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Am J Physiol Cell Physiol 277: C1008-C1018, 1999;
0363-6143/99 $5.00
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Vol. 277, Issue 5, C1008-C1018, November 1999

SPECIAL COMMUNICATION
Long-wavelength iodide-sensitive fluorescent indicators for measurement of functional CFTR expression in cells

Sujatha Jayaraman1, Leah Teitler1, Bohdan Skalski2, and A. S. Verkman1

1 Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California, 94143-0521; and 2 Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland

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.

cystic fibrosis transmembrane conductance regulator; cystic fibrosis; 6-methoxy-N-(3-sulfopropyl)quinolinium; chloride transport; fluorescence; high-throughput screening


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