XB-ART-54560
Nature
2017 Jul 20;5477663:364-368. doi: 10.1038/nature22988.
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K 2P 2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site.
Lolicato M
,
Arrigoni C
,
Mori T
,
Sekioka Y
,
Bryant C
,
Clark KA
,
Minor DL
.
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Polymodal thermo- and mechanosensitive two-pore domain potassium (K 2P ) channels of the TREK subfamily generate 'leak' currents that regulate neuronal excitability, respond to lipids, temperature and mechanical stretch, and influence pain, temperature perception and anaesthetic responses. These dimeric voltage-gated ion channel (VGIC) superfamily members have a unique topology comprising two pore-forming regions per subunit. In contrast to other potassium channels, K 2P channels use a selectivity filter 'C-type' gate as the principal gating site. Despite recent advances, poor pharmacological profiles of K 2P channels limit mechanistic and biological studies. Here we describe a class of small-molecule TREK activators that directly stimulate the C-type gate by acting as molecular wedges that restrict interdomain interface movement behind the selectivity filter. Structures of K 2P 2.1 (also known as TREK-1) alone and with two selective K 2P 2.1 (TREK-1) and K 2P 10.1 (TREK-2) activators-an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402-define a cryptic binding pocket unlike other ion channel small-molecule binding sites and, together with functional studies, identify a cation-π interaction that controls selectivity. Together, our data reveal a druggable K 2P site that stabilizes the C-type gate 'leak mode' and provide direct evidence for K 2P selectivity filter gating.
???displayArticle.pubmedLink??? 28693035
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???displayArticle.grants??? [+]
R01 MH093603 NIMH NIH HHS
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Extended Data Figure 2. K2P2.1(TREK-1)cryst modulator binding pocket densities and K2P2.1(TREK-1)cryst functional propertiesa–e, Exemplar electron densities for the modulator binding pockets. a–c, 2Fo-Fc densities (blue) for a, K2P2.1(TREK-1):ML335 (1.5 σ), b, K2P2.1(TREK-1):ML402 (1.0 σ), and c, K2P 2.1(TREK-1) (1.0 σ). Offset angle for the cation-π interactions for Lys271:ML335 and Lys271:ML402 is shown and adopts an oblique geometry common to cation-π interactions35,36. d, and e, Fo-Fc densities (hotpink, 3.0 σ) for d, K2P2.1(TREK-1):ML335 and e, K2P2.1(TREK-1):ML402. Final models are shown in all panels and select residues are shown and labeled. f–h, Exemplar current traces for f, K2P2.1 (TREK-1)cryst (black) with 40 μM ML335 (yellow orange), g, K2P2.1 (TREK-1)cryst (black) with 80 μM ML402 (green), and h, K2P2.1 (TREK-1) G137I (black) with 80 μM ML335 (blue). i, Dose response curves for K2P2.1(TREK-1):ML335 (black), EC50=14.3 ± 2.7 μM (n≥5); K2P2.1 (TREK-1)cryst:ML335, EC50 = 10.5 ± 2.7 μM (n≥3) (yellow orange) K2P2.1 (TREK-1)cryst:ML402, EC50 = 14.9 ± 1.6 μM (n≥3); and K2P2.1(TREK-1) G137I:ML335 (blue ). |
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Extended Data Figure 3. Comparison of K2P modulator and VGIC antagonist sitesa, Superposition of the K2P2.1(TREK-1):ML335 complex (smudge and light orange) with the BacNaV ‘pore-only’ NaVMs structure18 (magenta and warm pink). Bromine site (Br) from labeled sodium channel antagonists is shown as a firebrick sphere. b, Superposition of the pore domains of the K2P2.1(TREK-1):ML335 complex (smudge and light orange) with the pore domain of the BacNaV CaVAb (5KMD) bound to the inhibitor amlodipine (AMLOD)19, a site normally occupied by lipid19,37. Select residues of the K2P modulator pocket are shown as sticks and are labeled. CaVAb subunits are colored cyan, marine, slate, and dark blue. ML335 (yellow) and amlodipine (cyan) are shown in space filling representation. |
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Extended Data Figure 4. K2P modulator pocket structure and conservationDetails of a, ML335, and b, ML402 interactions with K2P2.1(TREK-1). c and d, Representative K2P channel sequence comparisons for the c, M4 face and d, P1 face. Purple bar and orange shading on sequence identifiers denotes the thermo- and mechanosensitive K2P2.1(TREK-1) subfamily. Protein secondary structure is marked above the sequences. Selectivity filter region is in red. Residues involved in direct interactions with ML335 and ML402 are orange and marked with an orange asterisk. Conserved positions are highlighted. K2P2.1(TREK-1) is the mouse protein used for this study. K2P2.1H(TREK-1) is the human homolog. All other K2P sequences are human origin. Sequences and identifiers are as follows: K2P2.1(TREK-1) NP_034737.2; K2P2.1H(TREK-1), NP_001017424.1; K2P10.1(TREK-2), NP_612190.1; K2P4.1(TRAAK), NP_001304019.1; K2P3.1(TASK-1), NP_002237.1; K2P9.1(TASK-3), NP_001269463.1; K2P5.1(TASK-2), NP_003731.1; K2P1.1(TWIK-1), NP_002236.103812.2; K2P6.1(TWIK-2), NP_004823.1; K2P7.1(KCNK7), AAI03812.2; K2P16.1(TALK-1), NP_001128577.1; K2P17.1(TALK-2), NP_113648.2; K2P12.1(THIK-2), NP_071338.1; K2P12.1(THIK-1), NP_071337.2; K2P15.1(TASK-5), NP_071753.2; and K2P18.1(TRESK), NP_862823.1. ‘●’ in K2P16.1(TALK-1) sequence in ‘c’ denotes the following, non-conserved sequence that was removed to avoid a long alignment gap: NFITPSGLLPSQEPFQTPHGKPESQQIP. |
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Extended Data Figure 5. K2P structure comparisonsK2P modulator pocket views colored by B-factor for a, K2P2.1(TREK-1), b, K2P2.1(TREK-1):ML335, and c, K2P2.1(TREK-1):ML402. Bars show B-factor scale. |
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Extended Data Figure 6. K2P structure comparisonsa, Backbone atom superposition of K2P2.1(TREK-1) (smudge, up), K2P2.1(TREK-1):ML335 (yellow, up), K2P2.1(TREK-1):ML402 (cyan, up), K2P10.1(TREK-2) (4BW5) (pink, up)6, K2P10.1(TREK-2) (4XDJ) (magenta, down)6, K2P10.1(TREK-2):Norfluoxetine (4XDK)(violet purple, down)6, K2P4.1(TRAAK) (4I9W) (limon, up)13, K2P4.1(TRAAK) G124I (4RUE) (marine, down)15, and K2P4.1(TRAAK) W262S (4RUF) (lime, down)15. ‘up’ or ‘down’ denotes M4 conformation. Selectivity filter ions for K2P2.1(TREK-1) (smudge), K2P2.1(TREK-1):ML335 (yellow), and K2P2.1(TREK-1):ML402 (cyan) are shown as spheres. ML335 and ML402 are shown in sticks. Select channel elements are labeled. b–e, Superposition showing b, K2P2.1(TREK-1) chain A (smudge) and chain B (light orange). Sites of GOF mutations, G137I (orange)7, and Trp2758, are indicated. c, K2P2.1(TREK-1):ML335 chain A (pink) and chain B (deep salmon), d, K2P2.1(TREK-1):ML402 chain A (cyan) and chain B (deep teal). e, K2P2.1(TREK-1) chain A (smudge) and chain B (light orange), K2P10.1(TREK-2) (4BW5) (pink)6, K2P10.1(TREK-2) (4XDJ) (magenta)6, K2P10.1(TREK-2):Norfluoxetine (4XDK)(violet purple)6. f, K2P2.1(TREK-1):ML335 (deep salmon), K2P4.1(TRAAK) (4I9W) (limon)13, K2P4.1(TRAAK) G124I (4RUE) (marine)15, K2P4.1(TRAAK) W262S (4RUF) (lime)15. G124I from K2P4.1(TRAAK) G124I15 is shown in sticks. In b–f, Phe134, His126, Lys271, Trp275, their equivalents in K2P10.1(TREK-2), K2P4.1(TRAAK), and K2P4.1(TRAAK) G124I, are shown in sticks. ML335, ‘c’, and ML402, ‘d’, are shown as sticks. |
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Extended Data Figure 7. K2P activator responsesExemplar current traces for a, K2P10.1(TREK-2) (black) with 20 μM ML335 (yellow orange) and b, K2P10.1(TREK-2) (black) with 20 μM ML402 (cyan). c, Dose response curves for K2P10.1(TREK-2) with ML335 (EC50 = 5.2 ± 0.5 μM (n>3)) (yellow orange) and ML402 (EC50 = 5.9 ± 1.6 μM (n≥4)(cyan). Exemplar current traces for d, K2P2.1(TREK-1) K271Q (black) and with 20 μM ML335 (purple). e, K2P4.1(TREK-1) Q258K (black) and with 50 μM ML335 (orange). f, K2P2.1(TREK-1) K271Q (black) and with 50 μM ML402 (purple). g, K2P4.1(TREK-1) Q258K (black) and with 50 μM ML402 (orange). Currents were evoked from Xenopus oocytes expressing the indicated channels from a −80 mV holding potential followed by a 500 ms ramp from −150 mV to +50 mV. Compound structures are shown. h, and i, Exemplar current traces for HEK293 cell inside-out patches expressing h, K2P2.1(TREK-1) and i, K2P2.1(TREK-1) K271Q to stimulation by 10 μM arachidonic acid (AA) (green). j, Current potentiation measured in HEK cells at 0 mV in response to 10μM AA for K2P2.1(TREK-1) (n=5) and K2P2.1(TREK-1) K271Q (n=4). k, Current potentiation measured in Xenopus oocytes at 0 mV for K2P4.1(TRAAK) (white), K2P2.1(TREK-1) (black), K2P4.1(TRAAK) Q258K (cyan) and K2P2.1(TREK-1) K271Q (gray) in response to 10 μM BL-1249, 30 μM ML67-33, and 20 μM ML335. For all experiments (n≥4). Data are mean ± SEM. |
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Extended Data Figure 8. K2P channel patch clamp recordingsa, Dose response for K2P2.1(TREK-1) to ML335 (black circles) and ML402 (open circles) measured in HEK293 cells by whole cell patch clamp. EC50 values are 5.2 ± 0.8 μM and 5.9 ± 1.6 μM for ML335 and ML402, respectively (n≥3). b, and c, Representative current traces and voltage-current relationship from HEK293 inside-out patches for expressing b, K2P4.1(TRAAK) and c, K2P4.1(TRAAK) G124I elicited by a 350 ms-voltage step protocol from −100 mV to +100 mV in 150 mM K+[out]/150 mM Rb+[in]. d, Rectification coefficients (I+100mV/I-100mV) calculated from n≥3 current recordings obtained from the same conditions in ‘b’ and ‘c’. |
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Figure 1. K2P2.1(TREK-1) structuresa, K2P2.1(TREK-1)cryst cartoon (smudge and light orange) smudge subunit extracellular cap domain (CAP), M1–M4 transmembrane helices, C-tail, and V321 are labeled. b, K2P modulator pocket cutaway. Cutouts display ML335 and ML402 Fo-Fc densities (3.0σ). ML335, ML402, and Selectivity Filter 1 (SF1) are sticks. c, and d, Extracellular views excluding the CAP domain of c, ML335 and d, ML402 binding sites. ML335 and ML402 are space filling. Selectivity filter sidechains are sticks. e, Wire representation comparing K2P2.1(TREK-1):ML335 (smudge and light orange) and K2P10.1(TREK-2):norfluoxetine6 (light pink and magenta) binding sites. K2P2.1(TREK-1) P1 and P2 are cylinders. ML335 (yellow) and norfluoxetine (NorFx) (red) are space filling. In all panels select residues and channel elements are indicated, and where present, grey lines indicate the membrane. |
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Figure 2. K2P2.1(TREK-1) activator interactionsCartoon diagram of a, K2P2.1(TREK-1):ML335 and b, K2P2.1(TREK-1):ML402 interactions. Electrostatic and hydrogen bond interactions are black. Cation-π and π-π interactions are grey. c, and d, comparison of K2P2.1(TREK-1) (smudge) with c, K2P2.1(TREK-1):ML335 (deep salmon) and d, K2P2.1(TREK-1):ML402 (cyan). |
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Figure 3. K2P2.1(TREK-1) C-tail and lipid binding sitesa, K2P2.1(TREK-1) C-tail. Positively charged residues are blue. S300A and E306A, a site having slight distortion from helical geometry, are magenta. b, K2P2.1(TREK-1) Electrostatic surface potential. Orange box highlights M1/M2/M4 junction. Cytoplasmic view (left) indicates C-tail positively charged patch. c, Lipids L1, L2, and L3 (cyan and white) shown as space filling. ML335 (yellow) is indicated. Insets show lipid binding pocket details. |
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Figure 4. K2P2.1(TREK-1) activator functionExemplar current traces for a, K2P2.1(TREK-1) (black) with 20 μM ML335 (purple). b, K2P4.1(TRAAK) (black) with 50 μM ML335 (orange). c, ML335 dose response curves for K2P2.1(TREK-1) (black), EC50=14.3 ± 2.7 μM (n≥5); K2P2.1(TREK-1) K271Q (blue filled circles); K2P4.1(TRAAK) (orange); K2P4.1(TRAAK) Q258K (green) EC50=16.2 ± 3.0 μM (n≥4); and ML335a: K2P2.1(TREK-1) (black open triangles). Exemplar current traces for d, K2P2.1(TREK-1) (black) with 20 μM ML402 (purple). e, K2P4.1(TRAAK) (black) with 50 μM ML335 (orange). f, ML402 dose response curves for K2P2.1(TREK-1) (black), EC50=13.7 ± 7.0 μM (n≥3); K2P2.1(TREK-1) K271Q (blue); K2P2.1(TREK-1) (blue); K2P4.1(TRAAK) (orange); K2P4.1(TRAAK) Q258K (green) EC50=13.6 ± 1.5 μM (n≥3). g–j, Exemplar current traces and voltage-current relationships for indicated K2Ps in HEK293 inside-out patches in 150 mM K+[out]/150 mM Rb+[in] for g, K2P2.1(TREK-1), h, K2P2.1(TREK-1) with 5 μM ML335, I, K2P2.1(TREK-1) with 5 μM ML402, and j, K2P2.1(TREK-1) G137I. k, Rectification coefficients (I+100mV/I–100mV) from recordings (n≥3) made in ‘g–j’. l, TREK activation model. Grey lines indicate mobile P1 (tan) and M4 (blue). C-type activators (orange), stabilize the selectivity filter and channel ‘leak mode’. Potassium ions are purple. Gap in arrows indicates current flow intensity. Membrane is grey. |
References [+] :
Adams,
PHENIX: a comprehensive Python-based system for macromolecular structure solution.
2010, Pubmed
Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed
Bagnéris, Prokaryotic NavMs channel as a structural and functional model for eukaryotic sodium channel antagonism. 2014, Pubmed
Bagriantsev, A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels. 2013, 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
Brohawn, Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel. 2012, Pubmed
Brohawn, Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel. 2014, Pubmed
Brohawn, Domain-swapped chain connectivity and gated membrane access in a Fab-mediated crystal of the human TRAAK K+ channel. 2013, Pubmed
Chemin, A phospholipid sensor controls mechanogating of the K+ channel TREK-1. 2005, Pubmed
Chemin, Up- and down-regulation of the mechano-gated K(2P) channel TREK-1 by PIP (2) and other membrane phospholipids. 2007, Pubmed
Chen, MolProbity: all-atom structure validation for macromolecular crystallography. 2010, 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
Collaborative Computational Project, Number 4, The CCP4 suite: programs for protein crystallography. 1994, Pubmed
Crowley, Cation-pi interactions in protein-protein interfaces. 2005, Pubmed
Devilliers, Activation of TREK-1 by morphine results in analgesia without adverse side effects. 2013, Pubmed
Diederichs, Better models by discarding data? 2013, Pubmed
Dong, K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac. 2015, Pubmed
Drew, GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. 2008, Pubmed
Eguchi, Studies on hypotensive agents. Synthesis of 1-substituted 3-(2-chlorophenyl)-6-ethoxycarbonyl-5,7-dimethyl-2,4(1H,3H)-quinazoline diones. 1991, Pubmed
Emsley, Coot: model-building tools for molecular graphics. 2004, Pubmed
Evans, How good are my data and what is the resolution? 2013, Pubmed
Feliciangeli, The family of K2P channels: salient structural and functional properties. 2015, Pubmed
Fink, Cloning, functional expression and brain localization of a novel unconventional outward rectifier K+ channel. 1996, Pubmed , Xenbase
Guizouarn, Multiple transport functions of a red blood cell anion exchanger, tAE1: its role in cell volume regulation. 2001, Pubmed , Xenbase
Hardy, Searching for new allosteric sites in enzymes. 2004, Pubmed
Hibbs, Principles of activation and permeation in an anion-selective Cys-loop receptor. 2011, Pubmed
Honoré, An intracellular proton sensor commands lipid- and mechano-gating of the K(+) channel TREK-1. 2002, Pubmed
Kabsch, XDS. 2010, Pubmed
Karplus, Linking crystallographic model and data quality. 2012, Pubmed
Kawate, Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. 2006, Pubmed
Kirchhofer, Modulation of protein properties in living cells using nanobodies. 2010, Pubmed
Lolicato, Transmembrane helix straightening and buckling underlies activation of mechanosensitive and thermosensitive K(2P) channels. 2014, Pubmed , Xenbase
Maingret, TREK-1 is a heat-activated background K(+) channel. 2000, Pubmed , Xenbase
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
Murbartián, Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels. 2005, Pubmed
Newstead, High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae. 2007, Pubmed
Pascale, New N-(phenoxydecyl)phthalimide derivatives displaying potent inhibition activity towards alpha-glucosidase. 2010, Pubmed
Patel, A mammalian two pore domain mechano-gated S-like K+ channel. 1998, Pubmed
Payandeh, Bacterial voltage-gated sodium channels (BacNa(V)s) from the soil, sea, and salt lakes enlighten molecular mechanisms of electrical signaling and pharmacology in the brain and heart. 2015, Pubmed
Piechotta, The pore structure and gating mechanism of K2P channels. 2011, Pubmed
Rapp, Cation-π interactions of methylated ammonium ions: a quantum mechanical study. 2014, Pubmed
Sandoz, Extracellular acidification exerts opposite actions on TREK1 and TREK2 potassium channels via a single conserved histidine residue. 2009, Pubmed , Xenbase
Schewe, A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels. 2016, Pubmed
Shaya, Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins. 2011, Pubmed
Su, Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels. 2016, Pubmed
Tang, Structural basis for inhibition of a voltage-gated Ca2+ channel by Ca2+ antagonist drugs. 2016, 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
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
Yelshanskaya, Structural Bases of Noncompetitive Inhibition of AMPA-Subtype Ionotropic Glutamate Receptors by Antiepileptic Drugs. 2016, Pubmed
Zhou, Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution. 2001, Pubmed