Green fluorescent protein-based halide indicators with improved chloride and iodide affinities.
The green fluorescent protein YFP-H148Q is sensitive to halides by a mechanism involving halide binding and a shift in pK(a). However, a limitation of YFP-H148Q is its low halide sensitivity, with K(d)>100 mM for Cl(-). Indicators with improved sensitivities are needed for cell transport studies, particularly in drug discovery by high-throughput screening, and for measurement of Cl(-) concentration in subcellular organelles. YFP-H148Q libraries were generated in which pairs of residues in the vicinity of the halide binding site were randomly mutated. An automated procedure was developed to screen bacterial colonies for improved halide sensitivity. Analysis of 1536 clones revealed improved anion sensitivities with K(d) down to 2 mM for I(-) (I152L), 40 mM for Cl(-) (V163S), and 10 mM for NO(3)(-) (I152L). The anion-sensitive mechanism of these indicators was established and their utility in cells was demonstrated using transfected cells expressing the cystic fibrosis transmembrane conductance regulator chloride channel.
Galietta
L J
LJ
Department of Medicine, Cardiovascular Research Institute, 1246 Health Sciences East Tower, University of California, San Francisco, CA 94143-0521, USA.
Haggie
P M
PM
Verkman
A S
AS
D016475:3T3 Cells; D000818:Animals; D002712:Chlorides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins; D007454:Iodides / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial
D002712:Chlorides; D007454:Iodides; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins