Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding
The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (usin...
| Main Authors: | , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
eLife Sciences
2019
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| _version_ | 1797073768381677568 |
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| author | Puljung, M Vedovato, N Usher, S Ashcroft, F |
| author_facet | Puljung, M Vedovato, N Usher, S Ashcroft, F |
| author_sort | Puljung, M |
| collection | OXFORD |
| description | The response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg2+ was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount. |
| first_indexed | 2024-03-06T23:26:46Z |
| format | Journal article |
| id | oxford-uuid:6a9f7194-fe39-4c57-8dc9-f094863004f0 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T23:26:46Z |
| publishDate | 2019 |
| publisher | eLife Sciences |
| record_format | dspace |
| spelling | oxford-uuid:6a9f7194-fe39-4c57-8dc9-f094863004f02022-03-26T18:58:44ZActivation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide bindingJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:6a9f7194-fe39-4c57-8dc9-f094863004f0EnglishSymplectic Elements at OxfordeLife Sciences2019Puljung, MVedovato, NUsher, SAshcroft, FThe response of ATP-sensitive K+ channels (KATP) to cellular metabolism is coordinated by three classes of nucleotide binding site (NBS). We used a novel approach involving labeling of intact channels in a native, membrane environment with a non-canonical fluorescent amino acid and measurement (using FRET with fluorescent nucleotides) of steady-state and time-resolved nucleotide binding to dissect the role of NBS2 of the accessory SUR1 subunit of KATP in channel gating. Binding to NBS2 was Mg2+-independent, but Mg2+ was required to trigger a conformational change in SUR1. Mutation of a lysine (K1384A) in NBS2 that coordinates bound nucleotides increased the EC50 for trinitrophenyl-ADP binding to NBS2, but only in the presence of Mg2+, indicating that this mutation disrupts the ligand-induced conformational change. Comparison of nucleotide-binding with ionic currents suggests a model in which each nucleotide binding event to NBS2 of SUR1 is independent and promotes KATP activation by the same amount. |
| spellingShingle | Puljung, M Vedovato, N Usher, S Ashcroft, F Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding |
| title | Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding |
| title_full | Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding |
| title_fullStr | Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding |
| title_full_unstemmed | Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding |
| title_short | Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding |
| title_sort | activation mechanism of atp sensitive k channels explored with real time nucleotide binding |
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