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Green fluorescent protein‐based halide indicators with improved chloride and iodide affinities
Abstract
The green fluorescent protein YFP‐H148Q is sensitive to halides by a mechanism involving halide binding and a shift in p K 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.
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Cited by
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Verkman, A. S., Anderson, M. O., & Papadopoulos, M. C. (2014) · Nature Reviews Drug Discovery · doi:10.1038/nrd4226 · Cited by 586
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Qiu, Z., Dubin, A. E., Mathur, J., Tu, B., Reddy, K., Miraglia, L. J., Reinhardt, J., Orth, A. P., & Patapoutian, A. (2014) · Cell · doi:10.1016/j.cell.2014.03.024 · Cited by 571
Chloride channels as drug targets
Verkman, A. S., & Galietta, L. J. V. (2009) · Nature Reviews Drug Discovery · doi:10.1038/nrd2780 · Cited by 469
Self-citationAnion transport and supramolecular medicinal chemistry
Gale, P. A., Davis, J. T., & Quesada, R. (2017) · Chemical Society Reviews · doi:10.1039/c7cs00159b · Cited by 359
Planells‐Cases, R., Lutter, D., Guyader, C., Gerhards, N. M., Ullrich, F., Elger, D. A., Kucukosmanoglu, A., Xu, G., Voss, F. K., Reincke, S. M., et al. (2015) · The EMBO Journal · doi:10.15252/embj.201592409 · Cited by 256
Metadata
- Authors
- Galietta, L. J. V., Haggie, P. M., & Verkman, A. S.
- Venue
- FEBS Letters
- Publisher
- Wiley
- Published
- 2001-06-18
- Volume
- 499
- Issue
- 3
- Pages
- 220-224
- ISSN
- 0014-5793 (ISSN-L)
- 1873-3468
- OpenAlex
- W1983746433
- Cited by
- 371
- References
- 0
- FWCI
- 3.73
- Type
- journal article
- Language
- en
- Open access
- closed
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- Galietta, L. J. V., Haggie, P. M., & Verkman, A. S. (2001). Green fluorescent protein‐based halide indicators with improved chloride and iodide affinities. FEBS Letters. https://doi.org/10.1016/s0014-5793(01)02561-3
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