Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/00000033910.33594/000000339 © 2021 The Author(s).© 2021 Published The Author(s) by and Biochemistry Published online: online: 6 6March March 2021 2021 Cell Physiol BiochemPublished Press GmbH&Co. by Cell Physiol KG Biochem 87 Press GmbH&Co. KG, Duesseldorf Decher et al.: Molecular Pharmacology of K Channels Accepted: 12 January 2021 2P www.cellphysiolbiochem.com

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Molecular Pharmacology of K2P Potassium Channels

Niels Dechera Susanne Rinnéa Mauricio Bedoyab,c Wendy Gonzalezb,c Aytug K. Kipera aVegetative Physiology, Institute for Physiology and Pathophysiology, Philipps-University Marburg, Marburg, Germany, bCentro de Bioinformática y Simulación Molecular, Universidad de Talca, Talca, Chile, cMillennium Nucleus of Ion Channels-Associated Diseases (MiNICAD), Universidad de Talca, Talca, Chile

Key Words

Drug binding sites • K2P potassium channels • Ion channels • Molecular pharmacology

Abstract

Potassium channels of the tandem of two-pore-domain (K2P) family were among the last potassium channels cloned. However, recent progress in understanding their physiological relevance and molecular pharmacology revealed their therapeutic potential and thus these channels evolved as major drug targets against a large variety of diseases. However, after the initial cloning of the fifteen family members there was a lack of potent and/or selective modulators. By now a large variety of K2P channel modulators (activators and blockers) have been described, especially for TASK-1, TASK-3, TREK-1, TREK2, TRAAK and TRESK channels.

Recently obtained crystal structures of K2P channels, alanine scanning approaches to map drug binding sites, in silico experiments with molecular dynamics simulations (MDs) combined with electrophysiological studies to reveal the mechanism of channel inhibition/activation, yielded a good understanding of the molecular pharmacology of these channels. Besides summarizing drugs that were identified to modulate 2PK channels, the main focus of this article is on describing the differential binding sites and mechanisms of channel modulation that are utilized by the different 2PK channel blockers and activators. © 2021 The Author(s). Published by Cell Physiol Biochem Press GmbH&Co. KG Introduction

Tandem of two-pore-domain potassium (K2P) channels belong to the latest family of potassium channels cloned, with TOK1 from Saccharomyces cerevisiae discovered in 1995 [1]. The mammalian K2P potassium channel family contains 15 members with different subfamilies, characterized by mechanistic hallmarks like acid as inhibition, the first channel stretch activation, alkaline activation and halothane inhibition (shown in Fig. 1a). K2P channels have

N. Decher and S. Rinné contributed equally to this work. Prof. Dr. Niels Decher Vegetative Physiology Institute for Physiology and Pathophysiology, Philipps-University Marburg Deutschhausstraße 1-2, 35037 Marburg (Germany) Tel. +49 6421-28-62148, E-Mail [email protected] Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 88

Decher et al.: Molecular Pharmacology of K2P Channels four transmembrane domains and contain two pore loops and thus assemble as dimer in channel families (shown in Fig. 1b, 1c). The channels have a large extracellular M1-P1 loop order that four pore loops form the potassium selectivity filter, similar as in other potassium

which has been identified in early studies as self-interacting domain (‘SID’)2P and channels is relevant (shown for the dimerization of the channels (shown in Fig. 1b) [2]. In fact, the extracellular M1-P1 loop toxinsforms thefrom so binding called ‘cap’ to the structure pore region which [4]. is aHowever, structural whether hallmark there of the are K other physiological functionsin Fig. 1b, that 1c) can [3-9]. be assigned It has been to this postulated unique structure that the has extracellular not been addressed ‘cap’ prevents yet. Although classical

K2P appeared to solely obey the Goldman-Hodgkin-Katz equation for a potassium selective hole, we know channels in the were meantime initially that described these channels as leak are channels highly withregulated an outward by a plethora rectification of different that stimuli (shown in Fig. 1a). Moreover, these channels are in fact voltage sensitive with potassium acting as the actual voltage sensor, meaning similar to the CLC chloride channel family [10], the permeating ion actually also gates the channel [11]. Thus, K2P channels are of the channel at depolarized potentials. potassium gated2P channelspotassium were channels described with toa potassium have a unique efflux pharmacology increasing the compared open probability to other potassium channel families, as they were less sensitive to the classical potassium channel + 2+ blockersInitially, like KTEA, 4-AP, Cs or Ba . Channels of the K2P family appeared to be drug resistant. However, classical open channel blockers of ion channels share a conserved binding site scheme with about two or more interacting residues of the pore forming helices and an additional binding to residues of the pore signature sequence (shown in Fig. 1d). This binding

Fig. 1. The K2P 2P channels with their physiological or pharmacological + K+ channel family. (a) Dendrogram of K + keyK+ modulators. TWIK: Tandem of P-domains in a weak+ inward rectifying K channel, TREK: TWIK-related+ channel, TRAAK: TWIK-related arachidonic+ acid activated K channel, TASK: TWIK-related acid-sensitive channel. (b) Membrane topology of K2P channels depicting transmembrane channel, TALK: domains TWIK-related in blue, alkaline the pore activated helices in K red channel, and the THIK: extracellular TWIK-related cap structurehalothane in inhibited purple. (c)K channel, TRESK: TWIK-related spinal cord K 2P are illustrated in blue, the pore helices in red and the extracellular cap ‘ ’ Illustrationof the conserved of the scheme crystal structureof drug binding of the Ksites channel in voltage TWIK-1 gated (PDB ion channels.ID: 3UKM). The Transmembrane typical binding domains sites of ‘ ’ structure in purple. (d) Illustration formula of quaternary ammonium compounds, which are classical pore blockers with different alkyl side chainclassical lengths. pore blockers are indicated with purple stars. CC: central cavity; SF: selectivity filter (e) Chemical Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 89

Decher et al.: Molecular Pharmacology of K2P Channels

site pattern can be found in different potassium channel families, but also in voltage-gated

sodium and calcium channels [12-20]. Thus, it appears unlikely that K2P channels in general 2P channels are also highly sensitive to classical pore block by quaternary ammonium compounds (QA) (shownshould be in resistantFig. 1e), however, to classical presumably pore blockers. due to In differences the meantime, in the we architecture know that of K the central

alkyl side chains, like TPenA or THexA (shown in Fig. 1e), to be stabilized underneath the cavity providing more lateral space underneath the selectivity filter, the QAs need longer

selectivity filter by interact with the pore forming helices [21]. While the sensitivity of

TASK-3 channels to extracellular polyamines and ruthenium red (RR) has 2Pbeen channels described and very early after cloning of the TASK channels [22-29],2P there was for a longer time a lack of potent small compound inhibitors. A decade after cloning of the first K functionalTASK‑1 [23, studies 30, 31], of we the described channel inthe native first potenttissue [33],K channel leading blocker for instance A293 to[32]. the Strikingly, isolation

the development of a potent and selective TASK-1 channel blocker opened the door for2P channels there are still no highly potent and selective blockers or activators available (shown ofin Tablea whole 1-8). cell Thus, TASK-1 we eagerlycurrent anticipatein ventricular the developmentmyocytes [32]. of However,small compound for most modulators of the K

for all K2P channels as this will, besides the generation and study of transgenic mouse models, 2P channels in different organs. definitely foster our research aiming to understand the physiological role of K and TALK channels, there are in the meantime many potent activators and blockers described However, while we still have a poor understanding of the pharmacology of TWIK, THIK separate sections, especially by focusing on the mechanistic models of channel modulation andfor the the channelsdifferential of bindingthe TASK sites and utilized TREK-subfamily, by the drugs. which we would like to summarize in

Table 1. 2P channel subfamilies  List of inhibitors of the TWIK, THIK and TALK K ͷͲ Šƒ‡Žˆƒ‹Ž› Šƒ‡Ž •—„•–ƒ ‡ ƒ–‡‰‘”› ‹Š‹„‹–‘”  ȋρȌ ”‡ˆ‡”‡ ‡             Ǧͳ        Žƒ•• ƒ–‹ƒ””Š›–Š‹ • “—‹‹†‹‡ ≈95 ‡•ƒ‰‡‡–ƒŽǤǡͳͻͻ͸ ͺ͸Ͳͷͺ͸ͻ   Ž‘ ƒŽƒ‡•–Š‡–‹ • „—’‹˜ƒ ƒ‹‡ ‹†Ž‡”‡–ƒŽǤǡͳͻͻͻ ͳͲʹͲͳ͸ͺʹ    ƒŽƒ”‹ƒ–”‡ƒ–‡–• “—‹‹‡ ≈50 ‡•ƒ‰‡‡–ƒŽǤǡͳͻͻ͸ ͺ͸Ͳͷͺ͸ͻ   Ǧʹ        Žƒ•• ƒ–‹ƒ””Š›–Š‹ • “—‹‹†‹‡ ƒ–‡Ž‡–ƒŽǤǡʹͲͲͲ ͳͲͺͺ͹ͳͺ͹    ƒŽƒ”‹ƒ–”‡ƒ–‡–• “—‹‹‡ ƒ–‡Ž‡–ƒŽǤǡʹͲͲͲ ͳͲͺͺ͹ͳͺ͹    •ƒŽŽ‘Ž‡ —Ž‡• ǦǦͳ —‡–ƒŽǤǡʹͲͳ͸ ʹ͹Ͳͻͳͻͻ͹    ‹Š‹„‹–‘”• ‰‡‹•–‡‹ ‹‡”–‡‡–ƒŽǤǡʹͲͲͺ ͳͺͷͳ͸Ͳ͸ͻ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠŽ‘”‘ˆ‘” ƒ–‡Ž‡–ƒŽǤǡʹͲͲͲ ͳͲͺͺ͹ͳͺ͹    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡ ƒ–‡Ž‡–ƒŽǤǡʹͲͲͲ ͳͲͺͺ͹ͳͺ͹            Ǧͳ        •ƒŽŽ‘Ž‡ —Ž‡• ͳͺͻͻ ≈2 –”‡‹–‡–ƒŽǤǡʹͲͳͳ ʹͳ͵͸ʹ͸ͳͻ   ‹Š‹„‹–‘”• ‰‡‹•–‡‹ ‹‡”–‡‡–ƒŽǤǡʹͲͲͺ ͳͺͷͳ͸Ͳ͸ͻ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡ ≈2800 ƒŒƒ‡–ƒŽǤǡʹͲͲͳ ͳͳͲ͸Ͳ͵ͳ͸   Ǧʹ        Žƒ•• ƒ–‹ƒ””Š›–Š‹ • “—‹‹†‹‡ ‡‹‰—–ƒ‡–ƒŽǤǡʹͲͳͶ ʹͶʹͻ͹ͷʹʹ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡ ‡‹‰—–ƒ‡–ƒŽǤǡʹͲͳͶ ʹͶʹͻ͹ͷʹʹ          Ǧͳ        ƒ”†‹ƒ ‰Ž› ‘•‹†‡• †‹‰‹–‘š‹  Š‹†–‡–ƒŽǤǡʹͲͳͺ ʹͻ͸Ͷ͵ʹͷͶ    Žƒ•• ƒ–‹ƒ””Š›–Š‹ • “—‹‹†‹‡ ƒ‡–ƒŽǤǡʹͲͲ͵ ͳʹ͹ʹͶͳͶʹ    Ž‘š›“—‹ƒƒŽ‘‰• ʹ͹͸Ͷ ‡‰›‡Ž‡–ƒŽǤǡʹͲͳͻ ͵Ͳͻ͹ͻͺͳʹ    ƒŽƒ”‹ƒ–”‡ƒ–‡–• “—‹‹‡ ‹”ƒ”†‡–ƒŽǤǡʹͲͲͳ ͳͳʹ͸͵ͻͻͻ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠŽ‘”‘ˆ‘” ‹”ƒ”†‡–ƒŽǤǡʹͲͲͳ ͳͳʹ͸͵ͻͻͻ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡ ‹”ƒ”†‡–ƒŽǤǡʹͲͲͳ ͳͳʹ͸͵ͻͻͻ   Ǧʹ        ƒ–‹’•› Š‘–‹ • ˆŽ—‘š‡–‹‡ ≈17 —•–‘•‡–ƒŽǤǡʹͲʹͲ ͵ͳͻͶ͹͸͹ͻ   Žƒ•• ƒ–‹ƒ””Š›–Š‹ • “—‹‹†‹‡ ‡›‡•‡–ƒŽǤǡͳͻͻͺ ͻͺͳʹͻ͹ͺ    Žƒ•• ƒ–‹ƒ””Š›–Š‹ • Ž‘ˆ‹Ž‹— ≈25 ‹‡‡›‡”‡–ƒŽǤǡʹͲͲͳ ͳͳͷ͸Ͳͻ͵Ͷ   Ž‘ ƒŽƒ‡•–Š‡–‹ • „—’‹˜ƒ ƒ‹‡ ‹†Ž‡”‡–ƒŽǤǡʹͲͲ͵ ͳʹ͸͸Ͳ͵ͳͳ    Ž‘ ƒŽƒ‡•–Š‡–‹ • Ž‹†‘ ƒ‹‡ ‹†Ž‡”‡–ƒŽǤǡʹͲͲ͵ ͳʹ͸͸Ͳ͵ͳͳ    Ž‘ ƒŽƒ‡•–Š‡–‹ • ”‘’‹˜ƒ ƒ‹‡ ‹†Ž‡”‡–ƒŽǤǡʹͲͲ͵ ͳʹ͸͸Ͳ͵ͳͳ    ƒŽƒ”‹ƒ–”‡ƒ–‡–• “—‹‹‡ ≈22 ‡›‡•‡–ƒŽǤǡͳͻͻͺ ͻͺͳʹͻ͹ͺ   •ƒŽŽ‘Ž‡ —Ž‡• ͳͺͻͻ ≈12 –”‡‹–‡–ƒŽǤǡʹͲͳͳ ʹͳ͵͸ʹ͸ͳͻ   •ƒŽŽ‘Ž‡ —Ž‡• ʹͻ͵ ≈8 —–œ‡‡–ƒŽǤǡʹͲͲ͹ ͳ͹͵ͺͻͳͶʹ ǦͶȀ  ȋǦʹȌ        „‡–ƒ„Ž‘ ‡”• •‘–ƒŽ‘Ž  –ƒ—†ƒ Š‡”‡–ƒŽǤǡʹͲͳͺ ͵ͲͲͲͺͲͺʹ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ˜‡”ƒ’ƒ‹Ž –ƒ—†ƒ Š‡”‡–ƒŽǤǡʹͲͳͺ ͵ͲͲͲͺͲͺʹ    Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ƒ‹‘†ƒ”‘‡ –ƒ—†ƒ Š‡”‡–ƒŽǤǡʹͲͳͺ ͵ͲͲͲͺͲͺʹ    •ƒŽŽ‘Ž‡ —Ž‡• ͳͺͻͻ ≈8 –”‡‹–‡–ƒŽǤǡʹͲͳͳ ʹͳ͵͸ʹ͸ͳͻ   •ƒŽŽ‘Ž‡ —Ž‡• ʹͻ͵ ≈20 —–œ‡‡–ƒŽǤǡʹͲͲ͹ ͳ͹͵ͺͻͳͶʹ   •‘†‹— Šƒ‡Ž„Ž‘ ‡”• ”ƒ‘Žƒœ‹‡ –ƒ—†ƒ Š‡”‡–ƒŽǤǡʹͲͳͺ ͵ͲͲͲͺͲͺʹ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠŽ‘”‘ˆ‘” ‹”ƒ”†‡–ƒŽǤǡʹͲͲͳ ͳͳʹ͸͵ͻͻͻ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡ ‹”ƒ”†‡–ƒŽǤǡʹͲͲͳ ͳͳʹ͸͵ͻͻͻ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ‹•‘ˆŽ—”ƒ‡  ‹”ƒ”†‡–ƒŽǤǡʹͲͲͳ ͳͳʹ͸͵ͻͻͻ

Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 90

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Table 2.  2P channel subfamilies

 List of activators of the TWIK, THIK and TALK K ͷͲ Šƒ‡Žˆƒ‹Ž› Šƒ‡Ž •—„•–ƒ ‡ ƒ–‡‰‘”› ƒ –‹˜ƒ–‘”  ȋρȌ ‘ƒ –‹˜ƒ–‹‘ ”‡ˆ‡”‡ ‡              Ǧͳ          ǤƒǤ       Ǧʹ          ǤƒǤ                Ǧͳ         ˆ‡ƒƒ–‡†‡”‹˜ƒ–‡• ǦͳʹͶͻ ≈450  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵    Ž‹’‹†• ƒ”ƒ Š‹†‘‹ ƒ ‹† ≈1000 ƒŒƒ‡–ƒŽǤǡʹͲͲͳ ͳͳͲ͸Ͳ͵ͳ͸    Ǧʹ         •ƒŽŽ‘Ž‡ —Ž‡• ǦͳʹͶͻ ≈300  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵           Ǧͳ         ˆ‡ƒƒ–‡†‡”‹˜ƒ–‡• ǦͳʹͶͻ ≈200  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵    ‰ƒ•‡‘—•ƒ‡•–Š‡–‹ • ‹–”‘—•‘š‹†‡ —’”ƒ–‡–ƒŽǤǡʹͲͲͷ ͳͷͷͳ͵ͻͶ͸    Ǧʹ         ˆ‡ƒƒ–‡• ˆŽ—ˆ‡ƒ‹ ƒ ‹† ‡ƒŽ‡‡–ƒŽǤǡʹͲͳ͸ ʹ͸ͻͳͶͳͷ͸     ’Žƒ–‡š–”ƒ –• ƒ”‹•–‘Š‘Ž‹ ƒ ‹† ‡ƒŽ‡‡–ƒŽǤǡʹͲͳ͸ ʹ͸ͻͳͶͳͷ͸     ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡ ”ƒ›‡–ƒŽǤǡʹͲͲͲ ͳͲͺ͵ͻͻʹͶ     ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ‹•‘ˆŽ—”ƒ‡ ”ƒ›‡–ƒŽǤǡʹͲͲͲ ͳͲͺ͵ͻͻʹͶ     ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠŽ‘”‘ˆ‘”   ”ƒ›‡–ƒŽǤǡʹͲͲͲ ͳͲͺ͵ͻͻʹͶ ǦͶȀ        ȋǦʹȌ   „‡–ƒ„Ž‘ ‡”• ‡–‘’”‘Ž‘Ž –ƒ—†ƒ Š‡”‡–ƒŽǤǡʹͲͳͺ ͵ͲͲͲͺͲͺʹ     „‡–ƒ„Ž‘ ‡”• ’”‘’”ƒ‘Ž‘Ž –ƒ—†ƒ Š‡”‡–ƒŽǤǡʹͲͳͺ ͵ͲͲͲͺͲͺʹ     Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ‡š‹Ž‡–‹‡ –ƒ—†ƒ Š‡”‡–ƒŽǤǡʹͲͳͺ ͵ͲͲͲͺͲͺʹ     Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ’”‘’ƒˆ‡‘‡ –ƒ—†ƒ Š‡”‡–ƒŽǤǡʹͲͳͺ ͵ͲͲͲͺͲͺʹ     Žƒ•• ƒ–‹ƒ””Š›–Š‹ • “—‹‹†‹‡ –ƒ—†ƒ Š‡”‡–ƒŽǤǡʹͲͳͺ ͵ͲͲͲͺͲͺʹ     Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ˜‡”ƒƒŽƒ– ≈40 ‡›Ž‡”‡–ƒŽǤǡʹͲͳͶ ʹͷͳͲͺͳͷͷ    ˆ‡ƒƒ–‡†‡”‹˜ƒ–‡• ǦͳʹͶͻ ≈60  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵    ‰ƒ•‡‘—•ƒ‡•–Š‡–‹ • ‹–”‘—•‘š‹†‡ —’”ƒ–‡–ƒŽǤǡʹͲͲͷ ͳͷͷͳ͵ͻͶ͸     ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ‹•‘ˆŽ—”ƒ‡ Ǧͳ ‹”ƒ”†‡–ƒŽǤǡʹͲͲͳ ͳͳʹ͸͵ͻͻͻ  

 Pharmacology of K2P channels of the TWIK, THIK and TALK subfamilies  For K 2P 

 K2P channels channelsthese subfamilies of the TWIK, are also THIK sensitive and TALK to volatile subfamilies anesthetics a low (shown potency in Table block 1, was 2). observed for instance by local anesthetics or quinidine (shown in Table 1). Similar as for other albeit also with a very low potency (shown in Table 1). Thus, for the K  2P In addition, many anti-arrhythmic drugs were reported to block TASK-4/(TALK-2) channels, or blockers reported that are active in the submicromolar range. channels of the TWIK, THIK and TALK subfamilies there are to our best knowledge no small compound activators

 Pharmacology of the TRESK channel 

 2P channel that was reported to be primarily or almost exclusively expressed  TRESK is a K in the spinal cord and DRG neurons [34, 35] which posed this channel on the list of novel promising drugs targeting pain sensation. Noteworthy, TRESK was also found in other tissue like the heart and lung [36]. As a channel sharing 65% identity with human TRESK was isolated from mouse testis, the authors termed this as TRESK-2 [34], however later on it channel within this subfamily of K2P  became clear, that this channel is the mouse orthologue of TRESK and that there is only one channels. In contrast to the channels of the TWIK, THIK  and TALK subfamilies, TRESK appears to be more50 ‘druggable’values (shown since in many Table blockers 3), for instance and/or activators were already described (shown in Table50 of 3, about 4). TRESK 1 µM [37].is blocked Also many by many activators small havecompound been describedinhibitors (shown and also in withTable fairly 4), here low someIC compounds exhibit even EC50 values in bythe the submicromolar antihistaminic range. drug Forloratadine, instance, with acetyl-B-methylcholine, an IC oxotremorine and OXA-22  50 of about 700 nM, 300 nM and 100 nM, respectively [37] (shown  activate TRESK with an EC  in Table 4). Why it appears more feasible to identify modulators of TRESK channels than for members of the TWIK, THIK and TALK subfamilies is an open question. 



















 Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 91

Decher et al.: Molecular Pharmacology of K2P Channels

Table 3. 2P channel subfamily  List of inhibitors of the TRESK K ͷͲ Šƒ‡Ž •—„•–ƒ ‡ ƒ–‡‰‘”› ‹Š‹„‹–‘”  ȋρȌ ‘‹Š‹„‹–‹‘ ”‡ˆ‡”‡ ‡           ƒ–‹„‹‘–‹ • ‡˜ƒ•–ƒ–‹ ≈9 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒ–‹„‹‘–‹ • ‘Ž‹‰‘› ‹ ≈4 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒ–‹ ‘˜—Ž•ƒ–• ‰ƒ„ƒ’‡–‹ ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ    ƒ–‹ ‘˜—Ž•ƒ–• Žƒ‘–”‹‰‹‡ ƒ‰‡–ƒŽǤǡʹͲͲͺ ͳͺͳͻͲ͹ͺͶ    ƒ–‹†‹ƒ„‡–‹ • ‰Ž›„—”‹†‡   ƒ‘‡–ƒŽǤǡʹͲͲ͵ ͳʹ͹ͷͶʹͷͻ  ƒ–‹‹–‘–‹ • ’‘†‘’Š›ŽŽ‘–‘š‹ ≈7 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒ–‹’•› Š‘–‹ • ˆŽ—‘š‡–‹‡ ƒ‰‡–ƒŽǤǡʹͲͲͺ ͳͺͳͻͲ͹ͺͶ    ƒ–‹’•› Š‘–‹ • ˜ƒ‘š‡”‹‡ ≈15 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ͳƒ‰‹‘–‡•‹ ƒ‰‘‹•–• Ǧͳ͸ʹǡ͵ͳ͵ ≈12 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   „ƒ”„‹–—”ƒ–‡• ’‡–‘„ƒ”„‹–ƒŽ ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ    ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ƒˆˆ‡‹‡ ≈22 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ƒŽ‹†ƒœ‘Ž‹— ≈28 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ‰ƒŽŽ‘’ƒ‹Ž ƒ”‡–ƒŽǤǡʹͲͳͺ ʹͻͻ͹͵ͷͶͺ    ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ʹͻ͹Ͷ ≈27 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ‹„‡ˆ”ƒ†‹Ž ≈3 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ‹ ƒ”†‹’‹‡ ≈22 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ‹‘†‹’‹‡ ≈17 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ‹–”‡†‹’‹‡ ≈20 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ˜‡”ƒ’ƒ‹Ž ≈5  Ǧͳ ƒ”‡–ƒŽǤǡʹͲͳͺ ʹͻͻ͹͵ͷͶͺ  ƒƒ„‹‘‹†”‡ ‡’–‘”ƒ‰‘‹•–•  ǦͲͳͷ ≈6 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒƒ„‹‘‹†”‡ ‡’–‘”ƒ‰‘‹•–•  ͷͷǡʹͳʹǦʹ ≈18 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ’”‘’ƒˆ‡‘‡ ƒ‘‡–ƒŽǤǡʹͲͲ͵ ͳʹ͹ͷͶʹͷͻ    Žƒ•• ƒ–‹ƒ””Š›–Š‹ • “—‹‹†‹‡ ƒ‰‡–ƒŽǤǡʹͲͲͶ ͳͷͳʹ͵͸͹Ͳ    Ž‘š›“—‹ƒƒŽ‘‰• ʹ͹͸Ͷ ≈12 ‡‰›‡Ž‡–ƒŽǤǡʹͲͳͻ ͵Ͳͻ͹ͻͺͳʹ   Ž‘š›“—‹ƒƒŽ‘‰• ʹ͹ͻ͵ ≈7 ‡‰›‡Ž‡–ƒŽǤǡʹͲͳͻ ͵Ͳͻ͹ͻͺͳʹ   ʹƒ–ƒ‰‘‹•–• ‘ –‘ Ž‘–Š‡’‹ ≈7 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ‹ˆ‡†‹’‹‡ ≈64 ƒ”‡–ƒŽǤǡʹͲͳͺ ʹͻͻ͹͵ͷͶͺ   ƒ–ƒ‰‘‹•–• ‰—‰‰Ž‡•–‡”‘‡ ≈12 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ‰‡‡”ƒŽƒ‡•–Š‡–‹ • ƒŽ’ŠƒšƒŽ‘‡ ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ    ‰‡‡”ƒŽƒ‡•–Š‡–‹ • ‡–‘‹†ƒ–‡ ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ    ‰‡‡”ƒŽƒ‡•–Š‡–‹ • ‡–ƒ‹‡ ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ    ‰—ƒ›Ž›Ž › Žƒ•‡ƒ –‹˜ƒ–‘”• Ǧͳ ≈17 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ͳƒ–ƒ‰‘‹•–• Ž‘”ƒ–ƒ†‹‡ ≈0.7 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   Ž‡—‘–”‹‡‡ƒ–ƒ‰‘‹•–• Ǧͺͺ͸ ≈10 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   Ž‹’‹†• ƒ”ƒ Š‹†‘‹ ƒ ‹† ≈7 ƒ‰‡–ƒŽǤǡʹͲͲͶ ͳͷͳʹ͵͸͹Ͳ   Ž‘ ƒŽƒ‡•–Š‡–‹ • „—’‹˜ƒ ƒ‹‡ ≈80 ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ   Ž‘ ƒŽƒ‡•–Š‡–‹ • Ž‹†‘ ƒ‹‡ ≈3400 ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ   Ž‘ ƒŽƒ‡•–Š‡–‹ • ‡’‹˜ƒ ƒ‹‡ ≈ͳ͵ͲͲ ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ   Ž‘ ƒŽƒ‡•–Š‡–‹ • ”‘’‹˜ƒ ƒ‹‡ ≈610 ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ   Ž‘ ƒŽƒ‡•–Š‡–‹ • –‡–”ƒ ƒ‹‡ ≈500 ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ   ƒŽƒ”‹ƒ–”‡ƒ–‡–• “—‹‹‡ ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ    ‹Š‹„‹–‘”• ͻͺǡͲͷͻ ≈15 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ‡Žƒ–‘‹‡ ‡’–‘”„Ž‘ ‡”• ͳͺͷ ≈22 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   Šƒ–ƒ‰‘‹•–•  ‡–ƒ ≈19 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ‡—”‘’”‘–‡ –‹˜‡ƒ‰‡–• •‹’ƒ–”‹‰‹‡ ‡ƒ†‘™•‡–ƒŽǤǡʹͲͲͳ ͳͳͳ͹ʹ͹ͷ͵    ͳƒ–ƒ‰‘‹•–• Ǧ͹Ͳ͵ǡ͸Ͳ͸ ≈9 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ    ‹Š‹„‹–‘”• –”‡“—‹•‹ ≈8 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   Ƭƒ ‹Š‹„‹–‘”• ”‘––Ž‡”‹ ≈16 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ’Žƒ–‡š–”ƒ –• ͸Ǧ ‹‰‡”‘Ž ≈155 •—„ˆƒ‹Ž› ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͳʹͻͳʹ  ’Žƒ–‡š–”ƒ –• ƒ”‹•–‘Š‘Ž‹ ƒ ‹† ‡ƒŽ‡‡–ƒŽǤǡʹͲͳ͸ ʹ͸ͻͳͶͳͷ͸    ’Žƒ–‡š–”ƒ –• ƒ’•ƒ‹ ‹ ≈70 •—„ˆƒ‹Ž› ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͳʹͻͳʹ  ’Žƒ–‡š–”ƒ –•  —ŽŽ‡—†ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͷʹ͹Ͳͳͳ    ’Žƒ–‡š–”ƒ –• ’‹’‡”‹‡ ≈230 •—„ˆƒ‹Ž› ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͳʹͻͳʹ  ’Žƒ–‡š–”ƒ –• ’‘Ž›‰‘†‹ƒŽ •—„ˆƒ‹Ž› ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͳʹͻͳʹ   ’Žƒ–‡š–”ƒ –• •ƒ•Š‘‘Ž ≈50 ƒ—–‹•–ƒ‡–ƒŽǤǡʹͲͲͺ ͳͺͷ͸ͺͲʹʹ   ’›”‡–Š”‘‹†• –‡–”ƒ‡–Š”‹ ƒ•–‡ŽŽƒ‘•‡–ƒŽǤǡʹͲͳͺ ʹͺͻ͵͹ͷ͹ͻ    “—ƒ–‡”ƒ”›ƒ‘‹—‹‘• —– ≈7 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  ≈800 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  ‡’ ≈0.5 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  ‡š ≈0.5 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  – ≈6 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘• ‡ ≈0.3 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   •ƒŽŽ‘Ž‡ —Ž‡• ͳͺͻͻ ≈1 –”‡‹–‡–ƒŽǤǡʹͲͳͳ ʹͳ͵͸ʹ͸ͳͻ   •ƒŽŽ‘Ž‡ —Ž‡• ǦǦͳ —‡–ƒŽǤǡʹͲͳ͸ ʹ͹Ͳͻͳͻͻ͹    •ƒŽŽ‘Ž‡ —Ž‡• ǦǦʹ   —‡–ƒŽǤǡʹͲͳ͸ ʹ͹Ͳͻͳͻͻ͹  –”‹‘Ž• –”‹‡–Šƒ‘Žƒ‹‡ ƒ‘‡–ƒŽǤǡʹͲͲ͵ ͳʹ͹ͷͶʹͷͻ   







































 Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 92

Decher et al.: Molecular Pharmacology of K2P Channels

Table 4. 2P channel subfamily

List of activators of the TRESK K  ͷͲ Šƒ‡Ž •—„•–ƒ ‡ ƒ–‡‰‘”› ƒ –‹˜ƒ–‘”  ȋρȌ ‘ƒ –‹˜ƒ–‹‘ ”‡ˆ‡”‡ ‡            Šƒ‰‘‹•–• ƒ”‡ ‘Ž‹‡ ≈3 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ    Šƒ‰‘‹•–• ƒ”„ƒ Š‘Ž ≈2 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ    ƒŽ ‘Š‘Ž• ‡–Šƒ‘Ž ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ    ǦʹǡǦ͵ǡ Ǧͳǡ   ƒ’Š‡–ƒ‹‡• ͶǦ ≈9 ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ ǦͳǡǦʹ   ƒ–‹–—„‡” —Ž‘•‹ • Ž‘š›“—‹ ≈4 ”‹‰Š–‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵ͺ͵Ͳ͹͹    ʹƒ–ƒ‰‘‹•–• •’‹’‡”‘‡ ≈3 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ    ˆ‡ƒƒ–‡†‡”‹˜ƒ–‡• ǦͳʹͶͻ ≈6 ǦʹǡǦ͵ǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ   ˆ‡ƒƒ–‡• ˆŽ—ˆ‡ƒ‹ ƒ ‹† ≈474 ‘–‡‹ŽŽ‹‡”‡–ƒŽǤǡʹͲͳ͸ ʹ͹͸ͶͳͶ͹ʹ    ‰ƒ•‡‘—•ƒ‡•–Š‡–‹ • ‹–”‘—•‘š‹†‡ ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ     ƒ–ƒ‰‘‹•–• ‘–‡Ž—ƒ•– Ǧ͵ǡǦͳǡǦʹ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ    ƒ–ƒ‰‘‹•–• ’”ƒŽ—ƒ•– ≈6 Ǧʹǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ   ƒ–ƒ‰‘‹•–• œƒˆ‹”Ž—ƒ•– Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ    Šƒ‰‘‹•–• ƒ ‡–›ŽǦǦ‡–Š›Ž Š‘Ž‹‡ ≈0.7 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ    Šƒ‰‘‹•–• Ǧʹʹ ≈0.1 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ    Šƒ‰‘‹•–• ‘š‘–”‡‘”‹‡ ≈0.3 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ    ’Š‘•’Šƒ–ƒ•‡• ƒŽ ‹‡—”‹ œ‹”Œž‡–ƒŽǤǡʹͲͲͶ ͳͶͻͺͳͲͺͷ     ƒ –‹˜ƒ–‘”•  ≈0.03 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ    •ƒŽŽ‘Ž‡ —Ž‡• Ǧͷ͵Ͳͳͷͻ ≈5 ǦʹǡǦ͵ǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ   •ƒŽŽ‘Ž‡ —Ž‡• ǦʹǦͳ ǦʹǡǦ͵ǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ    •ƒŽŽ‘Ž‡ —Ž‡• ǦʹǦʹ ǦʹǡǦ͵ǡǦͳǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • †‡•ˆŽ—”ƒ‡ ≈660 ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡ ≈300 ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ‹•‘ˆŽ—”ƒ‡ ≈160 ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • •‡˜‘ˆŽ—”ƒ‡ ≈225 ‹—‡–ƒŽǤǡʹͲͲͶ ͳͷͷ͸ʹͲ͸Ͳ      Pharmacology of channels from the TREK/TRAAK subfamily  TREK/TRAAK activators    2P channels is the   [38, A39] hallmark (shown in of Fig. the 2a members and Table of 5). the Analyses TREK/TRAAK of deletion subfamily constructs of K demonstrated that channel modulation by polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA)   the C-terminus of TREK-1 is crucial for the response to AA [39]. In addition, replacing  responsethe C-terminus to AA. Theseof TREK-2 results with show the that C-terminus the mechanism of TASK of‑3 activation abolished of the TRAAK sensitivity and TREK to AA. by fattyHowever, acids replacing may be different the C-terminus [40]. of TRAAK with that of TASK-1 or TASK-3 did not affect the   activate TREK-1 and TREK-2 (but not TRAAK) channels which requires the C-terminus of  Volatile anesthetics, including diethyl ether, halothane, isoflurane or chloroform,   (shownthe channels in Fig. [41] 2a and(shown Table in 5,Table 6). On 5). theChloroform contrary, specifically diethyl ether and increased reversibly TREK-1 activates whereas TREK channels [41], whereas halothane or isoflurane activates both, TASK and TREK channels [41]  and cyclopropane activate TREK-1 channels in clinically relevant concentrations, whereas it decreased TASK-1 currents [41]. In addition, anesthetic gases, as nitrous oxide, xenon  modulation by AA, stretch or internal pH, was shown to be important for TREK-1 channel activationTASK-3 is insensitiveby these anesthetic [42] (shown gases in [42]. Table 5). The Glu306 residue, also critical for TREK-1  et al. that the neuroprotective agent riluzole activates TREK‑1 and TRAAK channels (shown in Fig. 2a and Table 5). As TREK-1 is inhibited by  increasedIn 2000 cAMP it was levels shown via PKAby Duprat phosphorylation and riluzole has the capacity of increasing   lack a PKA inhibition, are permanently activated [43].  cAMP levels, the activation of TREK-1 is only transient. In contrast, TRAAK channels, which  et al. demonstrated, that TREK-1 contributes to morphine-induced analgesia in mice. The channelTREK-1 was channelsdirectly activatedare discussed (independent as novel drugof µ targetsopioid againstreceptor pain activation) [44, 45]. leading Devilliers to  analgesia without adverse effects [46].  Another TREK-1 activator with therapeutic potential is BL-1249 [47] (shown in Fig. 2a   et al  mutation, an and Table 5). Using a whole exome sequencing approach in a patient withI267T right ventricular  outflow tract tachycardia (RVOT-VT), Decher . identified the TREK-1 amino acid exchange located directly before the selectivity filter of the second pore loop [47].                        Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 93

Decher et al.: Molecular Pharmacology of K2P Channels

Fig. 2. 2P channels and different binding sites subfamily. Drugs (b) Bindingthat modulate site of thechannels NCA of the TREK subfamily of K identified. (a) Chemical formula ofcryptic the most binding important site of TREK-1 activators determined for channels by a co-crystallization of the TREK/(TRAAK) study ‘ ’ BL-1249 mapped in the TREK-2 crystal structure template (PDB ID: (e)4XDJ) Binding [48]. (c)site ML402 of ruthenium bound inred the at ‘the keystone’ binding site, determined by co-crystallization with TREK- (PDB1 ID: 6CQ9) [6]. (d) Binding site of norfluoxetine in TREK-2, see co-crystal structure PDB ID: 4XDL [5]. TRAAKI110D subfamily. (b-e) Potassium ions ‘are represented’ by black spheres. Red arrows indicate the positions of the (PDB respective ID: 6V3C) drugs [58]. in top (f) view.Chemical formula of the most important inhibitors for channels of the TREK/

The mutation almost completely abolished outward currents through the channel and application of BL-1249 rescued the potassium selectivity and loss-of-function of the TREK-1introduced achannel. strong The sodium fact permeabilitythat this fenamate-like to the potassium compound channel was able [47]. to Interestingly, rescue the I267T indicated that this drug or maybe other similar I267T et al negativelyselectivity charged filter defect activators of TREK-1 (NCAs) harboring a negatively charged tetrazole or carboxylate activators directly stabilize the selectivity filter. Consequently, Schewe . identified2P groupactivate (such most as of BL-1249, the K2P channels PD-118057 (shown and inNS11021) Table 2, 4,as 5)activators and also of activate the mechano-gated other selectivity K channels TREK-1 and TREK-2 [48] (shown in Fig. 2a and Table 5). Strikingly, these NCAs filter gated ion channels like hERG or BK channels with equal efficiency [48]. Thus, NCAs act negativewith a common charge mechanismpromotes K +on binding selectivity to the filter pore gated cavity channels, (shown providingin Fig. 2b). a This universal in turn ‘master alters key’ to unlock the selectivity filter gate by binding below the selectivity filter where their the ion occupancy in the selectivity filter in a way that is known to promote activation of the filter gate [11, 48]. sensitiveIn contrast, to modulation 2-aminoethoxydiphenyl by 2-APB compared borate with (2-APB) TREK-1 is or a nonTRAAK NCA [49] that (shown activates in channelsTable 5). of the TREK/TRAAK subfamily (shown in Fig. 2a and Table 5). TREK-2 is much more

2-APB does not bind to either the binding site of NCAs or the ‘cryptic’ binding site described Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 94

Decher et al.: Molecular Pharmacology of K2P Channels

Table 5. 2P channel subfamily  List of activators of the TREK/(TRAAK) K ͷͲ  Šƒ‡Ž •—„•–ƒ ‡ ƒ–‡‰‘”› ƒ –‹˜ƒ–‘”  ȋρȌ ‘ƒ –‹˜ƒ–‹‘ ”‡ˆ‡”‡ ‡   Ǧͳ        –Š‡”ƒ’‡—–‹ • ”‹Ž—œ‘Ž‡ȋ‡ƒ”Ž›‡ˆˆ‡ –Ȍ —’”ƒ–‡–ƒŽǤǡʹͲͲͲ ͳͲ͹͹ͻ͵͹͵    ƒˆˆ‡‹ ƒ ‹†‡•–‡”•  ƒ–Š‹‡–ƒŽǤǡʹͲͲͶ ͳͶͻ͹ͺʹ͵ͺ    ƒˆˆ‡‹ ƒ ‹†‡•–‡”•  ƒ–Š‹‡–ƒŽǤǡʹͲͲͶ ͳͶͻ͹ͺʹ͵ͺ    ‡–Šƒ ”›‹ ƒ ‹††‡”‹˜ƒ–‡•   ‹‹‡”‹‡–ƒŽǤǡʹͲͳ͵ ʹ͵Ͳ͹ʹ͵ͷ͸    ˆ‡ƒƒ–‡†‡”‹˜ƒ–‡• ǦͳʹͶͻ ≈7 ǦʹǡǦ͵ǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ  ˆ‡ƒƒ–‡• †‹ Ž‘ˆ‡ƒ  ‡ƒŽ‡‡–ƒŽǤǡʹͲͳͶ ʹͶͷͲͻͺͶͲ    ˆ‡ƒƒ–‡• ˆŽ—ˆ‡ƒ‹ ƒ ‹† ≈100 ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ   ˆ‡ƒƒ–‡• ‡ˆ‡ƒ‹ ƒ ‹† ≈100 ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ   ˆ‡ƒƒ–‡• ‹ˆŽ—‹ ƒ ‹† ≈100 ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ   ˆ—”›Ž†‡”‹˜ƒ–‡• ‘’‘—†͵͸   ‹˜‹‡”‡–ƒŽǤǡʹͲͳ͹ ʹͺͲͷͳͺ͸͵  ‰ƒ•‡‘—•ƒ‡•–Š‡–‹ • › Ž‘’”‘’ƒ‡ Ǧ͵ ”—••‡–ƒŽǤǡʹͲͲͶ ͳͶ͹Ͷʹ͸ͺ͹   ‰ƒ•‡‘—•ƒ‡•–Š‡–‹ • ‹–”‘—•‘š‹†‡ Ǧ͵ ”—••‡–ƒŽǤǡʹͲͲͶ ͳͶ͹Ͷʹ͸ͺ͹   ‰‡‡”ƒŽƒ‡•–Š‡–‹ • –”‹ ŠŽ‘”‘‡–Šƒ‘Ž ƒ”‹ƒ–Šƒ†‹†ƒ”ǡʹͲͲͶ ͳͶͻͻ͸ͷͷ͵    Š›†”‘š› ‘—ƒ”‹†‡”‹˜ƒ–‡• ‘•–”—–Š‹ ≈5 Ǧʹǡ Ǧͳ ‘•‡’Š‡–ƒŽǤǡʹͲͳͺ ͵ͲͳͳͲ͵ͷͶ  Ž‹’‹†• ƒ”ƒ Š‹†‘‹ ƒ ‹† ƒ–‡Ž‡–ƒŽǤǡͳͻͻͺ ͻ͸ͺ͹Ͷͻ͹    Ž‹’‹†• Ž›•‘’Š‘•’Š‘Ž‹’‹†• Š‡‹‡–ƒŽǤǡʹͲͲͷ ͳͷͷ͹ʹ͵͸ͷ    ƒ–ƒ‰‘‹•–• ’”ƒŽ—ƒ•– ≈6 Ǧʹǡ Ǧͳǡ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ  ‘’‹ƒ–‡• ‘”’Š‹‡ ‡˜‹ŽŽ‹‡”•‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͷ͸ʹ͵ͳ    ’Žƒ–‡š–”ƒ –• ƒ”‹•–‘Š‘Ž‹ ƒ ‹†  ‡ƒŽ‡‡–ƒŽǤǡʹͲͳ͸ ʹ͸ͻͳͶͳͷ͸   •ƒŽŽ‘Ž‡ —Ž‡• ʹǦ ≈500 ǦͳǡǦ͵ǡ ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹ͵͹ʹͲ͸ʹ͹  •ƒŽŽ‘Ž‡ —Ž‡• ͵ͲͲͳƒ ≈13 ǦͳǡǦ͵ǡ Ǧͳǡ ‹—‡–ƒŽǤǡʹͲʹͲ ͵ʹͳ͸ʹͷͳʹ  •ƒŽŽ‘Ž‡ —Ž‡• Ǧͷ͵Ͳͳͷͻ ≈1  ‘— ‹ˆ‡–ƒŽǤǡʹͲͳͺ ʹͻͳͷͲͺ͵ͺ  •ƒŽŽ‘Ž‡ —Ž‡• ‘Ž‘ǦǦͷǦ͸  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵    •ƒŽŽ‘Ž‡ —Ž‡• ǦʹǦͳ ǦʹǡǦ͵ǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ   •ƒŽŽ‘Ž‡ —Ž‡• ͵͵ͷ ≈14  ‘Ž‹ ƒ–‘‡–ƒŽǤǡʹͲͳʹ ʹͺ͸ͻ͵Ͳ͵ͷ  •ƒŽŽ‘Ž‡ —Ž‡• ͶͲʹ ≈14  ‘Ž‹ ƒ–‘‡–ƒŽǤǡʹͲͳʹ ʹͺ͸ͻ͵Ͳ͵ͷ  •ƒŽŽ‘Ž‡ —Ž‡• ͸͹Ǧ͵͵ ≈36 ƒ‰”‹ƒ–•‡˜‡–ƒŽǤǡʹͲͳ͵ ʹ͵͹͵ͺ͹Ͳͻ   •ƒŽŽ‘Ž‡ —Ž‡• ͳͳͲʹͳ  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵    •ƒŽŽ‘Ž‡ —Ž‡• ǦͳͳͺͲͷ͹  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵    •ƒŽŽ‘Ž‡ —Ž‡• Ǧ͵Ͳ͹ʹͶ͵  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵   ǦʹǡǦ͵ǡ Ǧͳǡ  •ƒŽŽ‘Ž‡ —Ž‡• ʹ͸ͶͲ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ  Ǧʹǡ  ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠŽ‘”‘ˆ‘” Ǧͳǡ ƒ–‡Ž‡–ƒŽǤǡͳͻͻͻ ͳͲ͵ʹͳʹͶͷ   ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • †‹‡–Š›Ž‡–Š‡” Ǧͳǡ ƒ–‡Ž‡–ƒŽǤǡͳͻͻͻ ͳͲ͵ʹͳʹͶͷ   ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡  ƒ–‡Ž‡–ƒŽǤǡͳͻͻͻ ͳͲ͵ʹͳʹͶͷ   ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ‹•‘ˆŽ—”ƒ‡  ƒ–‡Ž‡–ƒŽǤǡͳͻͻͻ ͳͲ͵ʹͳʹͶͷ  Ǧʹ        ‡–Šƒ ”›‹ ƒ ‹††‡”‹˜ƒ–‡•    Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵    ˆ‡ƒƒ–‡†‡”‹˜ƒ–‡• ǦͳʹͶͻ ≈6 ǦʹǡǦ͵ǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ  ˆ‡ƒƒ–‡• ˆŽ—ˆ‡ƒ‹ ƒ ‹† ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ    ˆ‡ƒƒ–‡• ‡ˆ‡ƒ‹ ƒ ‹† ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ    ˆ‡ƒƒ–‡• ‹ˆŽ—‹ ƒ ‹† ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ    Š›†”‘š› ‘—ƒ”‹†‡”‹˜ƒ–‡• ‘•–”—–Š‹ ≈4000 Ǧʹǡ Ǧͳ ‘•‡’Š‡–ƒŽǤǡʹͲͳͺ ͵ͲͳͳͲ͵ͷͶ  Ž‹’‹†• ƒ”ƒ Š‹†‘‹ ƒ ‹† ‹‡–ƒŽǤǡʹͲͲͳ ͳͳ͸ͺͲ͸ʹͻ    Ž‹’‹†• Ž›•‘’Š‘•’Š‘Ž‹’‹†• Š‡‹‡–ƒŽǤǡʹͲͲͷ ͳͷͷ͹ʹ͵͸ͷ    ƒ–ƒ‰‘‹•–• ’”ƒŽ—ƒ•– ≈6 Ǧʹǡ Ǧͳǡ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ  ƒ–ƒ‰‘‹•–• œƒˆ‹”Ž—ƒ•– Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ   ’Žƒ–‡š–”ƒ –• ƒ”‹•–‘Š‘Ž‹ ƒ ‹†  ‡ƒŽ‡‡–ƒŽǤǡʹͲͳ͸ ʹ͸ͻͳͶͳͷ͸   ’Žƒ–‡š–”ƒ –• „ƒ‹ ƒŽ‡‹ ‹‡–ƒŽǤǡʹͲͳͳ ʹͳ͵Ͳ͸ͷ͸ͺ    ’Žƒ–‡š–”ƒ –• ™‘‰‘‹ ‹‡–ƒŽǤǡʹͲͳͳ ʹͳ͵Ͳ͸ͷ͸ͺ   ǦʹǡǦ͵ǡ Ǧͳǡ  ’”‘•–ƒ‰Žƒ†‹• ͳͳǦ‡‘š› ʹƒ ≈5 ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ Ǧͳǡ  •ƒŽŽ‘Ž‡ —Ž‡• ʹǦ ≈1200 ǦͳǡǦ͵ǡ ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹ͵͹ʹͲ͸ʹ͹  •ƒŽŽ‘Ž‡ —Ž‡• ͵ͲͲͳƒ ≈11 ǦͳǡǦ͵ǡ Ǧͳǡ ‹—‡–ƒŽǤǡʹͲʹͲ ͵ʹͳ͸ʹͷͳʹ  •ƒŽŽ‘Ž‡ —Ž‡• Ǧͷ͵Ͳͳͷͻ ≈6 ǦʹǡǦ͵ǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ  •ƒŽŽ‘Ž‡ —Ž‡• ‘Ž‘ǦǦͷǦ͸  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵    •ƒŽŽ‘Ž‡ —Ž‡• ǦʹǦͳ ǦʹǡǦ͵ǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ  ǦͳǡǦʹǡǦ͵ǡ  •ƒŽŽ‘Ž‡ —Ž‡• ǦʹǦʹ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ   Ǧͳ  •ƒŽŽ‘Ž‡ —Ž‡• ͸͹Ǧ͵͵ ≈30 ǦͳǡǦʹǡǦ͵ǡ ƒ‰”‹ƒ–•‡˜‡–ƒŽǤǡʹͲͳ͵ ʹ͵͹͵ͺ͹Ͳͻ  •ƒŽŽ‘Ž‡ —Ž‡• ͳͳͲʹͳ  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵    •ƒŽŽ‘Ž‡ —Ž‡• ǦͳͳͺͲͷ͹  Š‡™‡‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͹ͻʹ͵Ͳ͵    •ƒŽŽ‘Ž‡ —Ž‡• ʹ͵ Ǧͳ ƒ†‹‡–ƒŽǤǡʹͲͳ͹ ʹ͹ͺͲͷͺͳͳ   ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠŽ‘”‘ˆ‘” ‡•ƒ‰‡‡–ƒŽǤǡʹͲͲͲ ͳͲͺͺͲͷͳͲ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡   ‡•ƒ‰‡‡–ƒŽǤǡʹͲͲͲ ͳͲͺͺͲͷͳͲ  ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ‹•‘ˆŽ—”ƒ‡   ‡•ƒ‰‡‡–ƒŽǤǡʹͲͲͲ ͳͲͺͺͲͷͳͲ         –Š‡”ƒ’‡—–‹ • ”‹Ž—œ‘Ž‡ ‹‡–ƒŽǤǡͳͻͻͺ ͻ͸ʹͺͺ͸͹    ˆ‡ƒƒ–‡†‡”‹˜ƒ–‡• ǦͳʹͶͻ ≈6 ǦʹǡǦ͵ǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ  ˆ‡ƒƒ–‡• ˆŽ—ˆ‡ƒ‹ ƒ ‹† ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ    ˆ‡ƒƒ–‡• ‡ˆ‡ƒ‹ ƒ ‹† ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ    ˆ‡ƒƒ–‡• ‹ˆŽ—‹ ƒ ‹† ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ    ‰‡‡”ƒŽƒ‡•–Š‡–‹ • –”‹ ŠŽ‘”‘‡–Šƒ‘Ž ƒ”‹ƒ–Šƒ†‹†ƒ”ǡʹͲͲͶ ͳͶͻͻ͸ͷͷ͵    Ž‹’‹†• ƒ”ƒ Š‹†‘‹ ƒ ‹† ‹‡–ƒŽǤǡͳͻͻͺ ͻ͸ʹͺͺ͸͹    Ž‹’‹†• Ž›•‘’Š‘•’Š‘Ž‹’‹†• Š‡‹‡–ƒŽǤǡʹͲͲͷ ͳͷͷ͹ʹ͵͸ͷ    •ƒŽŽ‘Ž‡ —Ž‡• ʹǦ ǦͳǡǦ͵ǡ ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹ͵͹ʹͲ͸ʹ͹   •ƒŽŽ‘Ž‡ —Ž‡• ͵ͲͲͳƒ ≈15 ǦͳǡǦ͵ǡ Ǧͳǡ ‹—‡–ƒŽǤǡʹͲʹͲ ͵ʹͳ͸ʹͷͳʹ  •ƒŽŽ‘Ž‡ —Ž‡• ͸͹Ǧ͵͵ ≈27 ƒ‰”‹ƒ–•‡˜‡–ƒŽǤǡʹͲͳ͵ ʹ͵͹͵ͺ͹Ͳͻ                      Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 95

Decher et al.: Molecular Pharmacology of K2P Channels

 Table 6. 2P channel subfamily  List of activators of the TASK K ͷͲ Šƒ‡Ž •—„•–ƒ ‡ ƒ–‡‰‘”› ƒ –‹˜ƒ–‘”  ȋρȌ ‘ƒ –‹˜ƒ–‹‘ ”‡ˆ‡”‡ ‡   Ǧͳ       ‰—ƒ›Žƒ–‡ › Žƒ•‡  ”‹‘ ‹‰—ƒ– —‹‰Šƒ‡–ƒŽǤǡʹͲͳͻ ͵Ͳ͵͸ͷͺ͹͹ ƒ –‹˜ƒ–‘”•    ’Š‘•’Š‘Ž‹’ƒ•‡‹Š‹„‹–‘”• ONO‐RS‐082 ƒ‡–ƒŽǤǡʹͲͳ͵ ʹ͵ͺͺ͵͵ͺͲ    ’”‘•–ƒ › Ž‹ƒƒŽ‘‰• –”‡’”‘•–‹‹Ž Ž• Š‡™•‹‡–ƒŽǤǡʹͲͲ͸ ͳ͸ͷ͹ͶͻͲͺ    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡  ƒ–‡Ž‡–ƒŽǤǡͳͻͻͻ ͳͲ͵ʹͳʹͶͷ   ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ‹•‘ˆŽ—”ƒ‡  ƒ–‡Ž‡–ƒŽǤǡͳͻͻͻ ͳͲ͵ʹͳʹͶͷ   ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • •‡˜‘ˆŽ—”ƒ‡ —–œ‡‡–ƒŽǤǡʹͲͲ͹ ͳ͹͸ͻͻ͸͵ͺ   Ǧ͵        ƒ–‹ˆ—‰ƒŽ• –‡”„‹ƒˆ‹‡ Ǧʹǡ ǦͳǡǦʹǡǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳ͹ ʹͺͺͺʹͷͻͶ   „‹‰—ƒ‹†‡†‡”‹˜ƒ–‡•  ͵ ≈1.4  Ǧͳǡ•—„ˆƒ‹Ž›ǡ ‹ƒ‘‡–ƒŽǤǡʹͲͳͻ ͵ͳ͹Ͷͺʹ͵ͳ  ˆ‡ƒƒ–‡• ˆŽ—ˆ‡ƒ‹ ƒ ‹† ‡ƒŽ‡‡–ƒŽǤǡʹͲͳͶ ʹͶ͵Ͷʹ͹͹ͳ    ‰ƒ•‡‘—•ƒ‡•–Š‡–‹ • › Ž‘’”‘’ƒ‡   ”—••‡–ƒŽǤǡʹͲͲͶ ͳͶ͹Ͷʹ͸ͺ͹  ‰ƒ•‡‘—•ƒ‡•–Š‡–‹ • ‹–”‘—•‘š‹†‡   ”—••‡–ƒŽǤǡʹͲͲͶ ͳͶ͹Ͷʹ͸ͺ͹  ‰ƒ•‡‘—•ƒ‡•–Š‡–‹ • š‡‘   ”—••‡–ƒŽǤǡʹͲͲͶ ͳͶ͹Ͷʹ͸ͺ͹  ƒ–ƒ‰‘‹•–• ‹ƒŽ—ƒ•– Ǧʹǡ ǦͳǡǦͳǡǦʹǡ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ   ƒ–ƒ‰‘‹•–• ’”ƒŽ—ƒ•– Ǧʹǡ Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ   ƒ–ƒ‰‘‹•–• œƒˆ‹”Ž—ƒ•– ≈6 Ǧͳ ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ  •ƒŽŽ‘Ž‡ —Ž‡•  ≈7 Ǧͳǡ ǦͳǡǦͳǡ ‹ƒ‡–ƒŽǤǡʹͲͳͻ ͵ͳͲͳͷʹͺ͵  ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ŠƒŽ‘–Šƒ‡ ”—••‡–ƒŽǤǡʹͲͲͶ ͳͶ͹Ͷʹ͸ͺ͹    ˜‘Žƒ–‹Ž‡ƒ‡•–Š‡–‹ • ‹•‘ˆŽ—”ƒ‡ ‡”‰‡–ƒŽǤǡʹͲͲͶ ͳͷʹͺʹʹ͹ʹ   Ǧͷ         ǤƒǤ        below. Zhuo et al. described that for TREK-2 channels the cytosolic C‑terminus plays a role in     controlling the stimulatory effects of the compound. In particular the proximal C-terminus, authorsincluding proposed His368 anas allosterica key residue, coupling was between required the for proximal channel C activation‑terminus andby 2-APB the selectivity [50]. In addition, specific mutations in the M4 segment reduced the 2-APB efficiency and thus the

[51].filter induced by 2-APB [51]. This allosteric coupling is facilitated by the movement of M4 and thus mutations that reduce the flexibility of the M4 movements impair 2-APB activation

Very recently a novel class of small molecule activators has been identified that utilizes formeda completely by the different, P1 pore helixthe ‘cryptic’, and M4 binding transmembrane site, which helix is clearly intrasubunit distinct interface. to that of The NCAs drugs [6] (shown in Fig. 2c). ML335 and ML402 bind to an L-shaped pocket behind the selectivity filter

activate the channels by acting as ‘molecular wedges‘, restricting the interdomain interface movement behind the selectivity filter [6]. Mechanistically, binding to the ‘cryptic’ binding site stabilizes the C-type gate in a more conductive ‘leak mode’ through a common set of hydrogen bonds, π-π, and cation-π interactions of ML335 and ML402 with the ‘P1 face’ and thatan ‘M4 selectively face’ reducing activated P1/TM4 TREK-2 interface [52]. These dynamics novel [6].compounds were subsequently proven to directlyA high-throughput activate TREK-2 fluorescence-based channels, using single thallium channel flux measurements screen identified in excised small membrane molecules

activated TREK-2 while they blocked TREK-1 channels [52] (shown in Table 5, 7). For these compoundspatches [52]. a region Strikingly, connecting 11-deoxy the PGF2αsecond orpore T2A3 loop which to the wereM4 segment described was in proposed this study, to determine the observed activation or inhibition [52].

TREK/TRAAK inhibitors

In terms of blockers for channels of the TREK/TRAAK subfamily,50 of spadin70 nM [53]is presumably (shown in the best known blocker of TREK channels described (shown in Fig. 2f). Spadin is a 17 amino siteacid and sortilin-derived mechanism ofpeptide inhibition targeting are not TREK-1 known channels so far. However, with an IC it has been postulated that Fig. 2f and Table 7). It is discussed as a putative antidepressant [53, 54], although the binding TREK-1 by AA, utilizing an allosteric mechanism of inhibition [55]. spadin shouldet al. bind to the ‘down state’ of the channel to specifically antagonize activation of

Pro198Dong of the M2 described segment, the Cys249 crystal andstructure Val253 of in TREK-2 M3, Phe316 in complex and Leu320with norfluoxetine in M4, as well [5] (shown in Fig. 2d, 2f). Here several residues in the side fenestrations, including Ile194 and Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 96

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Table 7. 2P channel subfamily   List of inhibitors of the TREK/(TRAAK) K ͷͲ Šƒ‡Ž •—„•–ƒ ‡ ƒ–‡‰‘”› ‹Š‹„‹–‘”  ȋρȌ ‘‹Š‹„‹–‹‘ ”‡ˆ‡”‡ ‡   Ǧͳ        –Š‡”ƒ’‡—–‹ • ”‹Ž—œ‘Ž‡ȋŽƒ–‡‡ˆˆ‡ –Ȍ  —’”ƒ–‡–ƒŽǤǡʹͲͲͲ ͳͲ͹͹ͻ͵͹͵   ƒ–‹’•› Š‘–‹ • ŠŽ‘”’”‘ƒœ‹‡ ≈3  ŠòŽ‡”‡–ƒŽǤǡʹͲͲ͹ ͳ͹ʹʹʹͺͲ͸  ƒ–‹’•› Š‘–‹ • ˆŽ—’‡–‹š‘Ž ≈2  ŠòŽ‡”‡–ƒŽǤǡʹͲͲ͹ ͳ͹ʹʹʹͺͲ͸  ƒ–‹’•› Š‘–‹ • ˆŽ—’Š‡ƒœ‹‡ ≈5  ŠòŽ‡”‡–ƒŽǤǡʹͲͲ͹ ͳ͹ʹʹʹͺͲ͸  ƒ–‹’•› Š‘–‹ • ŠƒŽ‘’‡”‹†‘Ž ≈6  ŠòŽ‡”‡–ƒŽǤǡʹͲͲ͹ ͳ͹ʹʹʹͺͲ͸  ƒ–‹’•› Š‘–‹ • Ž‘šƒ’‹‡ ≈20  ŠòŽ‡”‡–ƒŽǤǡʹͲͲ͹ ͳ͹ʹʹʹͺͲ͸  ƒ–‹’•› Š‘–‹ • ’‹‘œ‹†‡ ≈2  ŠòŽ‡”‡–ƒŽǤǡʹͲͲ͹ ͳ͹ʹʹʹͺͲ͸  ƒ–‹’•› Š‘–‹ • ˆŽ—‘š‡–‹‡ ≈19 ‡ƒ”†‡–ƒŽǤǡʹͲͲͷ ͳͷ͸ͺͷʹͳʹ   ƒ–‹’•› Š‘–‹ • ‘”ˆŽ—‘š‡–‹‡ ≈9 ‡ƒ”†‡–ƒŽǤǡʹͲͲͷ ͳͷ͸ͺͷʹͳʹ   „‡–ƒ„Ž‘ ‡”• ƒ”˜‡†‹‘Ž ≈20 ‹••‡Ž„ƒ Š‡–ƒŽǤǡʹͲͳͶ ʹͷͳ͸ͺ͹͸ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• †‹Ž–‹ƒœ‡ ≈180  ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ  ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ‹„‡ˆ”ƒ†‹Ž ›‡ƒ”–‡–ƒŽǤǡʹͲͲʹ ͳʹ͵͸ͺʹͺͻ    ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ’‡ˆŽ—”‹†‘Ž ›‡ƒ”–‡–ƒŽǤǡʹͲͲʹ ͳʹ͵͸ͺʹͺͻ    ƒƒ„‹‘‹†• ƒƒ†ƒ‹†‡ ≈5 ‹—‡–ƒŽǤǡʹͲͲ͹ ͳ͹͸ʹʹͷ͹Ͷ   Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ‡š‹Ž‡–‹‡ ≈170  Š‹†–‡–ƒŽǤǡʹͲͳ͵ ʹͶͲ͹Ͳͺͳ͵   Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ’”‘’ƒˆ‡‘‡ ≈8  Š‹†–‡–ƒŽǤǡʹͲͳ͵ ʹͶͲ͹Ͳͺͳ͵   Žƒ•• ƒ–‹ƒ””Š›–Š‹ • †”‘‡†ƒ”‘‡ ≈27  Š‹†–‡–ƒŽǤǡʹͲͳʹ ʹʹ͹ͻͲ͹ͻͶ   Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ˜‡”ƒƒŽƒ– ≈13 Ǧͳ ‡›Ž‡”‡–ƒŽǤǡʹͲͳͶ ʹͶ͵͹ͶͲͲͺ  Ž‘š›“—‹ƒƒŽ‘‰• ʹ͹͸Ͷ ‡‰›‡Ž‡–ƒŽǤǡʹͲͳͻ ͵Ͳͻ͹ͻͺͳʹ     ƒ–ƒ‰‘‹•–• ƒŽ‘†‹’‹‡ ≈0.5 ‹—‡–ƒŽǤǡʹͲͲ͹ ͳ͹͸ʹʹͷ͹Ͷ    ƒ–ƒ‰‘‹•–• ‹ˆ‡†‹’‹‡ ≈8 ‹—‡–ƒŽǤǡʹͲͲ͹ ͳ͹͸ʹʹͷ͹Ͷ    ƒ–ƒ‰‘‹•–• ‹‰—Ž†‹’‹‡ ≈0.8 ‹—‡–ƒŽǤǡʹͲͲ͹ ͳ͹͸ʹʹͷ͹Ͷ   †‹’Š‡Ž›†‹’‡”ƒœ‹‡• ˆŽ—ƒ”ƒœ‹‡ ≈3 ‹—‡–ƒŽǤǡʹͲͲ͹ ͳ͹͸ʹʹͷ͹Ͷ   Ž‘ ƒŽƒ‡•–Š‡–‹ • „—’‹˜ƒ ƒ‹‡ ≈10Ͳ Š‹‡–ƒŽǤǡʹͲͳͶ ʹ͵͹ͻ͹͸ʹͷ   Ž‘ ƒŽƒ‡•–Š‡–‹ • Ž‡˜‘„—’‹˜ƒ ƒ‹‡ ≈130 Š‹‡–ƒŽǤǡʹͲͳͶ ʹ͵͹ͻ͹͸ʹͷ   Ž‘ ƒŽƒ‡•–Š‡–‹ • Ž‹†‘ ƒ‹‡ ≈180 ƒ›ƒ‡–ƒŽǤǡʹͲͲͻ ͳͻ͸ʹʹ͹ͻͲ   Ž‘ ƒŽƒ‡•–Š‡–‹ • ”‘’‹˜ƒ ƒ‹‡ ≈400 Š‹‡–ƒŽǤǡʹͲͳͶ ʹ͵͹ͻ͹͸ʹͷ   ƒ–ƒ‰‘‹•–• œƒˆ‹”Ž—ƒ•– ”‹‰Š–‡–ƒŽǤǡʹͲͳͻ ͵ͳͷ͸ͶͶͳͶ    ‡—”‘’”‘–‡ –‹˜‡ƒ‰‡–•  ≈0.1 ‹‡–ƒŽǤǡʹͲͳͳ ʹͳʹͻ͵Ͷ͹Ͳ   ‡—”‘’”‘–‡ –‹˜‡ƒ‰‡–• •‹’ƒ–”‹‰‹‡ ≈4 ‡ƒ†‘™•‡–ƒŽǤǡʹͲͲͳ ͳͳͳ͹ʹ͹ͷ͵   ’”‘•–ƒ‰Žƒ†‹• ͳͳǦ‡‘š› ʹƒ Ǧʹ ƒ†‹‡–ƒŽǤǡʹͲͳ͹ ʹ͹ͺͲͷͺͳͳ   ’›”‡–Š”‘‹†• –‡–”ƒ‡–Š”‹ ƒ•–‡ŽŽƒ‘•‡–ƒŽǤǡʹͲͳͺ ʹͺͻ͵͹ͷ͹ͻ    ’›”‹‹†‹‡• Ͷͷ ≈20 ƒ‰”‹ƒ–•‡˜‡–ƒŽǤǡʹͲͳ͵ ʹ͵͹͵ͺ͹Ͳͻ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘• —– ≈2000 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  ≈90000 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  ‡š ≈1 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘• ‡ ≈11 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   •ƒŽŽ‘Ž‡ —Ž‡• ͳͺͻͻ ≈24 –”‡‹–‡–ƒŽǤǡʹͲͳͳ ʹͳ͵͸ʹ͸ͳͻ   •ƒŽŽ‘Ž‡ —Ž‡• ʹͻ͵ ≈10 —–œ‡‡–ƒŽǤǡʹͲͲ͹ ͳ͹͵ͺͻͳͶʹ   •ƒŽŽ‘Ž‡ —Ž‡• ʹ͵ Ǧʹ ƒ†‹‡–ƒŽǤǡʹͲͳ͹ ʹ͹ͺͲͷͺͳͳ   •ƒŽŽ‘Ž‡ —Ž‡•  ≈5 —‘‡–ƒŽǤǡʹͲͳ͹ ʹͺͺͷͳͺ͸ͺ   •‘”–‹Ž‹Ǧ†‡”‹˜ƒ–‡• •’ƒ†‹ ≈0.1 ƒœ‡ŽŽƒ‡–ƒŽǤǡʹͲͳͲ ʹͲͶͲͷͲͲͳ   ͹ƒ‰‘‹•–• ‹‹“—‹‘† ≈80 ‡‡‡–ƒŽǤǡʹͲͳʹ ʹʹʹ͵͵͸ͲͶ  Ǧʹ        ƒ–‹’•› Š‘–‹ • ‘”ˆŽ—‘š‡–‹‡ ‘‰‡–ƒŽǤǡʹͲͳͷ ʹͷ͹͸͸ʹ͵͸    „‡–ƒ„Ž‘ ‡”• ƒ”˜‡†‹‘Ž ≈25 ‹••‡Ž„ƒ Š‡–ƒŽǤǡʹͲͳͶ ʹͷͳ͸ͺ͹͸ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• †‹Ž–‹ƒœ‡ ≈330  ƒƒŠ‹”ƒ‡–ƒŽǤǡʹͲͲͷ ͳ͸Ͳ͹ͷʹͶͲ  ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ‡–Š‘š›˜‡”ƒ’ƒ‹Ž ƒ”‡–ƒŽǤǡʹͲͳͺ ʹͻͻ͹͵ͷͶͺ    ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ˜‡”ƒ’ƒ‹Ž  Ǧͳ ƒ”‡–ƒŽǤǡʹͲͳͺ ʹͻͻ͹͵ͷͶͺ   ƒ–‹‘‹ †›‡• ”—–Š‡‹—‡† ≈0.2 Ǧͳ ”ƒ—‡–ƒŽǤǡʹͲͳͷ ʹͷͶͲͻͷ͹ͷ  Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ˜‡”ƒƒŽƒ– ‡›Ž‡”‡–ƒŽǤǡʹͲͳͶ ʹͷͳͲͺͳͷͷ    ’›”‡–Š”‘‹†• –‡–”ƒ‡–Š”‹ ƒ•–ƒŽŽ‡‘•‡–ƒŽǤǡʹͲͳͺ ʹͺͻ͵͹ͷ͹ͻ    •ƒŽŽ‘Ž‡ —Ž‡• ͳͺͻͻ ≈8 –”‡‹–‡–ƒŽǤǡʹͲͳͳ ʹͳ͵͸ʹ͸ͳͻ   •ƒŽŽ‘Ž‡ —Ž‡•  ≈5 —‘‡–ƒŽǤǡʹͲͳ͹ ʹͺͺͷͳͺ͸ͺ          ƒ–‹‘‹ †›‡• ”—–Š‡‹—‡† Ǧͳ ”ƒ—‡–ƒŽǤǡʹͲͳͷ ʹͷͶͲͻͷ͹ͷ   ƒ–‹‘‹ †›‡• ”—–Š‡‹—‹‘Ž‡– ≈0.1 ”ƒ—‡–ƒŽǤǡʹͲͳͷ ʹͷͶͲͻͷ͹ͷ   Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ˜‡”ƒƒŽƒ–   ‡›Ž‡”‡–ƒŽǤǡʹͲͳͶ ʹͷͳͲͺͳͷͷ  ‡—”‘’”‘–‡ –‹˜‡ƒ‰‡–• •‹’ƒ–”‹‰‹‡ ‡ƒ†‘™•‡–ƒŽǤǡʹͲͲͳ ͳͳͳ͹ʹ͹ͷ͵    ’›”‡–Š”‘‹†• –‡–”ƒ‡–Š”‹ ƒ•–ƒŽŽ‡‘•‡–ƒŽǤǡʹͲͳͺ ʹͺͻ͵͹ͷ͹ͻ    •ƒŽŽ‘Ž‡ —Ž‡• ͳͺͻͻ –”‡‹–‡–ƒŽǤǡʹͲͳͳ ʹͳ͵͸ʹ͸ͳͻ    •ƒŽŽ‘Ž‡ —Ž‡• ǦǦͳ ≈0.4 Ǧͳǡ Ǧͳ —‡–ƒŽǤǡʹͲͳ͸ ʹ͹Ͳͻͳͻͻ͹  •ƒŽŽ‘Ž‡ —Ž‡• ǦǦʹ ≈0.7 —‡–ƒŽǤǡʹͲͳ͸ ʹ͹Ͳͻͳͻͻ͹   •ƒŽŽ‘Ž‡ —Ž‡•  ≈66 —‘‡–ƒŽǤǡʹͲͳ͹ ʹͺͺͷͳͺ͸ͺ   ͹ƒ‰‘‹•–• ‹‹“—‹‘† ≈180 ‡‡‡–ƒŽǤǡʹͲͳʹ ʹʹʹ͵͵͸ͲͶ       as Val276, Leu279 and Thr280 of the second pore helix, close to the selectivity filter, were  proposed to interact with the compound [5]. Norfluoxetine binds to the channel in the ‘down  state’, presumably impairing the transition to the ‘up state’ from which the channel opening  inhibitionpreferentially of TREK occurs channels [56]. Surprisingly,[57]. although norfluoxetine appears to preferentially  bindRR to inhibitsthe ‘down a number state’ [5] of thision channelsstate dependence including is members not reflected of the by K 2Pa channelvoltage-dependent family [28,    of29] a (shownRR sensitive in Table TREK-1 7, 8) withmutant E70 (TREK-1 in TASK-3 [29]) alone and or D135 complexed in TREK-2 with [28] RR as revealed key residue that  for RR action. In contrast, TREK-1 is not sensitiveI110D to RR [28]. Using X-ray crystal structures                 Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 97

Decher et al.: Molecular Pharmacology of K2P Channels

a negatively charged residue at this specific site provides the key inhibitor site in the extracellular ion pathway (‘EIP’) above the selectivity filter which is formed by the ‘cap’ structure (shown in Fig. 2e). Binding of RR to this site occludes the ‘EIP’ and thus the current flux [58]. novelThe and methanesulfonamide unusual allosteric mechanism. TKDC was describedLuo et al. as another small molecule inhibitor of the TREK/TRAAK subfamily (shown in Fig. 2f and Table 7). However, TKDC blocks with a to induce an allosteric conformational transition which identified ultimately an leadsallosteric to an ligand-binding obstruction of site located in the extracellular ‘cap’ of the channels. From this site the ligands are supposed the ‘EIP’ [59]. Pharmacology of TASK channel subfamily members

TASK-1 and TASK-3 blockers variants of KCNK3 KCNK3TASK-1 by a transcriptsdominant negative and currents viral areapproach upregulated suppresses under AF atrial in afibrillation pacemaker-induced (AF) [60, 61], AF , encoding TASK-1, are associated with AF [62] and genetic ablation of body and brain stem regions associated with respiratory control, and mice lacking these model of the pig [63]. Furthermore, both, TASK-1 and TASK-3, are expressed in the carotid channels have impaired carotid body function [64]. Thus, TASK channel inhibition is for instance a promisinget therapeutical approach for the treatment of AF (DOCTOS2P Trial) channel or breathing disorders like 50obstructive sleep apnea (OSA) (SANDMANXenopus Trial) oocytes [65-67]. (shown in In 2007 Putzke . described A293 (shown in Fig. 3a), the first potent2P channel,K the blockerI in rat, [32] mouse with anand IC human of 222 cardiomyocytes nM on TASK-1 [32, expressed 68, 69]. A in few years later we described Table 8), enabling the isolation of the first native whole cell current of a K TASK‑1 50 the35.1 first nM highly[70] (shown potent in and Fig. selective 3a and TableTASK-1 8). channel A1899 wasblocker in the being submicromolar active in the rangeone digit not nanomolaractive on a range.plethora The of IC ion of channels A1899 on tested TASK-1 [70] expressed and thus in A1899 CHO cell is an was even 7 nM more and promisingin oocytes

2P channel which helped understanding the pore structure of tool than A293 in terms of specificity. Subsequently, using this compound we described the mutagenesisfirst drug binding approach site ofwe a foundK that the M2 and M4 segments form the inner pore of K2P thesechannels channels, and which as these residues were actually not crystallized face into at the this central time [70].cavity Using [70]. anThe alanine A1899 scanning binding

site is formed by Thr93 of the first pore loop, Ile118 and Leu122 of the M2 segment,50 of Thr199 A1899 of the second pore loop, Ile235, Gly236, Leu239 and Asn240 of the M4 segment and Val243 and Met247 of the halothane response element (‘HRE’) [70]. Noteworthy, the IC for TASK-3 is 10-fold higher than that of TASK-1 [70] (shown in Table243VLRF 8). MTheT248 binding site andof A1899 243VLRF in LTASK-1T248 is fully conserved in the TASK-1/3/5 subfamily except for one residue. This amino acid variation is located in the ‘HRE’ ofM247L TASK-3, mutation which causes is a 3.3-fold increasefor TASK-1 in

50 for A1899 [70]. for TASK-3. This sequence variation might contribute to the different drug affinitiesA few of years TASK-1 later and we TASK-3, found sincethat blockersthe TASK-1 of the Kv1.5 channel which were developed ICas antiarrhythmic compounds to treat or prevent AF, are much more potent inhibitors of wasTASK-1 under than clinical Kv1.5 [65].investigation Note that against A1899 AFwas [65]. initially However, developed both bycompounds Sanofi as awere blocker about of Kv1.5 (S0200951) and A293 was initially described as the Kv1.5 blocker AVE1231 which are applied [65]. These data suggest that the real channel, effectively targeted against AF by 70-fold more potent on TASK-1, making them TASK selective when low doses of the compounds

Kv1.5 blockers was TASK-1 and not Kv1.5, further supporting the notion that TASK-1 might be a promising drug target against AF and OSA [65]. However, it also raised the question Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 98

Decher et al.: Molecular Pharmacology of K2P Channels

Fig. 3. 2P channels and different binding sites

Drugs that modulate channels of the TASK subfamily of K - identified. (a) Chemical formula of the most important inhibitors of channels of the TASK subfamily. (b)- Binding site of A1899 in a TASK-1 homology model [70, 71]. (c) Binding site of A293 in a TASK-1 homol ogy model [72]. (d) Binding site of PK-THPP in a TASK-3 homology model [74]. (e) Illustration of the allos teric bupivacaine binding site in the side fenestration of a TASK-1 homology model [79]. (f) Binding site of BAY1000493 in TASK-1 determined by co-crystallization (PDB ID: 6RV3) [7]. (g) Chemical formula of the most important activators of channels of the TASK subfamily. (b-f) Potassium ions are represented by black spheres. Red arrows indicate the positions of the respective drugs in top view. *: homology model.

in silico how different compounds can efficiently block both, TASK-1 and Kv1.5 channels, albeit they Forbelong both to channels, only very the remotely drug binding related sites families are formed of potassium by a ring channel. of threonine Surprisingly, residues at the analyses comparing the binding sites in TASK-1 and Kv1.5 revealed important similarities. signature sequence of the selectivity filter plus three layers of lipophilic residues facing the central cavity underneath the selectivity filter [65]. We proposed that the accessibility of the drug to the pore and the more lipophilic environment of TASK-1 might be the reason why

KvAP-basedmost of the Kv1.5 homology blockers model are thateven has more a fourfoldpotent on symmetric TASK-1 [65]. pore [70]. However, since K2P channelsThe bindingdo not havemode such of A1899 a fourfold to the symmetry TASK-1 channel in the porecentral was cavity, initially Ramirez modelled et al. on re- a

2P channels evaluated the A1899 binding site in TASK-1 using a pore homology model of TASK-1 based on TWIK-1 (shown in Fig 3b). TWIK-1 was, together with TRAAK, one of the first K crystallized and TWIK-1 is more closely related to TASK-1 than TRAAK. The TWIK-1 based homology model, combined with docking experiments and MD simulations revealed that closedA1899 state binds of tothe residues channel located[71] (shown in the in sideFig. 3b). fenestrations providing a physical ‘anchor’, reflecting an energetically favorable binding mode that after pore occlusion stabilizes the Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 99

Decher et al.: Molecular Pharmacology of K2P Channels

Table 8. 2P channel subfamily 

List of inhibitors of the TASK K ͷͲ Šƒ‡Ž •—„•–ƒ ‡ ƒ–‡‰‘”› ‹Š‹„‹–‘”  ȋρȌ ‘‹Š‹„‹–‹‘ ”‡ˆ‡”‡ ‡   Ǧͳ        ƒŽ’ŠƒƬ„‡–ƒ„Ž‘ ‡”• ƒ”˜‡†‹Ž‘Ž ≈4 –ƒ—†ƒ Š‡”‡–ƒŽǤǡʹͲͳͳ ʹͳͶͳͲͶͷͷ   ƒ–‹ ‘˜—Ž•ƒ–• ’Š‡›–‘‹ ‡‘‘—†ƒ‹•‡–ƒŽǤǡͳͻͻͺ ͻͶ͵͹ͲͲͺ    ƒ–‹’•› Š‘–‹ • ˆŽ—‘š‡–‹‡ ≈226 ƒŒ†ï‡–ƒŽǤǡʹͲͲ͵ ͳʹ͸͸͹ͻͶͷ  ǦʹǡǦ͵ǡ •—„ˆƒ‹Ž›ǡ  ƒƒ„‹‘‹†• ƒƒ†ƒ‹†‡ ƒ‹‰”‡–‡–ƒŽǤǡʹͲͲͳ ͳͳʹʹ͸ͳͷͶ  •—„ˆƒ‹Ž›  ƒƒ„‹‘‹†• ‡–Šƒ†ƒ‹†‡ ≈0.7 ƒ‹‰”‡–‡–ƒŽǤǡʹͲͲͳ ͳͳʹʹ͸ͳͷͶ  ǦʹǡǦ͵ǡǦͶǡ Ǧͳǡ  ƒ”†‹ƒ ‰Ž› ‘•‹†‡• †‹‰‹–‘š‹ ≈8  Š‹†–‡–ƒŽǤǡʹͲͳͺ ʹͻ͸Ͷ͵ʹͷͶ •—„ˆƒ‹Ž›ǡ ǦʹǡǦ͵ǡǦͶǡ Ǧͳǡ  ƒ”†‹ƒ ‰Ž› ‘•‹†‡• †‹‰‘š‹ ≈8  Š‹†–‡–ƒŽǤǡʹͲͳͺ ʹͻ͸Ͷ͵ʹͷͶ •—„ˆƒ‹Ž›ǡ  Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ‡š‹Ž‡–‹‡ ≈97  Š‹†–‡–ƒŽǤǡʹͲͳ͵ ʹͶͲ͹Ͳͺͳ͵   Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ’”‘’ƒˆ‡‘‡ ≈5  Š‹†–‡–ƒŽǤǡʹͲͳ͵ ʹͶͲ͹Ͳͺͳ͵   Žƒ•• ƒ–‹ƒ””Š›–Š‹ • “—‹‹†‹‡ ‡‘‘—†ƒ‹•‡–ƒŽǤǡͳͻͻͺ ͻͶ͵͹ͲͲͺ    Žƒ•• ƒ–‹ƒ””Š›–Š‹ • ƒ‹‘†ƒ”‘‡ ≈0.4 ‹‡”–‡‡–ƒŽǤǡʹͲͳͲ ͳͻ͹͹͹ʹͳͳ   Žƒ•• ƒ–‹ƒ””Š›–Š‹ • †”‘‡†ƒ”‘‡ ≈19 Ǧͳǡ  Š‹†–‡–ƒŽǤǡʹͲͳʹ ʹʹ͹ͻͲ͹ͻͶ  Ž‘š›“—‹ƒƒŽ‘‰• ʹ͹͸Ͷ ‡‰›‡Ž‡–ƒŽǤǡʹͲͳͻ ͵Ͳͻ͹ͻͺͳʹ    ‰‡‡”ƒŽƒ‡•–Š‡–‹ • ‡–‘‹†ƒ–‡ ≈120 —–œ‡‡–ƒŽǤǡʹͲͲ͹ ͳ͹͸ͻͻ͸͵ͺ   Ž‘ ƒŽƒ‡•–Š‡–‹ • „—’‹˜ƒ ƒ‹‡ ≈70 ‡‘‘—†ƒ‹•‡–ƒŽǤǡͳͻͻͺ ͻͶ͵͹ͲͲͺ   Ž‘ ƒŽƒ‡•–Š‡–‹ • ‡–‹†‘ ƒ‹‡ ≈40 ‹†Ž‡”‡–ƒŽǤǡͳͻͻͻ ͳͲʹͲͳ͸ͺʹ   Ž‘ ƒŽƒ‡•–Š‡–‹ • Ž‹†‘ ƒ‹‡ ‡‘‘—†ƒ‹•‡–ƒŽǤǡͳͻͻͺ ͻͶ͵͹ͲͲͺ    Ž‘ ƒŽƒ‡•–Š‡–‹ • ‡’‹˜ƒ ƒ‹‡ ≈700 ‹†Ž‡”‡–ƒŽǤǡͳͻͻͻ ͳͲʹͲͳ͸ͺʹ   Ž‘ ƒŽƒ‡•–Š‡–‹ • ”‘’‹˜ƒ ƒ‹‡ ≈50 ‹†Ž‡”‡–ƒŽǤǡͳͻͻͻ ͳͲʹͲͳ͸ͺʹ   Ž‘ ƒŽƒ‡•–Š‡–‹ • –‡–”ƒ ƒ‹‡ ≈670 ‹†Ž‡”‡–ƒŽǤǡͳͻͻͻ ͳͲʹͲͳ͸ͺʹ   ‘’‹‘‹†• Ǧ‘”’”‘’‘š›’Š‡‡ ≈170 ƒŒ†ï‡–ƒŽǤǡʹͲͲ͵ ͳʹ͸͸͹ͻͶͷ   ’Žƒ–‡š–”ƒ –• ͸Ǧ ‹‰‡”‘Ž ≈230 •—„ˆƒ‹Ž› ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͳʹͻͳʹ  ’Žƒ–‡š–”ƒ –• ƒ’•ƒ‹ ‹ ≈60 •—„ˆƒ‹Ž› ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͳʹͻͳʹ  ’Žƒ–‡š–”ƒ –• ’‹’‡”‹‡ ≈45 •—„ˆƒ‹Ž› ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͳʹͻͳʹ  ’Žƒ–‡š–”ƒ –• •ƒ•Š‘‘Ž ≈30 ƒ—–‹•–ƒ‡–ƒŽǤǡʹͲͲͺ ͳͺͷ͸ͺͲʹʹ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  ‡‘‘—†ƒ‹•‡–ƒŽǤǡͳͻͻͺ ͻͶ͵͹ͲͲͺ    ”‡•’‹”ƒ–‘”›•–‹—Žƒ–• †‘šƒ’”ƒ ≈0.4 ‘––‘‡–ƒŽǤǡʹͲͲ͸ ͳ͸Ͷͻʹͺʹͺ   •ƒŽŽ‘Ž‡ —Ž‡• ͳͺͻͻ ≈0.04 –”‡‹–‡–ƒŽǤǡʹͲͳͳ ʹͳ͵͸ʹ͸ͳͻ   •ƒŽŽ‘Ž‡ —Ž‡• ʹͻ͵ ≈0.2 —–œ‡‡–ƒŽǤǡʹͲͲ͹ ͳ͹͵ͺͻͳͶʹ   •ƒŽŽ‘Ž‡ —Ž‡• ͳͲͲͲͶͻ͵ ≈0.001 چ•–”ڏ‡–ƒŽǤǡʹͲʹͲ ͵ʹͶͻͻ͸Ͷʹ   •ƒŽŽ‘Ž‡ —Ž‡• ʹ͵Ͷͳʹ͵͹ ≈0.001 چ•–”ڏ‡–ƒŽǤǡʹͲʹͲ ͵ʹͶͻͻ͸Ͷʹ   •ƒŽŽ‘Ž‡ —Ž‡• ͳ͸Ǥͳ ≈21  ƒÀ”‡œ‡–ƒŽǤǡʹͲͳͻ ͵ͳͶʹ͸Ͷͻͳ  •ƒŽŽ‘Ž‡ —Ž‡• ͵͸ͷ ≈0.02 ŽƒŠ‡”–›‡–ƒŽǤǡʹͲͳͶ ʹͷͲͳ͹Ͳ͵͵   •ƒŽŽ‘Ž‡ —Ž‡•  ≈8 Ǧ͵ ‹ƒ‡–ƒŽǤǡʹͲͳͻ ͵ͳͲͳͷʹͺ͵  •ƒŽŽ‘Ž‡ —Ž‡• Ǧ  ͲǤ͵ ‘„—”‡–ƒŽǤǡʹͲͳʹ ʹͳͻͳ͸Ͳͳʹ   ‹Š‹„‹–‘”• ‰‡‹•–‡‹ ≈10 ‹‡”–‡‡–ƒŽǤǡʹͲͲͺ ͳͺͷͳ͸Ͳ͸ͻ  Ǧ͵        ƒ–‹„‹‘–‹ • ‡˜ƒ•–ƒ–‹ ≈160 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒ–‹„‹‘–‹ • ‘Ž‹‰‘› ‹ ≈48 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ƒŽ ‹— Šƒ‡Ž„Ž‘ ‡”• ‹„‡ˆ”ƒ†‹Ž ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ    ƒƒ„‹‘‹†• ƒƒ†ƒ‹†‡ ‡”‰‡–ƒŽǤǡʹͲͲͶ ͳͷʹͺʹʹ͹ʹ    ƒƒ„‹‘‹†• ‡–Šƒ†ƒ‹†‡ ‡”‰‡–ƒŽǤǡʹͲͲͶ ͳͷʹͺʹʹ͹ʹ    ƒ–‹‘‹ †›‡• ”—–Š‡‹—‡† ≈0.7 Ǧͳ œ‹”Œžƒ†›‡†‹ǡʹͲͲ͵ ͳʹ͸Ͳ͸͹͹͵  Žƒ•• ƒ–‹ƒ””Š›–Š‹ • “—‹‹†‹‡ ‹‡–ƒŽǤǡʹͲͲͲ ͳͲ͹͵ͶͲ͹͸    ʹƒ–ƒ‰‘‹•–• ‘ –‘ Ž‘–Š‡’‹ ≈74 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ͳƒ–ƒ‰‘‹•–• Ž‘”ƒ–ƒ†‹‡ ≈64 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ‰‡‡”ƒŽƒ‡•–Š‡–‹ • ‡–‘‹†ƒ–‡ ≈130  —–œ‡‡–ƒŽǤǡʹͲͲ͹ ͳ͹͸ͻͻ͸͵ͺ  Ž‹’‹†• ƒ”ƒ Š‹†‘‹ ƒ ‹† ‹‡–ƒŽǤǡʹͲͲͲ ͳͲ͹͵ͶͲ͹͸    Šƒ–ƒ‰‘‹•–•  ‡–ƒ ≈74 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ͳƒ–ƒ‰‘‹•–• Ǧ͹Ͳ͵ǡ͸Ͳ͸ ≈46 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   ’Žƒ–‡š–”ƒ –• ͸Ǧ ‹‰‡”‘Ž ≈385 •—„ˆƒ‹Ž› ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͳʹͻͳʹ  ’Žƒ–‡š–”ƒ –• ƒ’•ƒ‹ ‹ ≈42 •—„ˆƒ‹Ž› ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͳʹͻͳʹ  ’Žƒ–‡š–”ƒ –• ’‹’‡”‹‡ ≈135 •—„ˆƒ‹Ž› ‡Ž–”ž‡–ƒŽǤǡʹͲͳ͵ ʹͶ͵Ͳʹͻͳʹ  ’Žƒ–‡š–”ƒ –• •ƒ•Š‘‘Ž ≈450 ƒ—–‹•–ƒ‡–ƒŽǤǡʹͲͲͺ ͳͺͷ͸ͺͲʹʹ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘• —– ≈200 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  ≈3000 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  ‡’ ≈0.4 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  ‡š ≈0.3 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘•  – ≈3 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   “—ƒ–‡”ƒ”›ƒ‘‹—‹‘• ‡ ≈9 ‹‡ Š‘––ƒ‡–ƒŽǤǡʹͲͳͳ ʹͳͺʹʹʹͳͺ   ”‡•’‹”ƒ–‘”›•–‹—Žƒ–• †‘šƒ’”ƒ ≈0.04 ‘––‘‡–ƒŽǤǡʹͲͲ͸ ͳ͸Ͷͻʹͺʹͺ   •ƒŽŽ‘Ž‡ —Ž‡• ͳͺͻͻ ≈0.3 –”‡‹–‡–ƒŽǤǡʹͲͳͳ ʹͳ͵͸ʹ͸ͳͻ   •ƒŽŽ‘Ž‡ —Ž‡• ʹͻ͵ ≈1 —–œ‡‡–ƒŽǤǡʹͲͲ͹ ͳ͹͵ͺͻͳͶʹ   •ƒŽŽ‘Ž‡ —Ž‡• ͳ͸ ≈60 ƒÀ”‡œ‡–ƒŽǤǡʹͲͳͻ ͵ͳͶʹ͸Ͷͻͳ   •ƒŽŽ‘Ž‡ —Ž‡• ͳ͸Ǥͳ ≈14 ƒÀ”‡œ‡–ƒŽǤǡʹͲͳͻ ͵ͳͶʹ͸Ͷͻͳ   •ƒŽŽ‘Ž‡ —Ž‡• ʹͻ͹Ͷ ≈50 ”—‡”‡–ƒŽǤǡʹͲͳͶ ʹͶͻ͹ʹʹ͵ͻ   •ƒŽŽ‘Ž‡ —Ž‡• ͵͸ͷ ≈1 ŽƒŠ‡”–›‡–ƒŽǤǡʹͲͳͶ ʹͷͲͳ͹Ͳ͵͵   •ƒŽŽ‘Ž‡ —Ž‡• Ǧ  ≈0.04 ‘„—”‡–ƒŽǤǡʹͲͳʹ ʹͳͻͳ͸Ͳͳʹ   •ƒŽŽ‘Ž‡ —Ž‡• ǦǦͳ —‡–ƒŽǤǡʹͲͳ͸ ʹ͹Ͳͻͳͻͻ͹    •ƒŽŽ‘Ž‡ —Ž‡• ǦǦʹ —‡–ƒŽǤǡʹͲͳ͸ ʹ͹Ͳͻͳͻͻ͹    ‹Š‹„‹–‘”• ‰‡‹•–‡‹ ‹‡”–‡‡–ƒŽǤǡʹͲͲͺ ͳͺͷͳ͸Ͳ͸ͻ   Ǧͷ         ǤƒǤ     

  Although,Subsequently the A293 we binding reported site has the not binding been studied site of as the detailed antiarrhythmic as that of compoundsA1899, it appears A293 thatin TASK A293‑1 binds which at partiallya lower position overlaps within with the the central A1899 cavity, binding nearby site the [72] opening (shown of in the Fig. lateral 3c). fenestrations, however without parts of the drugs extending downwards to the halothane

A1899 and PK-THPP are effective breathing stimulants in rats and thus both compounds mayresponse have elementtherapeutic (‘T’ shapedpotential binding for treating mode breathingof A1899) disorders(compare [67].Fig. 3b PK-THPP versus Fig.which 3c). more

potently blocks TASK-3 than TASK-1 channels (shown in Fig. 3a and Table 8), also shared Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 100

Decher et al.: Molecular Pharmacology of K2P Channels several residues of the A1899 binding site in the central cavity [73-76] (shown in Fig. 3d).

(Q126,While L122, G231, L239, A237, G236 V242, and L244 L247 and (M247 T248) in TASK-1)[73, 74]. were On the identified other hand, to be other part of residues both binding were sites, there were also some novel residues identified as relevant for PK-THPP inhibition exclusivelyfound to be binds important to the exclusivelyopen state for[74]. A1899 Whether binding differences (I118, I235,in the N240state dependence and V243) [70]. and The binding of PK-THPP to TASK-3 depends on the state of the fenestration, as PK-THPP segmentaffinity towards and halothane TASK-1 response and TASK-3 element, (shown remains in Table an open8) depends question. on the different binding mode of PK-THPP and A1899, especially the different set of residues identified in the late M4

Following a high throughput fluorescent screen and structure50 activity relationship analysis of active compounds,50 of ML365,16 nM in a an bisamide, automated was electrophysiology identified as another assay promising [76]. The TASKsmall channelmolecule blocker inhibitor [76] displayed (shown inlittle Table or 8).no ML365effect on has more an IC distantly of 4 nM related in a thallium potassium flux fluorescent assay and an IC

50 channels like Kir2.1, KCNQ2 or hERG after application of 30 μM of the compound [76]. ML365 thatshowed was a described 62-fold more so far potent [76] (shownIC for TASK-1,in Table than 8). However, for the closely the molecular related TASK-3 explanation channel, for thisthus phenomenon displaying the was strongest not addressed ‘split’ in so pharmacology far. between TASK-1 and TASK-3 channels carvedilol, propafenone and amiodarone was also affected by mutations of the A1899 and Interestingly, channel inhibition by low-affinity antiarrhythmic compounds, such as channels [77, 78] (shown in Fig. 3a and Table 8) that acts at this common intracellular A293 binding site [70, 72]. Also, the respiratory stimulant doxapram is a blocker of TASK an overlap for residues in the TM2 and TM4 regions for different compounds arguing for a binding site, located in the inner vestibule of TASK-1 [76] and TASK-3 [73]. Hence, there is conservedRinné commonet al. described site of aaction, novel howeverbinding site with for substance the local specificanesthetic variations bupivacaine, in the differing binding modefrom those that appear described to modulate above which affinity results and/or in an specificity. allosteric and voltage-dependent inhibition

N240of TASK-1 in M4, and which TASK-3 were channels also part [79] of the (shown A1899 in binding Fig. 3a, site, 3e). however A large there alanine were scanning several mutagenesis approach identified residues that include I118 in M2 and I235, G236, L239 and (V234A and F238A). Bupivacaine was located laterally underneath the pore helices, in the sidenovel fenestrations, ‘hits’ in the M2 previously segment described(C110, M111, for A114,other KQ126,2P channels S127) (shownas well as in in Fig. the 3e, M4 top segment view). Thus, bupivacaine was proposed to act by an allosteric mechanism disrupting the voltage- dependent K+ second gate located-flux gating at the [11] entrance at the selectivityto the inner filter vestibule [79]. which was not observed in the structuresRecently, of otherthe TASK-1 K2P channels channel crystallized crystal structure so far [7] was (shown reported in Fig.revealing 3f). This an observationunexpected was very unexpected as K2P channels were thought to be exclusively gated at the selectivity filter. This lower gate was created by interaction of two M4 helices for which the C-terminal wasends previously crossed underneath described theto be central essential cavity, for promptingthe regulation us to of term the channelit ‘X-gate’ by [7]. Gq pathways,Strikingly, thevolatile ‘X-gate’ anesthetics is actually and formed drugs by [70, amino 80]. acidTwo residues bends can of thebe observed ‘HRE’ motif, in theV243 M4 to segment, T248, which one before the ‘X-gate’ which is supposedly a gating ‘hinge’ relevant for the positioning of the whichextended forms alpha extensive helix that interactions actually forms with the early‘X-gate’ M1 and and a latesecond M2 bendsegment observed [7]. This following region the ‘X-gate’ at residue Asn250, allowing the distal end of M4 to adopt an α-helical structure which stabilizes the ‘X-gate’ and thus the closed state of the channel was named ‘latch’. In the progress of this study our co-workers at Bayer identified by ultra-high throughput screening (uHTS) novel highly potent TASK blockers, namely BAY1000493 (shown in Fig. 3a, 3f and Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 101

Decher et al.: Molecular Pharmacology of K2P Channels

Table 8) and BAY2341237 (shown in Table 8), which were subsequently co-crystallized with

et al. for the binding of A1899TASK-1 [7, [7]. 70]. Both The drugs planar were orientation bound in a of planar the compound orientation is directlystabilized below by an the interaction selectivity of filter, the drugsinteracting with withL122 several of both key subunits. residues Rödström previously et al.described proposed, by thatStreit the blockers get trapped by the ‘X-gate’ within the inner vestibule, explaining the slow wash-out rates and almost irreversible inhibition of TASK-1 by these blockers [7]. Strikingly, mutations that are thought to destabilize the closed ‘X-gate’ did not only cause an increased open probability of the thechannel, T-shaped but bindingalso impaired mode ofthe the ability A1899 of (shown the ‘X-gate’ in Fig. to 3b) trap for BAY1000493which parts of [7]. the The compound higher potency of BAY1000493 in comparison to A1899 (shown in Table 8) might be reflected by extend all the way down to span the narrowest restriction point of the ‘X-gate’ (at residue withL244) a complementarity[7] and to interact between with M247 the shapesof the ‘HRE’of the motifinhibitor [70, and 71]. the This upper binding vestibule mode appear might prevent an efficient trapping of A1899. Compound trapping in the central cavity combined to beTASK-1 important and TASK-3for high-affinity activators drug binding in TASK-1 channels. against pulmonary arterial hypertension (PAH), Birk-Barel mental retardation syndrome In contrast to blockers, TASK channels activators would be of therapeutic interest modelingand some studiesmanifestations suggested of anpain anesthetic [81-83]. bindingHowever, pocket only a[84], few includingTASK-1 or the TASK-3 HRE [41,channel 80] inactivators the late areM4 describedand M159 so [85] far in(shown the late in M3Fig. segment3g and Table [84]. 6). Halogenated For TASK channels ether, alcohol, molecular and channels, in proximity to L239. However, mutating the pore facing L122 residue completely alkane anesthetics were reported to be positioned near the side fenestrations of TASK-3 caused by altered channel gating by the L122 mutant that is in close proximity to L239 and theeliminated side fenestrations. the activation This by hypothesis isoflurane is [86].supported The authors by mutations suggest at thatan equivalent these effects residue are

Alternatively,in TWIK-1 (Leu146) it could that be also activate possible TWIK-1 that andvolatile by molecular anesthetics dynamic are also studies bound which to residues suggest of that this region is important for pore hydration and/or the lipid access into the pore [87]. the central cavity and act from the pore on the selectivity filter, similar as described for the The‘master guanylate key’ mechanism cyclase stimulator [48]. riociguat, licensed for the treatment of PAH, enhances However, there are also examples of synthetic small molecule activators already: transfected tsA201 cells with the compound and a direct channel modulation has not been TASK-1 currents [82]. Albeit, this activation occurs after incubation of transiently demonstratedG236R mutation yet [82]. which In addition, causes TASK-3Birk-Barel for instancemental retardation is activated conducts by flufenamic only veryacid [83],little terbinafine [88], CHET3 [81] or NPBA [89] (shown in Fig. 3g and Table 6). Interestingly, the TASK-3 et al. demonstrated currents [90]. This electrophysiological phenotype could be partially rescued by the TASK activators flufenamic acid [83] or terbinafine [88]. Moreover, Garcia that intrathecal pre-treatment with terbinafine, reduced the formalin-induced flinching and allodynia/hyperalgesia in rat, further supporting the putative future clinical relevance of TASK activators [91]. In terms of the binding sites, amino acid residues in the early M2 and late M3 segment, which are not conserved in TASK-1, were important for TASK-3 channel activation by NPBA [89]. In contrast for CHET3 a binding site in TASK-3 was found underneath the selectivity filter close to the M2 and M4 segments altering channel gating by affecting the selectivity filter conformation [81]. Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 102

Decher et al.: Molecular Pharmacology of K2P Channels

Outlook - K2P channel modulators in human diseases

2P channel modulators carry a huge therapeutic potential, in

As briefly discussed above, K despitemany diseases, the multitude as became of K 2Pevident channel from modulators their involvement known by in now, inherited we clearly ‘channelopathies’ require more [47, K2P 90,modulating 92-94] and compounds, by the fact not that only ion to channels further increaseare known potency to be very and goodselectivity ‘druggable’. to avoid However, toxicity developmentand/or specific of side further effects, compounds but also tofrom have distinct compounds chemical with structural the right pharmacokineticsclasses, presumably of primarilyLiberation, possible Absorption, by pharmaceutical Distribution, Metabolism industry, one and can Excretion think of (LADME).many future Thus, applications given the of K2P KCNK3 loss- of-function channel mutation modulators as described in human by diseases. Ma et al. TASK-1 [94, 95] activators. might rescue PAH and be toeven rescue beneficial some in aspects those casesof the of Birk-Barel PAH in which mental the diseaseretardation was not[90], caused when byapplied a early in TASK‑3 activators might be able KCNK17 mutation juvenile development. TASK‑1 blockers are already under clinical investigations against OSA (DOCTOS Trial) and AF (SANDMAN Trial). Also, as a lesson learned from a causing progressive cardiac conduction disorder (PCCD) [93], TASK-4 blockers might be effectiveadvantageous against for pain the withouttreatment the of classical specific sideforms effects of PCCD. of opioids Furthermore, [46]. These TRESK putative modulators future therapeuticmight be beneficial applications in migraine are, as we [92, think, 96] a andstrong novel motivation potent to TREK-1 further modulators study the molecular could be pharmacology of K2P channels.

Conclusion

K2P drawback for studies aiming to address the physiological role of these channels. However, recent advances channels in notoriously the understanding suffered fromof the a molecular poor pharmacologic pharmacology profile of K which2P channels, was a provided an understanding about a large variety of complex mechanisms that can cause modulation of these potassium channels. While the gating and molecular pharmacology studies, we eagerly anticipate new drugs and more mechanistic insights for the molecular of TREK, TRESK and TASK channel subfamily members was the subject of many excellent modulation of channels of the TWIK, THIK and TALK subfamilies. Acknowledgements

The authors are grateful to all members of their laboratory and for useful discussion with all our collaborationAll authors contributed partners. to the draft of the manuscript and to the design of the figures.

Funding

A.K.K. is supported by the von-Behring-Röntgen Stiftung (67-0015) and by the P.E. Kempkes Stiftung (05/2020). N.D. is supported by the Deutsche Forschungsgemeinschaft DFG (DE 1482/4-1). W.G. is supported by Fondecyt (Fondo Nacional de Desarrollo Científico y Tecnológico; 1191133). Disclosure Statement

The authors have no conflicts of interest to declare. Cellular Physiology Cell Physiol Biochem 2021;55(S3):87-107 DOI: 10.33594/000000339 © 2021 The Author(s). Published by and Biochemistry Published online: 6 March 2021 Cell Physiol Biochem Press GmbH&Co. KG 103

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