TY - JOUR

T1 - The dynamic interplay of PIP2 and ATP in the regulation of the KATP channel

AU - Pipatpolkai, Tanadet

AU - Usher, Samuel G

AU - Vedovato, Natascia

AU - Ashcroft, Frances M

AU - Stansfeld, Phillip J

N1 - The peer review history is available in the Supporting Information section of this article (https://doi.org/10.1113/JP283345#support-information-section). This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as https://doi.org/10.1113/JP283345

PY - 2022

Y1 - 2022

N2 - KEY POINTS: The KATP channel is activated by the binding of phosphoinositol-bisphosphate (PIP2 ) lipids and inactivated by the binding of adenosine triphosphate (ATP). K39 has the potential to bind to both PIP2 and ATP. A mutation to this residue (K39R) results in neonatal diabetes. This study uses patch-clamp fluorometry, electrophysiology and molecular dynamics simulation. We show that PIP2 competes with ATP for K39, and this reduces channel inhibition by ATP. We show that K39R increases channel affinity to PIP2 by increasing the number of hydrogen bonds with PIP2 , when compared with the wild-type K39. This therefore decreases KATP channel inhibition by ATP.ABSTRACT: ATP-sensitive potassium (KATP ) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol-4,5-bisphosphate (PIP2 ). Here, we use molecular dynamics (MD) simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, simulations suggest that K39 shows a greater preference to co-ordinate with PIP2 than ATP. They also predict that a neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2 , potentially accounting for the reduced ATP inhibition observed in electrophysiological experiments. Our work suggests PIP2 and ATP interact allosterically to regulate KATP channel activity. Abstract figure legend In this study we have used electrophysiology, patch clamp fluorometry and molecular dynamics simulations to study the dynamic interplay of PIP2 and ATP in the regulation of the KATP channel, identifying K39 as a residue that engages with both ligands. This article is protected by copyright. All rights reserved.

AB - KEY POINTS: The KATP channel is activated by the binding of phosphoinositol-bisphosphate (PIP2 ) lipids and inactivated by the binding of adenosine triphosphate (ATP). K39 has the potential to bind to both PIP2 and ATP. A mutation to this residue (K39R) results in neonatal diabetes. This study uses patch-clamp fluorometry, electrophysiology and molecular dynamics simulation. We show that PIP2 competes with ATP for K39, and this reduces channel inhibition by ATP. We show that K39R increases channel affinity to PIP2 by increasing the number of hydrogen bonds with PIP2 , when compared with the wild-type K39. This therefore decreases KATP channel inhibition by ATP.ABSTRACT: ATP-sensitive potassium (KATP ) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol-4,5-bisphosphate (PIP2 ). Here, we use molecular dynamics (MD) simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, simulations suggest that K39 shows a greater preference to co-ordinate with PIP2 than ATP. They also predict that a neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2 , potentially accounting for the reduced ATP inhibition observed in electrophysiological experiments. Our work suggests PIP2 and ATP interact allosterically to regulate KATP channel activity. Abstract figure legend In this study we have used electrophysiology, patch clamp fluorometry and molecular dynamics simulations to study the dynamic interplay of PIP2 and ATP in the regulation of the KATP channel, identifying K39 as a residue that engages with both ligands. This article is protected by copyright. All rights reserved.

U2 - 10.1113/JP283345

DO - 10.1113/JP283345

M3 - Journal article

C2 - 36047384

VL - 600

SP - 4503

EP - 4519

JO - The Journal of Physiology

JF - The Journal of Physiology

SN - 0022-3751

IS - 20

ER -