Human two-pore domain potassium ion channel TREK2 (K2P10.1)

Target ID:

KCNK10C

Deposition Date:

Sunday 30th June 2013

Families:

Membrane proteins

Structure type:

apo

Download Coordinates:

4BW5

Authors:

A.C.W.PIKE,Y.Y.DONG,L.DONG,A.QUIGLEY,L.SHRESTHA, S.MUKHOPADHYAY,C.STRAIN-DAMERELL,S.GOUBIN,M.GRIEBEN, C.A.SHINTRE,A.MACKENZIE,M.VOLLMAR,F.VON DELFT, C.H.ARROWSMITH,A.M.EDWARDS,C.BOUNTRA,N.BURGESS-BROWN, E.P.CARPENTER

Site:

Oxford

4BW5

tabs

Structure Details

Read the article in Science (Open Access): K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac
" target="_blank">Abstract -- " target="_blank">Reprint -- Full text

KCNK10, also known as TREK-2, is a two-pore domain (K2P) potassium channel, found in the plasma membranes of neuronal cells. It provides a background leak current and is regulated by diverse stimuli including physical factors such as mechanical stretch, voltage and temperature, natural ligands such as polyunsaturated acids arachidonic acid and both intra- and extracellular pH [1-4]. Its activity can also be modulated by a variety of pharmacological agents such as volatile anesthetics [5], neuroprotective drugs [6] and antidepressants such as fluoxetine (Prozac) [7, 8] in vitro. Such diverse regulation allows these channels to couple cellular electrical activity to a remarkable variety of signaling pathways and consequently they represent important targets in pharmacology [9-11].

Here we present a structure of TREK-2 in the up state to 3.4Å resolution. TREK-2 has a classic K2P channel fold formed from a disulphide-linked dimer. Each monomer has two pore forming domains, giving an overall pseudo four-fold symmetry. K2P channels have four transmembrane helices (M1 to M4), with cytoplasmic N- and C-termini. The M1 helix is connected at an extracellular cap domain with two helices, a disulphide at the apex and a domain swap. This is followed by a pore helix and pore filter sequence, helices M2 and M3, a second pore helix and pore filter sequence, followed by the M4 helix. The pore filter provides a series of carbonyls which coordinate the potassium ions. In this structure we observed four potassium ions in the filter.

In the 4BW5 structure we observed an upwards movement of the M2 - M4 helices on the cytoplasmic side of the membrane, relative to the arrangement seen in the 'down' state. This structure could represent a stretch activated state of TREK-2, where mechanical force applied to the lipid membrane leads to an upward movement in transmembrane helices M2-M4 into this up conformation. In this conformation, the C-terminal portion of helix M4 from one protomer packs tightly against the M2 helix from the adjacent protomer. As a result, no side fenestrations are observed between the transmembrane helices and the inner vestibule.

Materials and Methods

KCNK10C

PDB Code: 4BW5

Material and Methods

Entry Clone Source: 4BW5

Entry Clone Accession: IMAGE: 30915621

SGC Construct ID: KCNK10C-c011

GI number: 54207

Vector: pFB-CT10HF-LIC

Amplified construct sequence:

ATGGGCTTGCAGACCGTCATGAAGTG
GAAGACGGTGGTTGCCATCTTTGTGG
TTGTGGTGGTCTACCTTGTCACTGGC
GGTCTTGTCTTCCGGGCATTGGAGCA
GCCCTTTGAGAGCAGCCAGAAGAATA
CCATCGCCTTGGAGAAGGCGGAATTC
CTGCGGGATCATGTCTGTGTGAGCCC
CCAGGAGCTGGAGACGTTGATCCAGC
ATGCTCTTGATGCTGACAATGCGGGA
GTCAGTCCAATAGGAAACTCTTCCAA
CAACAGCAGCCACTGGGACCTCGGCA
GTGCCTTTTTCTTTGCTGGAACTGTC
ATTACGACCATAGGGTATGGGAATAT
TGCTCCGAGCACTGAAGGAGGCAAAA
TCTTTTGTATTTTATATGCCATCTTT
GGAATTCCACTCTTTGGTTTCTTATT
GGCTGGAATTGGAGACCAACTTGGAA
CCATCTTTGGGAAAAGCATTGCAAGA
GTGGAGAAGGTCTTTCGAAAAAAGCA
AGTGAGTCAGACCAAGATCCGGGTCA
TCTCAACCATCCTGTTCATCTTGGCC
GGCTGCATTGTGTTTGTGACGATCCC
TGCTGTCATCTTTAAGTACATCGAGG
GCTGGACGGCCTTGGAGTCCATTTAC
TTTGTGGTGGTCACTCTGACCACGGT
GGGCTTTGGTGATTTTGTGGCAGGGG
GAAACGCTGGCATCAATTATCGGGAG
TGGTATAAGCCCCTAGTGTGGTTTTG
GATCCTTGTTGGCCTTGCCTACTTTG
CAGCTGTCCTCAGTATGATCGGAGAT
TGGCTACGGGTTCTGTCCAAAAAGAC
AAAAGAAGAGGTGGGTGAAGCAGAGA
ACCTCTACTTCCAATCGCACCATCAT
CACCATCACCATCACCACCATGATTA
CAAGGATGACGACGATAAGTGA

Expressed sequence (small letters refer to tag sequence):

MGLQTVMKWKTVVAIFVVVVVYLVTG
GLVFRALEQPFESSQKNTIALEKAEF
LRDHVCVSPQELETLIQHALDADNAG
VSPIGNSSNNSSHWDLGSAFFFAGTV
ITTIGYGNIAPSTEGGKIFCILYAIF
GIPLFGFLLAGIGDQLGTIFGKSIAR
VEKVFRKKQVSQTKIRVISTILFILA
GCIVFVTIPAVIFKYIEGWTALESIY
FVVVTLTTVGFGDFVAGGNAGINYRE
WYKPLVWFWILVGLAYFAAVLSMIGD
WLRVLSKKTKEEVGEAENLYFQ^SHH
HHHHHHHHDYKDDDDK ^ TEV cleavage site

Tags and additions: C-terminal, TEV cleavable decahistidine / FLAG tag.

Host: Spodoptera frugiperda (SF9) insect cells

Growth medium, induction protocol:

Insect cells with a density of 2x106 per litre of cell culture in SF900 medium (Invitrogen) were infected with recombinant baculovirus (5ml P2 virus per litre cell culture) and incubated for 65 hours. Cells were harvested by centrifugation and the pellet was flash frozen in liquid N2.

Extraction method: Frozen pellets were thawed and re-suspended in extraction buffer supplemented with 1% OGNG/ 0.1% CHS and incubated at 4°C on a tube rotator. The extracted protein was separated from insoluble membranes by centrifugation and collected for purification.

Extraction buffer:

50 mM HEPES, pH 7.5; 200 mM KCl;

Column 1: Co-affinity. Cobalt Talon (Clontech), 1 ml of 50 % slurry/ 1L of cells in 1.5 x 10 cm column, washed with extraction/ wash buffer.

Buffers:

Wash buffer: 50 mM HEPES, pH 7.5; 200 mM KCl; 20 mM imidazole; detergent at 3x CMC. Elution buffer: 50 mM HEPES, pH 7.5; 200 mM KCl; 250 mM imidazole; detergent at 3xCMC

Procedure:

The solubilised membrane protein was batch bound to Cobalt Talon resin equilibrated with extraction buffer at 4°C for one hour and subsequently poured into a gravity flow glass column. The column was then washed with 30CV of wash buffer followed by elution in 1CV fractions until all the protein was eluted.

Desalting:

IMAC purified protein was loaded onto PD10 columns (GE healthcare) pre- equilibrated in extraction buffer supplemented with detergent at 3x CMC, and eluted with the same buffer.

Enzymatic treatment: TEV protease (1:10, TEV:protein) and PNGaseF (1:20, PNGaseF:protein) was added overnight at 4°C to desalted protein. The enzymes were removed and tag cleaved protein was collected by binding to Cobalt talon and collecting the flow-through.

Column 2:Size Exclusion Chromatography. Superose 6 (GE healthcare).

SEC Buffer: 20 mM HEPES pH 7.5, 200 mM KCl, detergent at 2xCMC

Procedure: The protein was concentrated and applied to a Superose 6 gel filtration column equilibrated with gel filtration buffer using an AKTA purifier system.

Protein concentration: Protein was concentrated to ~20mg/ml using a 30kDa cut-off concentrator and back diluted to 9-11mg/ml.

Crystallization: Crystals grew at 4°C from sitting drops (195nl) comprising 130nl of concentrated protein and 65nl of reservoir equilibrated against 20?l of reservoir solution (0.1M HEPES pH 8.0, 1mM cadmium chloride, 2% (w/v) benzamidine, 31% (v/v) PEG400).

Cryocooling: Crystals were cryo-cooled by slow transfer into artificial mother liquor containing 0.2 M potassium chloride, 2 % (w/v) benzamidine, 0.2 % OGNG / 0.02 % CHS, 0.1 M HEPES, pH 8.0, 35 % (v/v) PEG400 followed by plunging into liquid nitrogen. Diffraction of the crystals was improved by slowly increasing the concentration of PEG400 to above 35 % (v/v) which resulted in a 10 % shrinkage in unit cell volume compared to untreated crystals.

Data Collection: Data were collected at 100K on the microfocus beamline I24 at Diamond Light Source using helical / straight line scans with a beamsize of 10?m x 10?m (wxh). Diffraction was anisotropic with spots to 3.2Å in the best direction and 4.1Å in the worst direction.

Structure Solution: Structure was phased by molecular replacement using the coordinates for the transmembrane regions of TRAAK (3UM7) and TWIK1 (3UKM). The spacegroup is P21 with two channel homodimers per asymmetric unit. Refinement was carried out using BUSTER using NCS and TLS restraints using all data to 3.2Å.

Resolution: 3.4Å

References

1. Enyedi, P. and G. Czirjak, Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol Rev, 2010. 90(2): p. 559-605.

2. Cohen, A., Y. Ben-Abu, and N. Zilberberg, Gating the pore of potassium leak channels. Eur Biophys J, 2009. 39(1): p. 61-73.

3. Dedman, A., et al., The mechano-gated K(2P) channel TREK-1. Eur Biophys J, 2009. 38(3): p. 293-303.

4. Noel, J., G. Sandoz, and F. Lesage, Molecular regulations governing TREK and TRAAK channel functions. Channels (Austin), 2011. 5(5): p. 402-9.

5. Patel, A.J. and E. Honore, 2P domain K+ channels: novel pharmacological targets for volatile general anesthetics. Adv Exp Med Biol, 2003. 536: p. 9-23.

6. Cadaveira-Mosquera, A., et al., Activation of TREK currents by the neuroprotective agent riluzole in mouse sympathetic neurons. J Neurosci, 2011. 31(4): p. 1375-85.

7. Heurteaux, C., et al., Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype. Nat Neurosci, 2006. 9(9): p. 1134-41.

8. Kennard, L.E., et al., Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine. Br J Pharmacol, 2005. 144(6): p. 821-9.

9. Bayliss, D.A. and P.Q. Barrett, Emerging roles for two-pore-domain potassium channels and their potential therapeutic impact. Trends Pharmacol Sci, 2008. 29(11): p. 566-75.

10. Es-Salah-Lamoureux, Z., D.F. Steele, and D. Fedida, Research into the therapeutic roles of two-pore-domain potassium channels. Trends Pharmacol Sci, 2010. 31(12): p. 587-95.

11. Mathie, A. and E.L. Veale, Therapeutic potential of neuronal two-pore domain potassium-channel modulators. Curr Opin Investig Drugs, 2007. 8(7): p. 555-62.