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 approximately 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 (ClO(4)(-) approximately I(-) > SCN(-) > NO(3)(-) > 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.

Highlights

  • From the Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco California 94143 and the ‡Institute of Molecular Biology and Department of Physics, University of Oregon, Eugene, Oregon 97403

  • We report the application of a targetable green fluorescent protein-based cellular halide indicator

  • The biophysical data suggest that YFP-H148Q anion sensitivity involves ground state anion binding to a site close to the triamino acid chromophore

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Summary

Introduction

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. Transport of ClϪ across cell plasma membranes is required in many cellular processes such as cell volume regulation and epithelial fluid absorption and secretion. ClϪ concentration in cytoplasm and ClϪ transport across cell plasma membranes has been measured using ClϪ sensitive fluorescent indicators. Our laboratory introduced quinolinium-type ClϪ indicators whose fluorescence is quenched by ClϪ by a collisional mechanism [1, 2] These indicators have been used widely to study ClϪ transport mechanisms in mammalian cells, including cells expressing mutant CFTR ClϪ channels and cells obtained from human subjects in CFTR gene therapy trials [1]. Various tailored GFP-based indicators have been introduced for measurement of calcium concentration [15, 16], membrane potential [17], and protease activity [18, 19]

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