Human potassium channel, subfamily K, member 10 [isoform 3]

Target ID:

KCNK10C

Deposition Date:

Friday 19th December 2014

Families:

Membrane proteins

Structure type:

protein-protein

Download Coordinates:

4XDJ

Authors:

Pike, A.C.W., Dong, Y.Y., Mackenzie, A., Mukhopadhyay, S., Arrowsmith, C.H., Edwards, A.M., Bountra, C., Burgess-Brown, N.A., Carpenter, E.P.

Site:

Oxford

4XDJ

tabs

Structure Details

KCNK10, also known as TREK-2, is a two-pore domain (K2P) potassium channel that is important in the regulating of the plasma membrane resting potential. KCNK10 is regulated by diverse array of stimuli including physical factors such as mechanical stretch, voltage and temperature, natural ligands such as polyunsaturated acids arachidonic acid, as well as 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]. 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 the structure of TREK-2 in the down state to 3.9Å resolution. In this conformation, the C-terminal M4 transmembrane helix moves away from the adjacent M2 helix towards the cytoplasmic side of the membrane. As a consequence, a side fenestration opens up between M2 and M4 that connects the inner vestibule to the membrane bilayer. In this structure, the side fenestrations are filled by elongated electron density, assumed to be an alkyl chain derived from a co-purified lipid. This putative lipid binds across the uppermost portion of inner vestibule across the ion conductance pathway. Interestingly, only 3 well defined K+ ions occupy the selectivity filter in this 'M4-down' structure.

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 (150nl) comprising 90nl of concentrated protein and 60nl of reservoir of solution containing 0.1 M sodium cacodylate, pH6.5, 3% (v/v) methanol, 22% (w/v) PEG1500

Cryocooling: Crystals were equilibrated against solutions containing increasing concentrations of PEG1500 (22-35 %) prior to cryo-cooling in liquid nitrogen

Data Collection: Data were collected at 100K from a single crystal on the microfocus beamline I24 at Diamond Light Source using helical / straight line scans with a beamsize of 10?m x 30?m (wxh).

Structure Solution: The structure was phased by molecular replacement using the coordinates of the 'M4-up' conformation of KCNK10C (4BW5). Crystals belong to spacegroup P21 with two channel homodimers per asymmetric unit. Structure refined using BUSTER with NCS and TLS restraints. All data to 3.8Å was used in refinement although, due to anisotropy, nominal resolution quoted below is based on Mn I/sI>2 criteria.

Resolution: 3.9Å

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.