Related Experiment Videos
Mechanism and cellular applications of a green fluorescent protein-based halide sensor
S Jayaraman1, P Haggie, R M Wachter
1Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco California 94143, USA.
The Journal of Biological Chemistry
|February 29, 2000
Summary
We developed a yellow fluorescent protein (YFP-H148Q) that functions as a cellular halide indicator. This biosensor accurately measures chloride (Cl(-)) transport in cells, aiding cystic fibrosis research.
Area of Science:
- Biophysics
- Molecular Biology
- Cellular Imaging
Background:
- Development of genetically encoded biosensors for ion dynamics is crucial for understanding cellular processes.
- Existing methods for halide sensing often lack the spatiotemporal resolution or cellular compatibility required for complex biological systems.
Purpose of the Study:
- To engineer and characterize a yellow fluorescent protein (YFP-H148Q) as a sensitive and targetable cellular halide indicator.
- To validate the sensor's performance in vitro and in live mammalian cells for measuring chloride transport.
- To demonstrate the utility of the sensor in studying ion channel function, specifically cystic fibrosis transmembrane conductance regulator (CFTR).
Main Methods:
- Fluorescence titrations, circular dichroism, absorption spectroscopy, and stopped-flow kinetics were used to characterize YFP-H148Q.
- Mammalian cells were transfected with YFP-H148Q for live-cell imaging and functional assays.
- Ionophore calibrations were performed to correlate in vitro and in cell measurements.
- Assays involved monitoring YFP-H148Q fluorescence changes during extracellular ion exchange to measure Cl(-) transport.
Main Results:
- YFP-H148Q exhibited pH-dependent pK(a) values and showed a significant fluorescence decrease with increasing chloride (Cl(-)) concentration (K(D) = 100 mM at pH 7.5).
- Spectroscopic and kinetic analyses confirmed a 1:1 binding mechanism between YFP-H148Q and Cl(-), with sensitivity to various anions.
- Transfected cells displayed bright cytoplasmic and nuclear fluorescence, with sensitivities comparable to aqueous solutions.
- The sensor successfully monitored cyclic AMP-regulated Cl(-) transport through CFTR channels with high sensitivity.
Conclusions:
- YFP-H148Q serves as a robust, genetically encoded biosensor for cellular halide and chloride (Cl(-)) dynamics.
- The sensor's performance in live cells enables sensitive real-time monitoring of ion transport.
- This YFP-based indicator has broad applications in anion channel research, drug screening for diseases like cystic fibrosis, and genetic screening.