Cryo-EM structure of the human THIK-1 K2P K+ channel reveals a lower Y gate regulated by lipids and anesthetics.

Nature structural & molecular biology (2025)

Authors:

Karin EJ Rödström, Bisher Eymsh, Peter Proks, Mehtab S Hayre, Sönke Cordeiro, Edward Mendez-Otalvaro, Christian Madry, Anna Rowland, Wojciech Kopec, Simon Newstead, Thomas Baukrowitz, Marcus Schewe, Stephen J Tucker

Abstract:

THIK-1 (KCNK13) is a halothane-inhibited and anionic-lipid-activated two-pore domain (K2P) K+ channel implicated in microglial activation and neuroinflammation, and a current target for the treatment of neurodegenerative disorders, for example Alzheimer's disease and amyothropic lateral sclerosis (ALS). However, compared to other K2P channels, little is known about the structural and functional properties of THIK-1. Here we present a 3.16-Å-resolution cryo-EM structure of human THIK-1 that reveals several distinct features, in particular, a tyrosine in M4 that contributes to a lower 'Y gate' that opens upon activation by physiologically relevant G-protein-coupled receptor and lipid signaling pathways. We demonstrate that linoleic acid bound within a modulatory pocket adjacent to the filter influences channel activity, and that halothane inhibition involves a binding site within the inner cavity, both resulting in conformational changes to the Y gate. Finally, the extracellular cap domain contains positively charged residues that line the ion exit pathway and contribute to the distinct biophysical properties of this channel. Overall, our results provide structural insights into THIK-1 function and identify distinct regulatory sites that expand its potential as a drug target for the modulation of microglial function.

Electronic Polarizability Tunes the Function of the Human Bestrophin 1 Cl<sup>-</sup> Channel.

Journal of chemical theory and computation American Chemical Society (ACS) (2025)

Authors:

Linda X Phan, Aaron P Owji, Tingting Yang, Jason Crain, Mark SP Sansom, Stephen J Tucker

Abstract:

Mechanisms of anion permeation within ion channels and nanopores remain poorly understood. Recent cryo-electron microscopy structures of the human bestrophin 1 Cl<sup>-</sup> channel (hBest1) provide an opportunity to evaluate ion interactions predicted by molecular dynamics (MD) simulations against experimental observations. Here, we implement the fully polarizable force field AMOEBA in MD simulations on different conformations of hBest1. This force field models multipole moments up to the quadrupole. Using this approach, we model key biophysical properties of the channel that can only be simulated when electronic polarization is included in the molecular models and show that Cl<sup>-</sup> permeation through the neck of the pore is achieved through hydrophobic solvation concomitant with partial ion dehydration. Furthermore, we demonstrate how such polarizable simulations can help determine the identity of ion-like densities within high-resolution cryo-EM structures and demonstrate that neglecting polarization places Cl<sup>-</sup> at positions that do not correspond to their experimentally resolved location. Overall, our results demonstrate the importance of including electronic polarization in realistic and physically accurate models of biological systems, especially channels and pores that selectively permeate anions.

Structures of TASK-1 and TASK-3 K2P channels provide insight into their gating and dysfunction in disease.

Structure (London, England : 1993) Elsevier 33:1 (2024) 115-122.e4

Authors:

Peter Rory Hall, Thibault Jouen-Tachoire, Marcus Schewe, Peter Proks, Thomas Baukrowitz, Elisabeth P Carpenter, Simon Newstead, Karin EJ Rödström, Stephen J Tucker

Abstract:

TASK-1 and TASK-3 are pH-sensitive two-pore domain (K2P/KCNK) K<sup>+</sup> channels. Their functional roles make them promising targets for treatment of multiple disorders including sleep apnea, pain, and atrial fibrillation. Mutations in these channels are also associated with neurodevelopmental and hypertensive disorders. A previous crystal structure of TASK-1 revealed a lower "X-gate" as a hotspot for missense gain-of-function (GoF) mutations associated with DDSA (developmental delay with sleep apnea). However, the mechanisms of gating in TASK channels are still not fully understood. Here, we resolve structures for both human TASK-1 and TASK-3 by cryoelectron microscopy (cryo-EM), as well as a recurrent TASK-3 variant (G236R) associated with KCNK9 imprinting syndrome (KIS) (formerly known as Birk-Barel syndrome). Combined with functional studies of the X-gating mechanism, we provide evidence for how a highly conserved gating mechanism becomes defective in disease, and also provide further insight into the pathway of conformational changes that underlie the pH-dependent inhibition of TASK channel activity.

Extracellular modulation of TREK-2 activity with nanobodies provides insight into the mechanisms of K2P channel regulation

Nature Communications Springer Nature 15:1 (2024) 4173

Authors:

Karin EJ Rödström, Alexander Cloake, Janina Sörmann, Agnese Baronina, Kathryn HM Smith, Ashley CW Pike, Jackie Ang, Peter Proks, Marcus Schewe, Ingelise Holland-Kaye, Simon R Bushell, Jenna Elliott, Els Pardon, Thomas Baukrowitz, Raymond J Owens, Simon Newstead, Jan Steyaert, Elisabeth P Carpenter, Stephen J Tucker

Abstract:

Potassium channels of the Two-Pore Domain (K2P) subfamily, KCNK1-KCNK18, play crucial roles in controlling the electrical activity of many different cell types and represent attractive therapeutic targets. However, the identification of highly selective small molecule drugs against these channels has been challenging due to the high degree of structural and functional conservation that exists not only between K2P channels, but across the whole K+ channel superfamily. To address the issue of selectivity, here we generate camelid antibody fragments (nanobodies) against the TREK-2 (KCNK10) K2P K+ channel and identify selective binders including several that directly modulate channel activity. X-ray crystallography and CryoEM data of these nanobodies in complex with TREK-2 also reveal insights into their mechanisms of activation and inhibition via binding to the extracellular loops and Cap domain, as well as their suitability for immunodetection. These structures facilitate design of a biparatropic inhibitory nanobody with markedly improved sensitivity. Together, these results provide important insights into TREK channel gating and provide an alternative, more selective approach to modulation of K2P channel activity via their extracellular domains.

CryoEM Structure of the human THIK-1 K2P K+Channel Reveals a Lower ‘Y-gate’ Regulated by Lipids and Anaesthetics

(2024)

Authors:

Karin EJ Rödström, Bisher Eymsh, Peter Proks, Mehtab Hayre, Christian Madry, Anna Rowland, Simon Newstead, Thomas Baukrowitz, Marcus Schewe, Stephen Tucker