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 K2P 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 K2P channel blockers and activators.
Introduction
Tandem of two-pore-domain potassium (K2P) channels belong to the latest family of potassium channels cloned, with TOK1 from Saccharomyces cerevisiae as the first channel discovered in 1995 [1]. The mammalian K2P potassium channel family contains 15 members with different subfamilies, characterized by mechanistic hallmarks like acid inhibition, stretch activation, alkaline activation and halothane inhibition (shown in Fig. 1a). K2P channels have four transmembrane domains and contain two pore loops and thus assemble as dimer in order that four pore loops form the potassium selectivity filter, similar as in other potassium channel families (shown in Fig. 1b, 1c). The channels have a large extracellular M1-P1 loop which has been identified in early studies as self-interacting domain (‘SID’) and is relevant for the dimerization of the channels (shown in Fig. 1b) [2]. In fact, the extracellular M1-P1 loop forms the so called ‘cap’ structure which is a structural hallmark of the K2P channels (shown in Fig. 1b, 1c) [3-9]. It has been postulated that the extracellular ‘cap’ prevents classical toxins from binding to the pore region [4]. However, whether there are other physiological functions that can be assigned to this unique structure has not been addressed yet. Although K2P channels were initially described as leak channels with an outward rectification that appeared to solely obey the Goldman-Hodgkin-Katz equation for a potassium selective hole, we know in the meantime that these channels are highly regulated by a plethora of different 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 potassium gated potassium channels with a potassium efflux increasing the open probability of the channel at depolarized potentials.
Initially, K2P channels were described to have a unique pharmacology compared to other potassium channel families, as they were less sensitive to the classical potassium channel blockers like TEA, 4-AP, Cs+ or Ba2+. 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 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 should be resistant to classical pore blockers. In the meantime, we know that K2P channels are also highly sensitive to classical pore block by quaternary ammonium compounds (QA) (shown in Fig. 1e), however, presumably due to differences in the architecture of the central cavity providing more lateral space underneath the selectivity filter, the QAs need longer alkyl side chains, like TPenA or THexA (shown in Fig. 1e), to be stabilized underneath the 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 been described very early after cloning of the TASK channels [22-29], there was for a longer time a lack of potent small compound inhibitors. A decade after cloning of the first K2P channels and TASK‑1 [23, 30, 31], we described the first potent K2P channel blocker A293 [32]. Strikingly, the development of a potent and selective TASK-1 channel blocker opened the door for functional studies of the channel in native tissue [33], leading for instance to the isolation of a whole cell TASK-1 current in ventricular myocytes [32]. However, for most of the K2P channels there are still no highly potent and selective blockers or activators available (shown in Table 1-8). Thus, we eagerly anticipate the development of small compound modulators for all K2P channels as this will, besides the generation and study of transgenic mouse models, definitely foster our research aiming to understand the physiological role of K2P channels in different organs.
However, while we still have a poor understanding of the pharmacology of TWIK, THIK and TALK channels, there are in the meantime many potent activators and blockers described for the channels of the TASK and TREK-subfamily, which we would like to summarize in separate sections, especially by focusing on the mechanistic models of channel modulation and the differential binding sites utilized by the drugs.
Pharmacology of K2P channels of the TWIK, THIK and TALK subfamilies
For K2P channels of the TWIK, THIK and TALK subfamilies a low potency block was observed for instance by local anesthetics or quinidine (shown in Table 1). Similar as for other K2P channels these subfamilies are also sensitive to volatile anesthetics (shown in Table 1, 2). In addition, many anti-arrhythmic drugs were reported to block TASK-4/(TALK-2) channels, albeit also with a very low potency (shown in Table 1). Thus, for the K2P channels of the TWIK, THIK and TALK subfamilies there are to our best knowledge no small compound activators or blockers reported that are active in the submicromolar range.
Pharmacology of the TRESK channel
TRESK is a K2P channel that was reported to be primarily or almost exclusively expressed 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 became clear, that this channel is the mouse orthologue of TRESK and that there is only one channel within this subfamily of K2P channels. In contrast to the channels of the TWIK, THIK and TALK subfamilies, TRESK appears to be more ‘druggable’ since many blockers and/or activators were already described (shown in Table 3, 4). TRESK is blocked by many small compound inhibitors and also with fairly low IC50 values (shown in Table 3), for instance by the antihistaminic drug loratadine, with an IC50 of about 1 µM [37]. Also many activators have been described (shown in Table 4), here some compounds exhibit even EC50 values in the submicromolar range. For instance, acetyl-B-methylcholine, oxotremorine and OXA-22 activate TRESK with an EC50 of about 700 nM, 300 nM and 100 nM, respectively [37] (shown 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.
Pharmacology of channels from the TREK/TRAAK subfamily
TREK/TRAAK activators
A hallmark of the members of the TREK/TRAAK subfamily of K2P channels is the channel modulation by polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA) [38, 39] (shown in Fig. 2a and Table 5). Analyses of deletion constructs demonstrated that the C-terminus of TREK-1 is crucial for the response to AA [39]. In addition, replacing the C-terminus of TREK-2 with the C-terminus of TASK‑3 abolished the sensitivity to AA. However, replacing the C-terminus of TRAAK with that of TASK-1 or TASK-3 did not affect the response to AA. These results show that the mechanism of activation of TRAAK and TREK by fatty acids may be different [40].
Volatile anesthetics, including diethyl ether, halothane, isoflurane or chloroform, activate TREK-1 and TREK-2 (but not TRAAK) channels which requires the C-terminus of the channels [41] (shown in Table 5). Chloroform specifically and reversibly activates TREK channels [41], whereas halothane or isoflurane activates both, TASK and TREK channels [41] (shown in Fig. 2a and Table 5, 6). On the contrary, diethyl ether increased TREK-1 whereas it decreased TASK-1 currents [41]. In addition, anesthetic gases, as nitrous oxide, xenon and cyclopropane activate TREK-1 channels in clinically relevant concentrations, whereas TASK-3 is insensitive [42] (shown in Table 5). The Glu306 residue, also critical for TREK-1 modulation by AA, stretch or internal pH, was shown to be important for TREK-1 channel activation by these anesthetic gases [42].
In 2000 it was shown by Duprat 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 increased cAMP levels via PKA phosphorylation and riluzole has the capacity of increasing cAMP levels, the activation of TREK-1 is only transient. In contrast, TRAAK channels, which lack a PKA inhibition, are permanently activated [43].
TREK-1 channels are discussed as novel drug targets against pain [44, 45]. Devilliers et al. demonstrated, that TREK-1 contributes to morphine-induced analgesia in mice. The channel was directly activated (independent of µ opioid receptor activation) leading to analgesia without adverse effects [46].
Another TREK-1 activator with therapeutic potential is BL-1249 [47] (shown in Fig. 2a and Table 5). Using a whole exome sequencing approach in a patient with right ventricular outflow tract tachycardia (RVOT-VT), Decher et al. identified the TREK-1I267T mutation, an amino acid exchange located directly before the selectivity filter of the second pore loop [47]. The mutation almost completely abolished outward currents through the channel and introduced a strong sodium permeability to the potassium channel [47]. Interestingly, application of BL-1249 rescued the potassium selectivity and loss-of-function of the TREK-1I267T channel. The fact that this fenamate-like compound was able to rescue the selectivity filter defect of TREK-1I267T indicated that this drug or maybe other similar activators directly stabilize the selectivity filter. Consequently, Schewe et al. identified negatively charged activators (NCAs) harboring a negatively charged tetrazole or carboxylate group (such as BL-1249, PD-118057 and NS11021) as activators of the mechano-gated K2P channels TREK-1 and TREK-2 [48] (shown in Fig. 2a and Table 5). Strikingly, these NCAs activate most of the K2P channels (shown in Table 2, 4, 5) and also activate other selectivity filter gated ion channels like hERG or BK channels with equal efficiency [48]. Thus, NCAs act with a common mechanism on selectivity filter gated channels, providing a universal ‘master key’ to unlock the selectivity filter gate by binding below the selectivity filter where their negative charge promotes K+ binding to the pore cavity (shown in Fig. 2b). This in turn alters the ion occupancy in the selectivity filter in a way that is known to promote activation of the filter gate [11, 48].
In contrast, 2-aminoethoxydiphenyl borate (2-APB) is a non NCA that activates channels of the TREK/TRAAK subfamily (shown in Fig. 2a and Table 5). TREK-2 is much more sensitive to modulation by 2-APB compared with TREK-1 or TRAAK [49] (shown in Table 5). 2-APB does not bind to either the binding site of NCAs or the ‘cryptic’ binding site described 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, including His368 as a key residue, was required for channel activation by 2-APB [50]. In addition, specific mutations in the M4 segment reduced the 2-APB efficiency and thus the authors proposed an allosteric coupling between the proximal C‑terminus and the selectivity 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 [51].
Very recently a novel class of small molecule activators has been identified that utilizes a completely different, the ‘cryptic’, binding site, which is clearly distinct to that of NCAs [6] (shown in Fig. 2c). ML335 and ML402 bind to an L-shaped pocket behind the selectivity filter formed by the P1 pore helix and M4 transmembrane helix intrasubunit interface. The drugs 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 an ‘M4 face’ reducing P1/TM4 interface dynamics [6].
A high-throughput fluorescence-based thallium flux screen identified small molecules that selectively activated TREK-2 [52]. These novel compounds were subsequently proven to directly activate TREK-2 channels, using single channel measurements in excised membrane patches [52]. Strikingly, 11-deoxy PGF2α or T2A3 which were described in this study, activated TREK-2 while they blocked TREK-1 channels [52] (shown in Table 5, 7). For these compounds a region connecting the second pore loop to the M4 segment was proposed to determine the observed activation or inhibition [52].
TREK/TRAAK inhibitors
In terms of blockers for channels of the TREK/TRAAK subfamily, spadin is presumably the best known blocker of TREK channels described (shown in Fig. 2f). Spadin is a 17 amino acid sortilin-derived peptide targeting TREK-1 channels with an IC50 of 70 nM [53] (shown in Fig. 2f and Table 7). It is discussed as a putative antidepressant [53, 54], although the binding site and mechanism of inhibition are not known so far. However, it has been postulated that spadin should bind to the ‘down state’ of the channel to specifically antagonize activation of TREK-1 by AA, utilizing an allosteric mechanism of inhibition [55].
Dong et al. described the crystal structure of TREK-2 in complex with norfluoxetine [5] (shown in Fig. 2d, 2f). Here several residues in the side fenestrations, including Ile194 and Pro198 of the M2 segment, Cys249 and Val253 in M3, Phe316 and Leu320 in M4, as well 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 preferentially occurs [56]. Surprisingly, although norfluoxetine appears to preferentially bind to the ‘down state’ [5] this state dependence is not reflected by a voltage-dependent inhibition of TREK channels [57].
RR inhibits a number of ion channels including members of the K2P channel family [28, 29] (shown in Table 7, 8) with E70 in TASK-3 [29] and D135 in TREK-2 [28] as key residue for RR action. In contrast, TREK-1 is not sensitive to RR [28]. Using X-ray crystal structures of a RR sensitive TREK-1 mutant (TREK-1I110D) alone or complexed with RR revealed that 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].
The methanesulfonamide TKDC was described as another small molecule inhibitor of the TREK/TRAAK subfamily (shown in Fig. 2f and Table 7). However, TKDC blocks with a novel and unusual allosteric mechanism. Luo et al. identified an allosteric ligand-binding site located in the extracellular ‘cap’ of the channels. From this site the ligands are supposed to induce an allosteric conformational transition which ultimately leads to an obstruction of the ‘EIP’ [59].
Pharmacology of TASK channel subfamily members
TASK-1 and TASK-3 blockers
TASK-1 transcripts and currents are upregulated under atrial fibrillation (AF) [60, 61], variants of KCNK3, encoding TASK-1, are associated with AF [62] and genetic ablation of KCNK3 by a dominant negative viral approach suppresses AF in a pacemaker-induced AF model of the pig [63]. Furthermore, both, TASK-1 and TASK-3, are expressed in the carotid body and brain stem regions associated with respiratory control, and mice lacking these channels have impaired carotid body function [64]. Thus, TASK channel inhibition is for instance a promising therapeutic approach for the treatment of AF (DOCTOS Trial) or breathing disorders like obstructive sleep apnea (OSA) (SANDMAN Trial) [65-67].
In 2007 Putzke et al. described A293 (shown in Fig. 3a), the first potent K2P channel blocker [32] with an IC50 of 222 nM on TASK-1 expressed in Xenopus oocytes (shown in Table 8), enabling the isolation of the first native whole cell current of a K2P channel, the ITASK‑1 in rat, mouse and human cardiomyocytes [32, 68, 69]. A few years later we described the first highly potent and selective TASK-1 channel blocker being active in the one digit nanomolar range. The IC50 of A1899 on TASK-1 expressed in CHO cell was 7 nM and in oocytes 35.1 nM [70] (shown in Fig. 3a and Table 8). A1899 was in the submicromolar range not active on a plethora of ion channels tested [70] and thus A1899 is an even more promising tool than A293 in terms of specificity. Subsequently, using this compound we described the first drug binding site of a K2P channel which helped understanding the pore structure of these channels, as these were not crystallized at this time [70]. Using an alanine scanning mutagenesis approach we found that the M2 and M4 segments form the inner pore of K2P channels and which residues actually face into the central cavity [70]. The A1899 binding site is formed by Thr93 of the first pore loop, Ile118 and Leu122 of the M2 segment, Thr199 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 IC50 of A1899 for TASK-3 is 10-fold higher than that of TASK-1 [70] (shown in Table 8). The binding site of A1899 in TASK-1 is fully conserved in the TASK-1/3/5 subfamily except for one residue. This amino acid variation is located in the ‘HRE’ of TASK-3, which is 243VLRFMT248 for TASK-1 and 243VLRFLT248 for TASK-3. This sequence variation might contribute to the different drug affinities of TASK-1 and TASK-3, since the TASK-1M247L mutation causes a 3.3-fold increase in IC50 for A1899 [70].
A few years later we found that blockers of the Kv1.5 channel which were developed as antiarrhythmic compounds to treat or prevent AF, are much more potent inhibitors of TASK-1 than Kv1.5 [65]. Note that A1899 was initially developed by Sanofi as a blocker of Kv1.5 (S0200951) and A293 was initially described as the Kv1.5 blocker AVE1231 which was under clinical investigation against AF [65]. However, both compounds were about 70-fold more potent on TASK-1, making them TASK selective when low doses of the compounds are applied [65]. These data suggest that the real channel, effectively targeted against AF by 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 how different compounds can efficiently block both, TASK-1 and Kv1.5 channels, albeit they belong to only very remotely related families of potassium channel. Surprisingly, in silico analyses comparing the binding sites in TASK-1 and Kv1.5 revealed important similarities. For both channels, the drug binding sites are formed by a ring of threonine residues at the 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 most of the Kv1.5 blockers are even more potent on TASK-1 [65].
The binding mode of A1899 to the TASK-1 channel pore was initially modelled on a KvAP-based homology model that has a fourfold symmetric pore [70]. However, since K2P channels do not have such a fourfold symmetry in the central cavity, Ramirez et al. re-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 K2P channels 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 A1899 binds to residues located in the side fenestrations providing a physical ‘anchor’, reflecting an energetically favorable binding mode that after pore occlusion stabilizes the closed state of the channel [71] (shown in Fig. 3b).
Subsequently we reported the binding site of the antiarrhythmic compounds A293 in TASK‑1 which partially overlaps with the A1899 binding site [72] (shown in Fig. 3c). Although, the A293 binding site has not been studied as detailed as that of A1899, it appears that A293 binds at a lower position within the central cavity, nearby the opening of the lateral fenestrations, however without parts of the drugs extending downwards to the halothane response element (‘T’ shaped binding mode of A1899) (compare Fig. 3b versus Fig. 3c).
A1899 and PK-THPP are effective breathing stimulants in rats and thus both compounds may have therapeutic potential for treating breathing disorders [67]. PK-THPP which more potently blocks TASK-3 than TASK-1 channels (shown in Fig. 3a and Table 8), also shared several residues of the A1899 binding site in the central cavity [73-76] (shown in Fig. 3d). While L122, L239, G236 and L247 (M247 in TASK-1) were identified to be part of both binding sites, there were also some novel residues identified as relevant for PK-THPP inhibition (Q126, G231, A237, V242, L244 and T248) [73, 74]. On the other hand, other residues were found to be important exclusively for A1899 binding (I118, I235, N240 and V243) [70]. The binding of PK-THPP to TASK-3 depends on the state of the fenestration, as PK-THPP exclusively binds to the open state [74]. Whether differences in the state dependence and affinity towards TASK-1 and TASK-3 (shown in Table 8) depends on the different binding mode of PK-THPP and A1899, especially the different set of residues identified in the late M4 segment and halothane response element, remains an open question.
Following a high throughput fluorescent screen and structure activity relationship analysis of active compounds, ML365, a bisamide, was identified as another promising TASK channel blocker [76] (shown in Table 8). ML365 has an IC50 of 4 nM in a thallium flux fluorescent assay and an IC50 of 16 nM in an automated electrophysiology assay [76]. The small molecule inhibitor displayed little or no effect on more distantly related potassium channels like Kir2.1, KCNQ2 or hERG after application of 30 μM of the compound [76]. ML365 showed a 62-fold more potent IC50 for TASK-1, than for the closely related TASK-3 channel, thus displaying the strongest ‘split’ in pharmacology between TASK-1 and TASK-3 channels that was described so far [76] (shown in Table 8). However, the molecular explanation for this phenomenon was not addressed so far.
Interestingly, channel inhibition by low-affinity antiarrhythmic compounds, such as carvedilol, propafenone and amiodarone was also affected by mutations of the A1899 and A293 binding site [70, 72]. Also, the respiratory stimulant doxapram is a blocker of TASK channels [77, 78] (shown in Fig. 3a and Table 8) that acts at this common intracellular binding site, located in the inner vestibule of TASK-1 [76] and TASK-3 [73]. Hence, there is an overlap for residues in the TM2 and TM4 regions for different compounds arguing for a conserved common site of action, however with substance specific variations in the binding mode that appear to modulate affinity and/or specificity.
Rinné et al. described a novel binding site for the local anesthetic bupivacaine, differing from those described above which results in an allosteric and voltage-dependent inhibition of TASK-1 and TASK-3 channels [79] (shown in Fig. 3a, 3e). A large alanine scanning mutagenesis approach identified residues that include I118 in M2 and I235, G236, L239 and N240 in M4, which were also part of the A1899 binding site, however there were several novel 'hits' in the M2 segment (C110, M111, A114, Q126, S127) as well as in the M4 segment (V234A and F238A). Bupivacaine was located laterally underneath the pore helices, in the side fenestrations, previously described for other K2P channels (shown in Fig. 3e, top view). Thus, bupivacaine was proposed to act by an allosteric mechanism disrupting the voltage-dependent K+-flux gating [11] at the selectivity filter [79].
Recently, the TASK-1 channel crystal structure was reported revealing an unexpected second gate located at the entrance to the inner vestibule which was not observed in the structures of other K2P channels crystallized so far [7] (shown in Fig. 3f). This observation 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 ends crossed underneath the central cavity, prompting us to term it ‘X-gate’ [7]. Strikingly, the ‘X-gate’ is actually formed by amino acid residues of the ‘HRE’ motif, V243 to T248, which was previously described to be essential for the regulation of the channel by Gq pathways, volatile anesthetics and drugs [70, 80]. Two bends can be observed in the M4 segment, one before the ‘X-gate’ which is supposedly a gating ‘hinge’ relevant for the positioning of the extended alpha helix that actually forms the ‘X-gate’ and a second bend observed following the ‘X-gate’ at residue Asn250, allowing the distal end of M4 to adopt an α-helical structure which forms extensive interactions with the early M1 and late M2 segment [7]. This region 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 Table 8) and BAY2341237 (shown in Table 8), which were subsequently co-crystallized with TASK-1 [7]. Both drugs were bound in a planar orientation directly below the selectivity filter, interacting with several key residues previously described by Streit et al. for the binding of A1899 [7, 70]. The planar orientation of the compound is stabilized by an interaction of the drugs with L122 of both subunits. Rödström et al. proposed, that 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 channel, but also impaired the ability of the ‘X-gate’ to trap BAY1000493 [7]. The higher potency of BAY1000493 in comparison to A1899 (shown in Table 8) might be reflected by the T-shaped binding mode of the A1899 (shown in Fig. 3b) for which parts of the compound extend all the way down to span the narrowest restriction point of the ‘X-gate’ (at residue L244) [7] and to interact with M247 of the ‘HRE’ motif [70, 71]. This binding mode might prevent an efficient trapping of A1899. Compound trapping in the central cavity combined with a complementarity between the shapes of the inhibitor and the upper vestibule appear to be important for high-affinity drug binding in TASK-1 channels.
TASK-1 and TASK-3 activators
In contrast to blockers, TASK channels activators would be of therapeutic interest against pulmonary arterial hypertension (PAH), Birk-Barel mental retardation syndrome and some manifestations of pain [81-83]. However, only a few TASK-1 or TASK-3 channel activators are described so far (shown in Fig. 3g and Table 6). For TASK channels molecular modeling studies suggested an anesthetic binding pocket [84], including the HRE [41, 80] in the late M4 and M159 [85] in the late M3 segment [84]. Halogenated ether, alcohol, and alkane anesthetics were reported to be positioned near the side fenestrations of TASK-3 channels, in proximity to L239. However, mutating the pore facing L122 residue completely eliminated the activation by isoflurane [86]. The authors suggest that these effects are caused by altered channel gating by the L122 mutant that is in close proximity to L239 and the side fenestrations. This hypothesis is supported by mutations at an equivalent residue in TWIK-1 (Leu146) that activate TWIK-1 and by molecular dynamic studies which suggest that this region is important for pore hydration and/or the lipid access into the pore [87]. Alternatively, it could be also possible that volatile anesthetics are also bound to residues of the central cavity and act from the pore on the selectivity filter, similar as described for the ‘master key’ mechanism [48].
However, there are also examples of synthetic small molecule activators already: The guanylate cyclase stimulator riociguat, licensed for the treatment of PAH, enhances TASK-1 currents [82]. Albeit, this activation occurs after incubation of transiently transfected tsA201 cells with the compound and a direct channel modulation has not been demonstrated yet [82]. In addition, TASK-3 for instance is activated by flufenamic acid [83], terbinafine [88], CHET3 [81] or NPBA [89] (shown in Fig. 3g and Table 6). Interestingly, the TASK-3G236R mutation which causes Birk-Barel mental retardation conducts only very little currents [90]. This electrophysiological phenotype could be partially rescued by the TASK activators flufenamic acid [83] or terbinafine [88]. Moreover, Garcia et al. demonstrated 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].
Outlook - K2P channel modulators in human diseases
As briefly discussed above, K2P channel modulators carry a huge therapeutic potential, in many diseases, as became evident from their involvement in inherited ‘channelopathies’ [47, 90, 92-94] and by the fact that ion channels are known to be very good ‘druggable’. However, despite the multitude of K2P channel modulators known by now, we clearly require more K2P modulating compounds, not only to further increase potency and selectivity to avoid toxicity and/or specific side effects, but also to have compounds with the right pharmacokinetics of Liberation, Absorption, Distribution, Metabolism and Excretion (LADME). Thus, given the development of further compounds from distinct chemical structural classes, presumably primarily possible by pharmaceutical industry, one can think of many future applications of K2P channel modulators in human diseases. TASK-1 activators might rescue PAH and be even beneficial in those cases of PAH in which the disease was not caused by a KCNK3 loss-of-function mutation as described by Ma et al. [94, 95]. TASK‑3 activators might be able to rescue some aspects of the Birk-Barel mental retardation [90], when applied early in 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 KCNK17 mutation causing progressive cardiac conduction disorder (PCCD) [93], TASK-4 blockers might be advantageous for the treatment of specific forms of PCCD. Furthermore, TRESK modulators might be beneficial in migraine [92, 96] and novel potent TREK-1 modulators could be effective against pain without the classical side effects of opioids [46]. These putative future therapeutic applications are, as we think, a strong motivation to further study the molecular pharmacology of K2P channels.
Conclusion
K2P channels notoriously suffered from a poor pharmacologic profile which was a drawback for studies aiming to address the physiological role of these channels. However, recent advances in the understanding of the molecular pharmacology of K2P channels, provided an understanding about a large variety of complex mechanisms that can cause modulation of these potassium channels. While the gating and molecular pharmacology of TREK, TRESK and TASK channel subfamily members was the subject of many excellent studies, we eagerly anticipate new drugs and more mechanistic insights for the molecular modulation of channels of the TWIK, THIK and TALK subfamilies.
Funding
A.K.K. is supported by the von-Behring-Röntgen Stiftung (67-0015). N.D. is supported by the Deutsche Forschungsgemeinschaft DFG (DE 1482/9-1). W.G. is supported by Fondecyt (1191133).
Disclosure Statement
The authors have no conflicts of interest to declare.
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