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Selective regulation of human TRAAK channels by biologically active phospholipids

Nature Chemical Biology volume 17, pages 89–95 (2021)Cite this article

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Abstract

TRAAK is an ion channel from the two-pore domain potassium (K2P) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K2P4.1 toward lipids remains poorly understood. Here we show the two isoforms of K2P4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K2P4.1. Liposome potassium flux assays with K2P4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K2P4.1.

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Fig. 1: Optimization of human K2P4.1 isoforms for high-resolution native MS studies.
Fig. 2: Determination of equilibrium dissociation constants (Kd) for individual lipid-binding events to K2P4.1a.
Fig. 3: Biophysical characterization of individual lipid-binding events to K2P4.1b.
Fig. 4: Liposome potassium flux assays of K2P4.1a reconstituted in defined lipid environments.

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Data availability

The native MS data are available on Zenodo (https://doi.org/10.5281/zenodo.3993214). Source data are provided with this paper.

Code availability

Python code is available at GitHub (https://github.com/LaganowskyLab/Laganowsky_Lab_Code.git).

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Acknowledgements

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) awarded to A.L. (grant no. DP2GM123486) and development of custom instrumentation supported by NIH (grant no. P41GM128577-01, D.R.).

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Author notes

  1. These authors contributed equally: Samantha Schrecke, Yun Zhu.

Authors and Affiliations

  1. Department of Chemistry, Texas A&M University, College Station, TX, USA

    Samantha Schrecke, Yun Zhu, Jacob W. McCabe, Mariah Bartz, Charles Packianathan, David Russell & Arthur Laganowsky

  2. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA

    Minglei Zhao

  3. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA

    Ming Zhou

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Contributions

S.S., Y.Z. and A.L. designed the research. S.S., Y.Z. and C.P. expressed and purified the protein. S.S. and M.B. carried out molecular biology experiments. S.S. and Y.Z. performed the native MS experiments and Y.Z. conducted functional assays. S.S., Y.Z. and A.L. analyzed the data. S.S., Y.Z. and A.L. wrote the manuscript with input from the other authors.

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Correspondence to Arthur Laganowsky.

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Extended data

Extended Data Fig. 1 Optimization of expression and purification of human K2P4.1 for native ion mobility mass spectrometry studies.

a, Shown are representative mass spectra of the K2P4.1b fusion protein acquired on a Synapt G1 HDMS instrument. Denoted in the inset is the detergent abbreviation: dodecyl maltoside (DDM); decyl maltoside (DM); lauryldimethylamine-N-oxide (LDAO); pentaethylene glycol monodecyl ether (C10E5); and octaethylene glycol monododecyl ether (C12E8). The polyethylene glycol detergents, C10E5 and C12E8, yielded well resolved mass spectra compared to the other detergents screened. b, Membrane proteins were expressed with affinity tags fused to both termini. Shown is the native mass spectrum for K2P4.1b with tags removed by TEV protease in C10E5 acquired on an Orbitrap. c, Native ion mobility mass spectra of the K2P4.1a fusion protein solubilized in (left) 2x CMC C10E5 and with the addition of (right) 50 mM spermidine acquired on Synapt G1. In the absence of charge-reducing molecule, the arrival time distributions are broad for high charge states indicating collisional activation (or partial unfolding) of the protein complex. d, Collision induced unfolding (CIU) plot of arrival time distribution for the 9+ charge state of K2P4.1a with tags removed in C10E5 and spermidine acquired under different collision voltages (10 V step size) on a Synapt G1 instrument. The arrival time distribution for the 9+ and other charge states of K2P4.1a remain constant showing that charge reduction aids retention of native-like structure, even at the highest collision voltage (240 V). e, Ion mobility mass spectrum for K2P4.1a in C10E5 and spermidine recorded on an Orbitrap UHMR equipped with a REIS and 1.5 m drift tube operating in the FT-mode. f, Collision cross section profiles for different charge states of K2P4.1a. The centroid CCS for the 10+, 9+, and 8+ are 4559, 4528, 4547 Å2, respectively. The calculated CCS for PDB 4WFE is 4656 Å2.

Extended Data Fig. 2 Native mass spectra for weakly (or no) binding lipids to K2P4.1a.

Shown are native mass spectra for K2P4.1a in the presence of 20 µM of lipid. Lipid abbreviations are provided in Supplementary Table 1.

Extended Data Fig. 3 Determination of equilibrium dissociation constants (KD) for lipids binding K2P4.1a.

Shown are representative native mass for K2P4.1a in the presence of 20 µM of lipid. Plots of mole fraction (right panel) for apo K2P4.1a and bound to different number of lipids determined from a titration series (dots with error bars to represent the three measurements) and resulting fit from a sequential lipid-binding model (solid lines). Lipid abbreviations are provided in Supplementary Table 1.

Extended Data Fig. 4 Determination of KD for lysolipids and PIPs binding K2P4.1a.

Shown as described in Extended Data Fig. 3. * and ** denote lipid concentrations of 15 μM and 10 μM, respectively.

Extended Data Fig. 5 Determination of KD for lipids binding K2P4.1b.

Shown as described for Extended Data Fig. 3. * and ** denote lipid concentrations of 15 μM and 10 μM, respectively.

Extended Data Fig. 6 Determination of KD for lysolipids, PIPs lipids binding K2P4.1b.

Shown as described for Extended Data Fig. 3. * and ** denote lipid concentrations of 15 μM and 10 μM, respectively.

Extended Data Fig. 7 Liposome potassium flux assays of K2P4.1a reconstituted in POPC.

a-d, Flux assay traces for K2P4.1a reconstituted in POPC and POPC doped with POPG or POPA. Empty liposomes of the same lipid composition are shown in gray. The mole fraction (%) of doped lipid is shown in the inset. e, Photograph of a silver stained SDS-PAGE of different proteoliposome mixtures used in the liposome potassium flux assays. Bands corresponding to monomer and dimer K2P4.1a are denoted by a single and double asterisk, respectively. The Precision Plus Protein Dual Color Standard (Bio-Rad) protein ladder was loaded in the first lane and molecular weight in kDa is shown to the left. The white scale bar (lower right corner) represents 1 cm. f, Densitometry analysis of K2P4.1a monomeric bands. No statistical differences were found between the different samples. Shown are the mean and standard deviation (n = 3) for three independent experiments (white dots).

Source data

Supplementary information

Source data

Source Data Fig. 2

Kd values for each replicate

Source Data Fig. 3

Kd values for each replicate

Source Data Fig. 4

Liposome flux for each experiment

Source Data Extended Data Fig. 7

Unprocessed stained gel, densitometry measurements

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Schrecke, S., Zhu, Y., McCabe, J.W. et al. Selective regulation of human TRAAK channels by biologically active phospholipids. Nat Chem Biol 17, 89–95 (2021). https://doi.org/10.1038/s41589-020-00659-5

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