Mechanism and cellular applications of a green fluorescent protein-based halide sensor

We report the application of a targetable green fluorescent protein-based c ellular halide indicator. Fluorescence titrations of the purified recombina nt yellow fluorescent protein YFP-H148Q indicated a pK(a) of 7.14 in the ab sence of Cl-, which increased to 7.86 at 150 mM Cl-. At pH 7.5, YFP-H148Q f luorescence decreased maximally by similar to 2-fold with a K-D of 100 mM C l-. YFP-H148Q had a fluorescence lifetime of 8.1 ns that was independent of pH and [Cl-]. Circular dichroism and absorption spectroscopy revealed dist inct Cl--dependent spectral changes indicating Cl-/YFP binding. Stopped-flo w kinetic analysis showed a biexponential time course of YFP-H148Q fluoresc ence (time constants <100 ms) in response to changes in pH or [Cl-], establ ishing a 1:1 YFP-H148Q/Cl- binding mechanism, Photobleaching analysis revea led a millisecond triplet state relaxation process that was insensitive to anions and aqueous-phase quenchers. The anion selectivity sequence for YFP- H148Q quenching (ClO4(-) similar to I- > SCN- > NO3- > Cl- > Br- > formate > acetate) indicated strong binding of weakly hydrated chaotropic ions, The biophysical data suggest that YFP-H148Q anion sensitivity involves ground state anion binding to a site close to the tri-amino acid chromophore. YFP- H148Q transfected mammalian cells were brightly fluorescent with cytoplasmi c/nuclear staining. Ionophore calibrations indicated similar YFP-H148Q pH a nd anion sensitivities in cells and aqueous solutions. Cyclic AMP-regulated Cl- transport through plasma membrane cystic fibrosis transmembrane conduc tance regulator Cl- channels was assayed with excellent sensitivity from th e time course of YFP-H148Q fluorescence in response to extracellular Cl-/I- exchange. The green fluorescent protein-based halide sensor described here should have numerous applications, such as anion channel cloning by screen ing of mammalian expression libraries and discovery of compounds that corre ct the cystic fibrosis phenotype by screening of combinatorial libraries.