Cell-permeable fluorescent indicator for cytosolic chloride.
A major limitation of quinolinium-based fluorescent indicators for cytosolic Cl- has been the necessity of invasive cell loading because the positively charged ring nitrogen confers high polarity and membrane impermeability. A novel approach to mask the positive nitrogen was developed and evaluated for rapid, noninvasive indicator loading into living cells and effective intracellular trapping. The nonpolar and lipophilic compound 6-methoxy-N-ethyl-1,2-dihydroquinoline (diH-MEQ) was Cl- insensitive but was readily oxidized to the membrane-impermeable and Cl(-)-sensitive fluorescent indicator 6-methoxy-N-ethylquinolium chloride (MEQ), MEQ had 344-nm absorbance and 440-nm emission maxima, 0.70 quantum yield, and 4100 M-1 cm-1 molar extinction coefficient. In aqueous buffers, the fluorescence of MEQ was quenched by Cl- by a collisional mechanism with a Stern-Volmer constant (KCl) of 145 M-1. MEQ fluorescence was quenched by other anions (KBr = 275 M-1, KI = 360 M-1, KSCN = 300 M-1) but not by NO3-, SO4(2-), cations, and pH. Swiss 3T3 fibroblasts and colonic T84 cells were loaded with MEQ by incubation at 37 degrees C with 25-50 microM diH-MEQ for 5-10 min followed by diH-MEQ-free buffer for 15 min. MEQ stained cells brightly and uniformly and was nontoxic in studies of cell growth, cAMP and Ca2+ signaling, and electrophysiological properties. MEQ leaked out of cells by less than 10% in 60 min and was sensitive to cytosolic Cl- with KCl = 19 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Biwersi
J
J
Department of Medicine, University of California, San Francisco 94143-0532.
Verkman
A S
AS
D000818:Animals; D001711:Biotransformation; D002460:Cell Line; D002463:Cell Membrane Permeability; D002712:Chlorides / Q000032:analysis* / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes; D006912:Hydroxyquinolines; D007700:Kinetics; D051379:Mice; D011806:Quinolinium Compounds / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry
D002712:Chlorides; D005456:Fluorescent Dyes; D006912:Hydroxyquinolines; D011806:Quinolinium Compounds; C070312:6-methoxy-N-ethyl-1,2-dihydroquinoline; C070311:6-methoxy-N-ethylquinolinium