Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry

Abstract

Pancreatic ATP-sensitive K⁺ channels (K_ATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts K_ATP. Mg-nucleotide binding to SUR1 stimulates K_ATP. In the absence of Mg²⁺, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that K_ATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit K_ATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit K_ATP. This suggests a dual role for SUR1 in K_ATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.

Data availability

All data and associated code are available on github (https://github.com/smusher/KATP_paper_2019) and have also been uploaded to Dryad (https://doi.org/10.5061/dryad.0vt4b8gtv).

The following data sets were generated

(2019) Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry
Dryad Digital Repository, doi:10.5061/dryad.0vt4b8gtv.

https://doi.org/10.5061/dryad.0vt4b8gtv

Article and author information

Author details

Samuel G 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 M 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.

"This ORCID iD identifies the author of this article:" 0000-0002-6970-1767
Michael C 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

Funding

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

Frances M Ashcroft
Michael C Puljung

John Fell Fund, University of Oxford

Michael C Puljung

Wellcome (203731/Z/16/A)

Samuel G Usher

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

Reviewing Editor

Gary Yellen, Harvard Medical School, United States

Publication history

Received: October 16, 2019
Accepted: January 7, 2020
Accepted Manuscript published: January 7, 2020 (version 1)
Version of Record published: February 6, 2020 (version 2)
Version of Record updated: February 27, 2020 (version 3)

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.