Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding

Abstract
Data availability
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Abstract

The response of ATP-sensitive K⁺ channels (K_ATP) 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 K_ATP in channel gating. Binding to NBS2 was Mg²⁺-independent, but Mg 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 K_ATP activation by the same amount.

Data availability

Data available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.6mh0sv3

The following data sets were generated

1. Puljung M
(2019) Data from: Activation mechanism of ATP-sensitive K+ channels explored with real-time nucleotide binding--Revised
Dryad Digital Repository, doi.org/10.5061/dryad.6mh0sv3.

https://doi.org/10.5061/dryad.6mh0sv3

Article and author information

Author details

Michael Puljung

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

For correspondence
michael.puljung@dpag.ox.ac.uk

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-9335-0936
Natascia Vedovato

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

Competing interests
The authors declare that no competing interests exist.
Samuel Usher

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-2487-6547
Frances Ashcroft

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom

For correspondence
frances.ashcroft@dpag.ox.ac.uk

Competing interests
The authors declare that no competing interests exist.

Funding

Biotechnology and Biological Sciences Research Council (BB/R002517/1)

Michael Puljung
Frances Ashcroft

H2020 European Research Council (322620)

Michael Puljung
Natascia Vedovato
Frances Ashcroft

Wellcome Trust Oxion Graduate Program

Samuel Usher

John Fell Fund, University of Oxford

Michael Puljung

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.