Long-wavelength iodide-sensitive fluorescent indicators for measurement offunctional CFTR expression in cells

Limitations of available indicators [such as 6-methoxy-N-(3-sulfopropyl)qui nolinium (SPQ)] for measurement of intracellular Cl- are their relatively d im fluorescence and need for ultraviolet excitation, A series of long-wavel ength polar fluorophores was screened to identify compounds with Cl- and/or I- sensitivity, bright fluorescence, low toxicity, uniform loading of cyto plasm with minimal leakage, and chemical stability in cells. The best compo und found was 7-(beta-D-ribofuranosylamino)-pyrido [2,1-h]-pteridin-11-ium- 5-olate (LZQ). LZQ is brightly fluorescent with excitation and emission max ima 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 collisiona l mechanism (Stern-Volmer constant 60 M-1) and is not affected by other hal ides, nitrate, cations, or pH changes (pH 5-8). After LZQ loading into cyto plasm by hypotonic shock or overnight incubation, LZQ remained trapped in c ells (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 qu enched selectively by I-(50% quenching at 38 mM I-). LZQ was used to measur e forskolin-stimulated I-/Cl- and I-/NO3- exchange in cystic fibrosis trans membrane 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 gen e delivery trials and high throughput screening of compounds for correction of the cystic fibrosis phenotype.