Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
| Autor: | Michael C. Puljung, Samuel Usher, Frances M. Ashcroft |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
chemistry.chemical_classification
General Immunology and Microbiology Nucleotides General Chemical Engineering General Neuroscience Voltage clamp Cell Membrane Gating Ligands Ligand (biochemistry) General Biochemistry Genetics and Molecular Biology HEK293 Cells Förster resonance energy transfer G Protein-Coupled Inwardly-Rectifying Potassium Channels chemistry Membrane protein Adenine nucleotide Biophysics Humans Nucleotide Ion channel |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/61401 |
| Popis: | We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. This method combines expression of proteins tagged with the fluorescent non-canonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged KATP ion channels measured in unroofed plasma membranes and excised, inside-out membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the ligand-dependent gating of KATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified. |
| Databáze: | OpenAIRE |
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