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TASK-1
Protein Expression and Purification
Vector: pFB-CT10HF-LIC (available from The Addgene Nonprofit Plasmid Repository)
Cell line: DH10Bac, Sf9 cells
Tags and additions: C-terminal TEV protease site, followed by 10x His and FLAG tags
Wild-type sequence:
MKRQNVRTLALIVCTFTYLLVGAAVFDALESEPELIERQRLELRQQELRARYNLSQGGYEELERVVLRLKPHKAGVQWRFAGSFYFAITVITTIGYGHAAPSTDGGKVFCMFYALLGIPLTLVMFQSLGERINTLVRYLLHRAKKGLGMRRADVSMANMVLIGFFSCISTLCIGAAAFSHYEHWTFFQAYYYCFITLTTIGFGDYVALQKDQALQTQPQYVAFSFVYILTGLTVIGAFLNLVVLRFMTMNAEDEKRDAENLYFQSHHHHHHHHHHDYKDDDDK
(underlined sequence contains vector encoded TEV protease cleavage site, His and FLAG tag)
Expression
The human KCNK3 gene (Genbank ID 4504849), which encodes the TASK-1 protein, was obtained from Origene. The crystallisation construct, comprising residues M1-E259 was subcloned into the pFB-CT10HF-LIC vector and baculovirus was generated using the Bac-to-Bac system. Briefly, this was performed by transforming into Escherichia coli strain DH10Bac to generate bacmid DNA, which was subsequently used to transfect Spodoptera frugiperda (Sf9) insect cells and generate recombinant baculovirus. Large scale grow-ups of Sf9 cells were infected with baculovirus and incubated for 72 h at 27 °C in shaker flasks.
Cell Lysis and detergent extraction of membrane proteins
Extraction Buffer (EXB): 50 mM HEPES pH 7.5, 200 mM KCl, 5% v/v glycerol, Roche protease inhibitors
The cell pellet from 1 litre of insect cell culture was resuspended in 40 ml of lysis buffer per litre pellet using a glass dounce homogeniser. Cells were lysed with an EmulsiFlex-C3 or C5 homogenizer (Avestin Inc.) at 15,000 psi, two passes. For solubilisation, 5 ml of 10%/1% w/v stock of DM/CHS was added per litre of cell culture and the volume was adjusted by the addition of EXB to a final volume of 50 ml/L of initial cell culture and rotated at 4oC for 1 hour. The insoluble fraction was removed by centrifugation at 35,000 g for 1 hour at 4oC.
Purification
Wash Buffer: 50 mM HEPES pH 7.5, 10 mM imidazole pH 8.0, 200 mM KCl, 5% w/v glycerol, 0.24% w/v DM, 0.024% w/v CHS
Elution Buffer: 50 mM HEPES pH 7.5, 250 mM imidazole pH 8.0, 200 mM KCl, 5% v/v glycerol, 0.24% w/v DM, 0.024% w/v CHS
PD10 Buffer: 50 mM HEPES pH 7.5, 200 mM KCl , 5% w/v glycerol, 0.24% w/v DM, 0.024% w/v CHS
Size exclusion buffer (SEC) Buffer: 20 mM HEPES pH 7.5, 200 mM KCl, 0.12% w/v DM, 0.012 % CHS
Column 1: Co2+ TALON resin (0.5 ml volume in a gravity-flow column)
The detergent-extracted membrane protein from each litre of cells was combined with 1 ml of pre-equilibrated slurry of 50% Co2+ TALON resin (previously washed twice with H2O and three times with EXB). Imidazole pH 8.0 was added to a final concentration of 5 mM and the sample was rotated for 1 hour at 4oC. It was then transferred to a gravity column and washed with 30 column volumes of wash buffer. The sample was eluted with two column volumes of elution buffer in 0.5-2 ml fractions. Peak fractions were combined and passed through PD10 columns, pre-equilibrated in PD10 buffer (one column per 5 litres of initial culture volume).
TEV protease cleavage and reverse purification
TEV protease and PNGaseF were added at ratios of 5:1 and 10:1 (TASK-1:enzyme, wt:wt), respectively, and incubated at 4oC overight. For each litre of initial cell culture volume, 0.2 ml of a 50 % slurry of TALON resin (pre-equilibrated as above) were added and the sample was rotated in the cold room for 1 hour. The sample was transferred to a gravity column and the flow-through was collected.
Column 2: Superose 6 Increase 10/300 GL column (GE Healthcare)
The protein sample was concentrated in a 100 kDa MWCO concentrator (pre-equilibrated in SEC buffer without detergent) at 4,000 g with mixing every 5 min, to a final volume of 500 μl. After centrifugation at 20,000 g for 30 min at 4oC, the sample was subjected to size exclusion chromatography on a Superose 6 Increase 10/300 column, previously equilibrated in SEC buffer. The peak fractions were pooled and concentrated in a 100 kDa MWCO concentrator as described previously. After concentration to 10-30 mg/ml, the protein was flash-frozen in liquid nitrogen.
Assays
Two-electrode voltage clamp assay
X. laevis oocytes were obtained and the TEVC measurements were recorded as described previously (7). Briefly, collected oocytes were stored at 18 °C in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES pH 7.5) supplemented with 50 mg/l gentamicin, 274 mg/l sodium pyruvate and 88 mg/l theophylline. Oocytes were injected with 5 ng of TASK-1 cRNA and incubated for 48 h at 18 ℃. ND96 was used as the recording solution. Oocytes were held at -80 mV and voltage was ramped from -120 to +45 mV within 3.5 s, using a sweep time interval of 4 s. Block was analysed with voltage steps from a holding potential of -80 mV. A first test pulse to 0 mV of 1 s duration was followed by a repolarising step to -80 mV for 1 s, directly followed by another 1 s test pulse to +40 mV. The sweep time interval was 10 s. All inhibitors were dissolved in DMSO, aliquoted, stored at -20 °C or room temperature and added to the external solution (ND96) just before the recordings. The EC50 was determined from Hill plots using four concentrations for each construct. The final DMSO concentration of 0.1 % was not exceed.
Structure Determination
Crystallisation
Crystals were grown using the HiLiDe method (16). Prior to crystallisation, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) in chloroform was dried down in a round-bottomed glass vial. A total amount of 1.5 µg lipid per µl protein was used. Subsequently, protein at 5.5-6.5 mg/ml was added along with 15 µg/µl DM and 1.5 µg/µl CHS (from a 10% w/v DM, 1% w/v CHS stock). The vial was incubated slowly shaking at 4°C for 16-24 h and centrifuged at 15,000 g for 2 hours at 4°C. Crystallisation trials were then set up in 24-well hanging drop plates with 2 µl drops and a 2:1 protein:reservoir ratio at 20°C. Crystallisation of TASK-1/compound complexes was undertaken in the same manner. Each compound was dissolved in 100% DMSO at 130 mM then added to the HiLiDe setup to a final concentration of 1.3 mM. Crystals grew over 1-12 weeks, in 0.1 M TRIS pH 8.5, 0.05 M KCl, 24-32% PEG400 and 3% w/v sucrose and were mounted at 6°C directly from the drop and vitrified in liquid nitrogen.
Data collection and structure determination
All data were collected at Diamond Light Source (beamline I24) to resolutions between 2.9-3.5 Å. Data were processed, reduced and scaled using XDS, Staraniso, and AIMLESS.
The crystals belonged to space group P22121 and contained two copies of TASK-1 homodimers in the asymmetric unit. Initial phases were obtained with molecular replacement, using Phaser. A truncated version of the TREK-2 structure (PDB: 4BW5) (13) was used as an initial search model, with the cap and TM domain separate. An initial TASK-1 model was built using a density modified prime-and-switch map calculated using phenix.autobuild as a guide and was improved by several rounds of manual model building and refinement in COOT. The final model was refined in BUSTER version 2.10.3.
The native structure was used for the first round of refinement against the BAY 1000493 and BAY 2341237 data. Only minor changes in the structures were observed. A large positive difference density was observed in the vestibule, under the pore, originating from the compounds. The BAY 1000493 compound was modelled in two alternative conformations, a consequence of the two-fold symmetry of the TASK-1 homodimer. This dual orientation was confirmed by anomalous data collected at the Bromine edge. The BAY 2341237 compound induced small changes in the conformations of vestibule residues, leading to a slight asymmetry between the two monomer chains and BAY 2341237 only binding in one orientation that was clearly resolved in the density.
- Ma, L., Roman-Campos, D., Austin, E. D., Eyries, M., Sampson, K. S., Soubrier, F., Germain, M., Tregouet, D. A., Borczuk, A., Rosenzweig, E. B., Girerd, B., Montani, D., Humbert, M., Loyd, J. E., Kass, R. S., and Chung, W. K. (2013) A novel channelopathy in pulmonary arterial hypertension. The New England journal of medicine 369, 351-361
- Duprat, F., Lesage, F., Fink, M., Reyes, R., Heurteaux, C., and Lazdunski, M. (1997) TASK, a human background K+ channel to sense external pH variations near physiological pH. EMBO J 16, 5464-5471
- Olschewski, A., Li, Y., Tang, B., Hanze, J., Eul, B., Bohle, R. M., Wilhelm, J., Morty, R. E., Brau, M. E., Weir, E. K., Kwapiszewska, G., Klepetko, W., Seeger, W., and Olschewski, H. (2006) Impact of TASK-1 in human pulmonary artery smooth muscle cells. Circ Res 98, 1072-1080
- Olschewski, A. (2010) Targeting TASK-1 channels as a therapeutic approach. Adv Exp Med Biol 661, 459-473
- Patel, A. J., Honore, E., Lesage, F., Fink, M., Romey, G., and Lazdunski, M. (1999) Inhalational anesthetics activate two-pore-domain background K+ channels. Nat Neurosci 2, 422-426
- Wilke, B. U., Lindner, M., Greifenberg, L., Albus, A., Kronimus, Y., Bunemann, M., Leitner, M. G., and Oliver, D. (2014) Diacylglycerol mediates regulation of TASK potassium channels by Gq-coupled receptors. Nat Commun 5, 5540
- Streit, A. K., Netter, M. F., Kempf, F., Walecki, M., Rinne, S., Bollepalli, M. K., Preisig-Muller, R., Renigunta, V., Daut, J., Baukrowitz, T., Sansom, M. S., Stansfeld, P. J., and Decher, N. (2011) A specific two-pore domain potassium channel blocker defines the structure of the TASK-1 open pore. J Biol Chem 286, 13977-13984
- O'Donohoe, P. B., Huskens, N., Turner, P. J., Pandit, J. J., and Buckler, K. J. (2018) A1899, PK-THPP, ML365, and Doxapram inhibit endogenous TASK channels and excite calcium signaling in carotid body type-1 cells. Physiol Rep 6, e13876
- Czirjak, G., and Enyedi, P. (2002) Formation of functional heterodimers between the TASK-1 and TASK-3 two-pore domain potassium channel subunits. J Biol Chem 277, 5426-5432
- Barel, O., Shalev, S. A., Ofir, R., Cohen, A., Zlotogora, J., Shorer, Z., Mazor, G., Finer, G., Khateeb, S., Zilberberg, N., and Birk, O. S. (2008) Maternally inherited Birk Barel mental retardation dysmorphism syndrome caused by a mutation in the genomically imprinted potassium channel KCNK9. American journal of human genetics 83, 193-199
- Miller, A. N., and Long, S. B. (2012) Crystal structure of the human two-pore domain potassium channel K2P1. Science 335, 432-436
- Brohawn, S. G., del Marmol, J., and MacKinnon, R. (2012) Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel. Science 335, 436-441
- Dong, Y. Y., Pike, A. C., Mackenzie, A., McClenaghan, C., Aryal, P., Dong, L., Quigley, A., Grieben, M., Goubin, S., Mukhopadhyay, S., Ruda, G. F., Clausen, M. V., Cao, L., Brennan, P. E., Burgess-Brown, N. A., Sansom, M. S., Tucker, S. J., and Carpenter, E. P. (2015) K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac. Science 347, 1256-1259
- Lolicato, M., Arrigoni, C., Mori, T., Sekioka, Y., Bryant, C., Clark, K. A., and Minor, D. L., Jr. (2017) K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site. Nature 547, 364-368
- Sediva, M., Lassuthova, P., Zamecnik, J., Sedlackova, L., Seeman, P., and Haberlova, J. (2019) Novel variant in the KCNK9 gene in a girl with Birk Barel syndrome. Eur J Med Genet
- Sitsel, O., Wang, K., Liu, X., and Gourdon, P. (2016) Crystallization of P-type ATPases by the High Lipid-Detergent (HiLiDe) Method. Methods Mol Biol 1377, 413-420
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