pH-Insensitive FRET voltage dyes.
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.
Maher
Michael P
MP
Johnson & Johnson Pharmaceutical Research and Development L.L.C., San Diego, California 92121, USA. mmaher1@prdus.jnj.com
Wu
Nyan-Tsz
NT
Ao
Hong
H
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002138:Calibration; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D006096:Gramicidin / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D015394:Molecular Structure; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013858:Thiobarbiturates / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D013858:Thiobarbiturates; C106875:bis(1,3-dihexyl-2-thiobarbiturate)trimethineoxonol; D006096:Gramicidin