Measuring nucleotide binding to intact, functional membrane proteins in real time
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 (trin...
| Main Authors: | , , |
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| Format: | Journal article |
| Language: | English |
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Journal of Visualized Experiments
2021
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| _version_ | 1826260357002297344 |
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| author | Usher, S Ashcroft, F Puljung, M |
| author_facet | Usher, S Ashcroft, F Puljung, M |
| author_sort | Usher, S |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-06T19:04:20Z |
| format | Journal article |
| id | oxford-uuid:149f0dea-c11c-451c-b33b-e7b95f4e6c6d |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T19:04:20Z |
| publishDate | 2021 |
| publisher | Journal of Visualized Experiments |
| record_format | dspace |
| spelling | oxford-uuid:149f0dea-c11c-451c-b33b-e7b95f4e6c6d2022-03-26T10:20:47ZMeasuring nucleotide binding to intact, functional membrane proteins in real timeJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:149f0dea-c11c-451c-b33b-e7b95f4e6c6dEnglishSymplectic ElementsJournal of Visualized Experiments2021Usher, SAshcroft, FPuljung, MWe 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. |
| spellingShingle | Usher, S Ashcroft, F Puljung, M Measuring nucleotide binding to intact, functional membrane proteins in real time |
| title | Measuring nucleotide binding to intact, functional membrane proteins in real time |
| title_full | Measuring nucleotide binding to intact, functional membrane proteins in real time |
| title_fullStr | Measuring nucleotide binding to intact, functional membrane proteins in real time |
| title_full_unstemmed | Measuring nucleotide binding to intact, functional membrane proteins in real time |
| title_short | Measuring nucleotide binding to intact, functional membrane proteins in real time |
| title_sort | measuring nucleotide binding to intact functional membrane proteins in real time |
| work_keys_str_mv | AT ushers measuringnucleotidebindingtointactfunctionalmembraneproteinsinrealtime AT ashcroftf measuringnucleotidebindingtointactfunctionalmembraneproteinsinrealtime AT puljungm measuringnucleotidebindingtointactfunctionalmembraneproteinsinrealtime |