Long-wavelength iodide-sensitive fluorescent indicators for measurement of functional CFTR expression in cells.
Limitations of available indicators [such as 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ)] for measurement of intracellular Cl(-) are their relatively dim fluorescence and need for ultraviolet excitation. A series of long-wavelength polar fluorophores was screened to identify compounds with Cl(-) and/or I(-) sensitivity, bright fluorescence, low toxicity, uniform loading of cytoplasm with minimal leakage, and chemical stability in cells. The best compound found was 7-(beta-D-ribofuranosylamino)-pyrido[2, 1-h]-pteridin-11-ium-5-olate (LZQ). LZQ is brightly fluorescent with excitation and emission maxima at 400-470 and 490-560 nm, molar extinction 11,100 M(-1). cm(-1) (424 nm), and quantum yield 0.53. LZQ fluorescence is quenched by I(-) by a collisional mechanism (Stern-Volmer constant 60 M(-1)) and is not affected by other halides, nitrate, cations, or pH changes (pH 5-8). After LZQ loading into cytoplasm by hypotonic shock or overnight incubation, LZQ remained trapped in cells (leakage <3%/h). LZQ stained cytoplasm uniformly, remained chemically inert, did not bind to cytoplasmic components, and was photobleached by <1% during 1 h of continuous illumination. Cytoplasmic LZQ fluorescence was quenched selectively by I(-) (50% quenching at 38 mM I(-)). LZQ was used to measure forskolin-stimulated I(-)/Cl(-) and I(-)/NO(-)(3) exchange in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing cell lines by fluorescence microscopy and microplate reader instrumentation using 96-well plates. The substantially improved optical and cellular properties of LZQ over existing indicators should permit the quantitative analysis of CFTR function in gene delivery trials and high-throughput screening of compounds for correction of the cystic fibrosis phenotype.
Jayaraman
S
S
Department of Medicine, Cardiovascular Research Institute, University of California, San Francisco, California, 94143-0521, USA.
Teitler
L
L
Skalski
B
B
Verkman
A S
AS
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D007454:Iodides / Q000494:pharmacology*; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010777:Photochemistry; D011621:Pteridines / Q000737:chemistry*; D011806:Quinolinium Compounds; D012266:Ribose / Q000031:analogs & derivatives* / Q000737:chemistry; D013873:Thionucleotides / Q000494:pharmacology
D002712:Chlorides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007454:Iodides; D010726:Phosphodiesterase Inhibitors; D011621:Pteridines; D011806:Quinolinium Compounds; D013873:Thionucleotides; C121386:luminarosine; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D012266:Ribose; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium; D000242:Cyclic AMP; D015056:1-Methyl-3-isobutylxanthine