Pope L et al. (2018), Protein and Chemical Determinants of BL-1249 Ac...

XB-ART-56591

ACS Chem Neurosci 2018 Dec 19;912:3153-3165. doi: 10.1021/acschemneuro.8b00337.

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Protein and Chemical Determinants of BL-1249 Action and Selectivity for K2P Channels.

Pope L , Arrigoni C , Lou H , Bryant C , Gallardo-Godoy A , Renslo AR , Minor DL .

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K2P potassium channels generate leak currents that stabilize the resting membrane potential of excitable cells. Various K2P channels are implicated in pain, ischemia, depression, migraine, and anesthetic responses, making this family an attractive target for small molecule modulator development efforts. BL-1249, a compound from the fenamate class of nonsteroidal anti-inflammatory drugs is known to activate K2P2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily; however, its mechanism of action and effects on other K2P channels are not well-defined. Here, we demonstrate that BL-1249 extracellular application activates all TREK subfamily members but has no effect on other K2P subfamilies. Patch clamp experiments demonstrate that, similar to the diverse range of other chemical and physical TREK subfamily gating cues, BL-1249 stimulates the selectivity filter "C-type" gate that controls K2P function. BL-1249 displays selectivity among the TREK subfamily, activating K2P2.1(TREK-1) and K2P10.1(TREK-2) ∼10-fold more potently than K2P4.1(TRAAK). Investigation of mutants and K2P2.1(TREK-1)/K2P4.1(TRAAK) chimeras highlight the key roles of the C-terminal tail in BL-1249 action and identify the M2/M3 transmembrane helix interface as a key site of BL-1249 selectivity. Synthesis and characterization of a set of BL-1249 analogs demonstrates that both the tetrazole and opposing tetralin moieties are critical for function, whereas the conformational mobility between the two ring systems impacts selectivity. Together, our findings underscore the landscape of modes by which small molecules can affect K2P channels and provide crucial information for the development of better and more selective K2P modulators of the TREK subfamily.

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Species referenced: Xenopus laevis
Genes referenced: kcnk4

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	Figure 1. External application of BL-1249 selectively activates mechanosensitive K2P channels. (A) Exemplar current traces for specified K2P channels (black) with 10 μM BL-1249 (light blue) as measured via TEVC in Xenopus oocytes. (B) K2P channel phylogenetic tree. Stars denote assayed representative K2P channels. Blue stars indicate BL-1249 responsive channels. (C) BL-1249 dose–response curves for K2P2.1(TREK-1) (blue circles), K2P10.1(TREK-2) (black triangles), and K2P4.1(TRAAK) (orange squares). EC50 = 5.5 ± 1.2 μM, 8.0 ± 0.8 μM, and 48 ± 10 μM, respectively. (D) BL-1249 responses of indicated K2P channels. Inset shows expanded view of poorly responsive K2P channels. Error bars are SEM.
	Figure 2. BL-1249 activates the K2P2.1(TREK-1) C-type gate. (A, B) Exemplar current traces for (A) K2P2.1(TREK-1) and (B) K2P2.1(TREK-1) with 1 μM BL-1249 in HEK293 inside-out patches in 150 mM K+[out]/150 mM Rb+[in]. Inset shows voltage protocol. (C, D) Current–voltage relationships for (C) K2P2.1(TREK-1) and (D) K2P2.1(TREK-1) with 1 μM BL-1249. (E) Rectification coefficients (I+100mV/I–100mV) from recordings (n ≥ 3) made in panels A–D. (F) Dose–response curves in Xenopus oocytes for K2P2.1(TREK-1) (blue circles), K2P2.1(TREK-1) G137I (purple squares), and K2P2.1(TREK-1) W275S (orange triangles). K2P2.1(TREK-1), 5.5 ± 1.2 μM; G137I and W275S, >60 μM. K2P2.1(TREK-1) data are from Figure 1C. Error bars are SEM.
	Figure 3. K2P2.1(TREK-1) C-terminus affects BL-1249 response. (A) Exemplar current traces for K2P2.1(TREK-1)GGG (black) with 20 μM BL-1249 (green) and BL-1249 dose–response curves for K2P2.1(TREK-1) (blue circles) and K2P2.1(TREK-1)GGG (green circles) (EC50 = 5.5 ± 1.2 μM and 19 ± 1 μM, respectively). Green star in cartoon indicates site of GGG mutation. (B) Exemplar current traces for K2P2.1(TREK-1)μ322 (magenta) and K2P2.1(TREK-1)μ308 (purple) with 20 μM BL-1249 and BL-1249 dose–response curves for K2P2.1(TREK-1) (blue circles), K2P2.1(TREK-1)Δ322 (purple squares), and K2P2.1(TREK-1)Δ308 (magenta triangles). (EC50 = 5.5 ± 1.2 μM, 26 ± 8 μM, and 35 ± 8 μM, respectively). Magenta and purple lines in cartoon indicate sites of Δ322 and Δ308 truncations, respectively. (C) Exemplar current traces for TREK-1/AAK_T alone (black) and with 20 μM BL-1249 (cyan). BL-1249 dose–response curves for K2P2.1(TREK-1) (blue circles), K2P4.1(TRAAK) (light orange squares), and TRAAK/EK-1_T (light blue triangles). (EC50 = 5.5 ± 1.2 μM, 48 ± 10 μM, and 7.7 ± 0.6 μM, respectively). Cartoon indicates TREK-1/AAK_T channel regions from K2P2.1 (blue) and K2P4.1 (yellow). (D) Exemplar current traces for TRAAK/EK_T alone (black) and with 20 μM BL-1249 (orange). BL-1249 dose–response curves for K2P2.1(TREK-1) (blue circles), K2P4.1(TRAAK) (light orange squares), and TRAAK/EK_T (orange triangles) (EC50 = 5.5 ± 1.2 μM, 48 ± 10 μM, and 23 ± 4 μM, respectively). Cartoon indicates TRAAK/EK_T channel regions from K2P2.1 (blue) and K2P4.1 (light orange). In panels A–D, K2P2.1(TREK-1) and K2P4.1(TRAAK) data are from Figure 1C. Error bars are SEM.
	Figure 4. BL-1249 responses of TREK-1/TRAAK chimeras (A) Exemplar current traces for of BL-1249 responses for K2P2.1(TREK-1) (blue), K2P4.1(TRAAK) (light orange), and chimeras TREK-1/AAK M4-C (light blue), TREK-1/AAK M3-C (green), TREK-1/AAK M2-C (light green), TRAAK/EK-1 M4-C (light green), TRAAK/EK-1 M3-C (green), and TRAAK/EK-1 M2-C (light blue). Black and colored traces show basal and currents with 20 μM BL-1249, respectively. Cartoon schematics show channel portions from K2P2.1(TREK-1) (blue) and K2P4.1(TRAAK) (light orange). (B, C) Dose–response curves for K2P2.1(TREK-1), K2P4.1(TRAAK), and the indicated chimeras. (EC50 = 5.5 ± 1.2 μM, 48 ± 10 μM, 19 ± 3 μM, 28 ± 3 μM, 39 ± 9 μM, 45 ± 2 μM, 18 ± 2 μM, and 28 ± 5 μM for K2P2.1(TREK-1), K2P4.1(TRAAK), TREK-1/AAK M4-C, TREK-1/AAK M3-C, TREK-1/AAK M2-C, TRAAK/EK-1 M4-C, TRAAK/EK-1 M3-C, and TRAAK/EK-1 M2-C, respectively). K2P2.1(TREK-1) and K2P4.1(TRAAK) data are from Figure 1C. Error bars are SEM.
	Figure 5. M2 residues contribute to BL-1249 selectivity between K2P2.1(TREK-1) and K2P4.1(TRAAK). (A) Exemplar current traces for TREK-1/TRAAK M2 (light blue) and TRAAK/TREK-1 M2 (orange) with 20 μM BL-1249 (left). Insets depict M2 helix swap. BL-1249 dose–response curves (right) for K2P2.1(TREK-1) (blue circles), TREK-1/TRAAK M2 (light blue squares), K2P4.1(TRAAK) (light orange circles), and TRAAK/TREK-1 M2 (orange triangles). (EC50 = 5.5 ± 1.2 μM, 26 ± 8 μM, 48 ± 10 μM, and 43 ± 11 μM, respectively). (B) Alignment of K2P2.1(TREK-1) and K2P4.1(TRAAK) M2 sequences. Nonconserved residues are highlighted in yellow. (C) K2P2.1(TREK-1) (PDB 6CQ6)6 structure. Residues that differ from K2P4.1(TRAAK) are highlighted yellow. Panel insets show the environment surrounding the highlighted M2 residues. (D) Exemplar current traces for TREK-1/F172M (pink) and TREK-1/F185L (light blue), with 20 μM BL-1249. BL-1249 dose–response curves for K2P2.1(TREK-1) (blue circles), TREK-1/F172M (pink circles), K2P2.1 (F185L) (light blue squares), and K2P4.1(TRAAK) (light orange circles). (EC50 = 5.5 ± 1.2 μM, 15 ± 2 μM, 27 ± 5 μM, and 48 ± 10 μM, respectively). (E) Exemplar current traces for TRAAK/M134F (orange triangles) and TRAAK/L147F (olive green squares) with 20 μM BL-1249. BL-1249 dose–response curves for K2P2.1(TREK-1) (blue circles), TRAAK/M134F (orange triangles), TRAAK/L147F (olive green squares), and K2P4.1(TRAAK) (light orange circles). (EC50 = 5.5 ± 1.2 μM, 58 ± 34 μM, 27 ± 4 μM, and 48 ± 10 μM, respectively). K2P2.1(TREK-1) and K2P4.1(TRAAK) data are from Figure 1C. Inset compares responses at 35 μM BL-1249: *** indicates p < 0.001 for a one-way ANOVA test; ns indicates no significant difference. Error bars are SEM.
	Figure 6. Structure–activity relationships of BL-1249 analogues. (A) Chemical structures of BL-1249 and analogues. “I” and “II” indicate the substitution sites. (B) Dose–response of K2P2.1(TREK-1) for BL-1249 (black) (from Figure 1C), BL-1249-amide (blue), BL-1249-acid (light blue), and BL-1249-Ph (red) (EC50= 5.5 ± 1.2 μM, 22 ± 8 μM, 44 ± 10 μM, and >100 μM, respectively). K2P2.1(TREK-1) and K2P4.1(TRAAK) data are from Figure 1C. (C–E) Exemplar current traces for K2P2.1(TREK-1) with 25 μM BL-1249-amide (blue), BL-1249-acid (light blue), and BL-1249-Ph (red), respectively (top), chemical structures of BL-1249 analogues (middle), and dose response of K2P2.1(TREK-1) (blue) and K2P4.1(TRAAK) (light orange) (bottom). (F) Chemical structure of BL-1249-tricycle. (G) Exemplar current traces for K2P2.1(TREK-1) and K2P4.1(TRAAK) with 25 μM BL-1249-tricycle (purple). (H) Dose–response of K2P2.1(TREK-1) (blue) and K2P4.1(TRAAK) (light orange) for BL-1249-tricycle (EC50= 34 ± 6 μM and 42 ± 9 μM, respectively). Error bars are SEM.

References [+] :

Alloui, TREK-1, a K+ channel involved in polymodal pain perception. 2006, Pubmed

Alloui, TREK-1, a K+ channel involved in polymodal pain perception. 2006, Pubmed
Andres-Enguix, Functional analysis of missense variants in the TRESK (KCNK18) K channel. 2012, Pubmed
Aryal, Bilayer-Mediated Structural Transitions Control Mechanosensitivity of the TREK-2 K2P Channel. 2017, Pubmed
Bagriantsev, Metabolic and thermal stimuli control K(2P)2.1 (TREK-1) through modular sensory and gating domains. 2012, Pubmed , Xenbase
Bagriantsev, Multiple modalities converge on a common gate to control K2P channel function. 2011, Pubmed
Bagriantsev, A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels. 2013, Pubmed
Bayliss, Emerging roles for two-pore-domain potassium channels and their potential therapeutic impact. 2008, Pubmed
Braun, Differential sensitivity of TREK-1, TREK-2 and TRAAK background potassium channels to the polycationic dye ruthenium red. 2015, Pubmed , Xenbase
Brohawn, Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel. 2014, Pubmed
Brohawn, Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel. 2012, Pubmed
Brohawn, Domain-swapped chain connectivity and gated membrane access in a Fab-mediated crystal of the human TRAAK K+ channel. 2013, Pubmed
Chemin, Up- and down-regulation of the mechano-gated K(2P) channel TREK-1 by PIP (2) and other membrane phospholipids. 2007, Pubmed
Chemin, A phospholipid sensor controls mechanogating of the K+ channel TREK-1. 2005, Pubmed
Chokshi, Breathing Stimulant Compounds Inhibit TASK-3 Potassium Channel Function Likely by Binding at a Common Site in the Channel Pore. 2015, Pubmed
Coburn, Discovery of a pharmacologically active antagonist of the two-pore-domain potassium channel K2P9.1 (TASK-3). 2012, Pubmed
Cohen, A novel mechanism for human K2P2.1 channel gating. Facilitation of C-type gating by protonation of extracellular histidine residues. 2008, Pubmed , Xenbase
Dadi, Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx. 2017, Pubmed
Decher, Sodium permeable and "hypersensitive" TREK-1 channels cause ventricular tachycardia. 2017, Pubmed
Devilliers, Activation of TREK-1 by morphine results in analgesia without adverse side effects. 2013, Pubmed
Dong, K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac. 2015, Pubmed
Enyedi, Molecular background of leak K+ currents: two-pore domain potassium channels. 2010, Pubmed
Feliciangeli, The family of K2P channels: salient structural and functional properties. 2015, Pubmed
Gibson, Enzymatic assembly of DNA molecules up to several hundred kilobases. 2009, Pubmed
Heurteaux, TREK-1, a K+ channel involved in neuroprotection and general anesthesia. 2004, Pubmed
Heurteaux, Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype. 2006, Pubmed
Honoré, An intracellular proton sensor commands lipid- and mechano-gating of the K(+) channel TREK-1. 2002, Pubmed
Kennard, Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine. 2005, Pubmed
Lafrenière, A dominant-negative mutation in the TRESK potassium channel is linked to familial migraine with aura. 2010, Pubmed
Laigle, Deletion of TRAAK potassium channel affects brain metabolism and protects against ischemia. 2012, Pubmed
Lazarenko, Anesthetic activation of central respiratory chemoreceptor neurons involves inhibition of a THIK-1-like background K(+) current. 2010, Pubmed
Lolicato, Transmembrane helix straightening and buckling underlies activation of mechanosensitive and thermosensitive K(2P) channels. 2014, Pubmed , Xenbase
Lolicato, K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site. 2017, Pubmed , Xenbase
Lotshaw, Biophysical, pharmacological, and functional characteristics of cloned and native mammalian two-pore domain K+ channels. 2007, Pubmed
Loucif, GI-530159, a novel, selective, mechanosensitive two-pore-domain potassium (K2P ) channel opener, reduces rat dorsal root ganglion neuron excitability. 2018, Pubmed
Luethy, Halogenated Ether, Alcohol, and Alkane Anesthetics Activate TASK-3 Tandem Pore Potassium Channels Likely through a Common Mechanism. 2017, Pubmed
Maingret, Molecular basis of the voltage-dependent gating of TREK-1, a mechano-sensitive K(+) channel. 2002, Pubmed
Mathie, Two-pore domain potassium channels: potential therapeutic targets for the treatment of pain. 2015, Pubmed
McClenaghan, Polymodal activation of the TREK-2 K2P channel produces structurally distinct open states. 2016, Pubmed , Xenbase
Miller, Crystal structure of the human two-pore domain potassium channel K2P1. 2012, Pubmed
Monteillier, Investigation of the structure activity relationship of flufenamic acid derivatives at the human TRESK channel K2P18.1. 2016, Pubmed
Murbartián, Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels. 2005, Pubmed
Patel, Inhalational anesthetics activate two-pore-domain background K+ channels. 1999, Pubmed
Patel, A mammalian two pore domain mechano-gated S-like K+ channel. 1998, Pubmed
Piechotta, The pore structure and gating mechanism of K2P channels. 2011, Pubmed
Renigunta, Much more than a leak: structure and function of K₂p-channels. 2015, Pubmed
Rice, EMBOSS: the European Molecular Biology Open Software Suite. 2000, Pubmed
Rodrigues, Synthesis and structure-activity relationship study of substituted caffeate esters as antinociceptive agents modulating the TREK-1 channel. 2014, Pubmed , Xenbase
Schewe, A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels. 2016, Pubmed
Su, Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels. 2016, Pubmed
Takahira, Fenamates and diltiazem modulate lipid-sensitive mechano-gated 2P domain K(+) channels. 2005, Pubmed
Tertyshnikova, BL-1249 [(5,6,7,8-tetrahydro-naphthalen-1-yl)-[2-(1H-tetrazol-5-yl)-phenyl]-amine]: a putative potassium channel opener with bladder-relaxant properties. 2005, Pubmed
Thümmler, Antipsychotics inhibit TREK but not TRAAK channels. 2007, Pubmed
Veale, Influence of the N terminus on the biophysical properties and pharmacology of TREK1 potassium channels. 2014, Pubmed
Vivier, Development of the First Two-Pore Domain Potassium Channel TWIK-Related K+ Channel 1-Selective Agonist Possessing in Vivo Antinociceptive Activity. 2017, Pubmed
Vivier, Perspectives on the Two-Pore Domain Potassium Channel TREK-1 (TWIK-Related K(+) Channel 1). A Novel Therapeutic Target? 2016, Pubmed
Wu, Involvement of TREK-1 activity in astrocyte function and neuroprotection under simulated ischemia conditions. 2013, Pubmed
Yekkirala, Breaking barriers to novel analgesic drug development. 2017, Pubmed
Zilberberg, KCNKØ: opening and closing the 2-P-domain potassium leak channel entails "C-type" gating of the outer pore. 2001, Pubmed , Xenbase