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

Sujatha Jayaraman, Peter Haggie, Rebekka M. Wachter, S. James Remington, A. S. Verkman

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Abstract

We report the application of a targetable green fluorescent protein- based cellular halide indicator. Fluorescence titrations of the purified recombinant yellow fluorescent protein YFP-H148Q indicated a pK(a) of 7.14 in the absence of Cl-, which increased to 7.86 at 150 mM Cl-. At pH 7.5, YFP- H148Q fluorescence decreased maximally by ~2-fold with a K(D) of 100 mM Cl. YFP-H148Q had a fluorescence lifetime of 3.1 ns that was independent of pH and [Cl-]. Circular dichroism and absorption spectroscopy revealed distinct Cl- dependent spectral changes indicating Cl-/YFP binding. Stopped-flow kinetic analysis showed a biexponential time course of YFP-H148Q fluorescence (time constants <100 ms) in response to changes in pH or [Cl-], establishing a 1:1 YFP-H148Q/Cl- binding mechanism. Photobleaching analysis revealed a millisecond triplet state relaxation process that was insensitive to anions and aqueous-phase quenchers. The anion selectivity sequence for YFP-H148Q quenching (ClO4/- ~ 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 cytoplasmic/nuclear staining. Ionophore calibrations indicated similar YFP- H148Q pH and anion sensitivities in cells and aqueous solutions. Cyclic AMP- regulated Cl- transport through plasma membrane cystic fibrosis transmembrane conductance regulator Cl- channels was assayed with excellent sensitivity from the 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 screening of mammalian expression libraries and discovery of compounds that correct the cystic fibrosis phenotype by screening of combinatorial libraries.

Original languageEnglish (US)
Pages (from-to)6047-6050
Number of pages4
JournalJournal of Biological Chemistry
Volume275
Issue number9
DOIs
StatePublished - Mar 3 2000
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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