indicators of cell membrane potential that use fluorescence resonance energy transfer. Chem Biol1997;4:269-277.
9.
González JE, Maher MP: Cellular fluorescent indicators and voltage/ion probe reader (VIPR) tools for ion channel and receptor drug discovery. Receptors Channels2002;8:283-295.
10.
González JE, Tsien RY: Voltage sensing by fluorescence resonance energy-transfer in single cells . Biophys J1995;69:1272-1280.
11.
Farinas J., Chow AW, Wada HG: A microfluidic device for measuring cellular membrane potential. Anal Biochem2001;295:138-142.
12.
Fromherz P.: Lipid coumarin dye as a probe of interfacial electrical potential in biomembranes . Methods Enzymol1989;171:376-387.
13.
Pal R., Petri WA Jr, Ben-Yashar V., Wagner RR, Barenholz Y.: Characterization of the fluorophore 4-heptadecyl-7-hydroxycoumarin: a probe for the head-group region of lipid bilayers and biological membranes . Biochemistry1985;24:573-581.
14.
Kraayenhof R. , Sterk GJ, Sang HW: Probing biomembrane interfacial potential and pH profiles with a new type of float-like fluorophores positioned at varying distance from the membrane surface. Biochemistry1993;32:10057-10066.
15.
Mingotaud C. , Chauvet JP, Patterson LK: The behavior of nitrobenz-2-oxa-1,3-diazol and coumarin dye indicators in lipid monolayers: a study of interfacial effects on polarity and pH. Thin Solid Films1994;242:243-248.
16.
Kachel K., Asuncion-Punzalan E., London E.: The location of fluorescence probes with charged groups in model membranes . Biochim Biophys Acta1998;1374:63-76.
17.
González JE , Oades K., Leychkis Y., Harootunian A., Negulescu PA: Cell-based assays and instrumentation for screening ion-channel targets. Drug Discov Today1999;4:431-439.
18.
Kriechbaum M., Wolfbeis OS, Koller E.: Evaluation of critical micelle concentrations of non-ionic detergents using new superpolar lipid probes. Chem Phys Lipids1987;44:19-29.
19.
Jiang B., Sun X., Cao K., Wang R.: Endogenous Kv channels in human embryonic kidney (HEK-293) cells. Mol Cell Biochem2002;238:69-79.
20.
Yu SP, Kerchner GA: Endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells. J Neurosci Res1998;52:612-617.
21.
Whiteaker KL , Gopalakrishnan SM, Groebe D., Shieh CC, Warrior U., Burns DJ, et al: Validation of FLIPR membrane potential dye for high throughput screening of potassium channel modulators. J Biomol Screen2001;6:305-312.
22.
Epps DE, Wolfe ML, Groppi V.: Characterization of the steady-state and dynamic fluorescence properties of the potential-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol (Dibac4(3)) in model systems and cells. Chem Phys Lipids1994;69:137-150.
23.
Sguilla FS, Tedesco AC, Bendhack LM: A membrane potential-sensitive dye for vascular smooth muscle cells assays . Biochem Biophys Res Commun2003;301:113-118.
24.
Tsien RY, Pozzan T., Rink TJ: T-cell mitogens cause early changes in cytoplasmic free Ca2+ and membrane potential in lymphocytes. Nature1982;295:68-71.
25.
Hoffman JF, Laris PC: Determination of membrane potentials in human and Amphiuma red blood cells by means of fluorescent probe. J Physiol1974 ;239:519-552.
26.
Dall'Asta V. , Gatti R., Orlandini G., Rossi PA, Rotoli BM, Sala R., et al: Membrane potential changes visualized in complete growth media through confocal laser scanning microscopy of bis-oxonol-loaded cells. Exp Cell Res1997;231:260-268.
Avila G., Sandoval A., Felix R.: Intramembrane charge movement associated with endogenous K+ channel activity in HEK-293 cells. Cell Mol Neurobiol2004;24:317-330.
29.
Zhu G., Zhang Y., Xu H., Jiang C.: Identification of endogenous outward currents in the human embryonic kidney (HEK 293) cell line. J Neurosci Methods1998 ;81:73-83.
30.
Waldmann R. , Champigny G., Bassilana F., Heurteaux C., Lazdunski M.: A proton-gated cation channel involved in acid-sensing. Nature1997;386: 173-177.
31.
Krishtal O. : The ASICs: signaling molecules? Modulators? Trends Neurosci2003;26:477-483.
32.
Hesselager M., Timmermann DB, Ahring PK: pH dependency and desensitization kinetics of heterologously expressed combinations of acid-sensing ion channel subunits . J Biol Chem2004;279:11006-11015.
33.
Gunthorpe MJ , Smith GD, Davis JB, Randall AD: Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells. Pflugers Arch2001;442:668-674.
34.
Wu P., Brand L.: Resonance energy transfer: methods and applications. Anal Biochem1994;218:1-13.
35.
Gibson GA, Loew LM: Phospholipid vesicle fusion monitored by fluorescence energy transfer . Biochem Biophys Res Commun1979;88:135-140.
36.
Fung BK, Stryer L.: Surface density determination in membranes by fluorescence energy transfer . Biochemistry1978;17:5241-5248.
