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J Biol Chem, Vol. 275, Issue 9, 6047-6050, March 3, 2000
From the Departments of Medicine and Physiology, Cardiovascular
Research Institute, University of California, San Francisco California
94143 and the 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 pKa of 7.14 in the absence of
Cl
ACCELERATED PUBLICATION
Mechanism and Cellular Applications of a Green Fluorescent
Protein-based Halide Sensor*
,, and Institute of Molecular Biology and
Department of Physics, University of Oregon, Eugene, Oregon 97403
, which increased to 7.86 at 150 mM
Cl. At pH 7.5, YFP-H148Q fluorescence decreased maximally
by ~2-fold with a KD 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.
*
This work was supported by Research Development Grant R613
from the National Cystic Fibrosis Foundation and Grants DK43840, DK35124, HL59198, and HL60288 from the National Institutes of Health
(to A. S. V.) and Grant MCB9728162 from the National Science Foundation (to S. J. R.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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