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Phosphatidylinositol-3,5-bisphosphate lipid-binding-induced activation of the human two-pore channel 2

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Abstract

Mammalian two-pore channels (TPCs) are activated by the low-abundance membrane lipid phosphatidyl-(3,5)-bisphosphate (PI(3,5)P2) present in the endo-lysosomal system. Malfunction of human TPC1 or TPC2 (hTPC) results in severe organellar storage diseases and membrane trafficking defects. Here, we compared the lipid-binding characteristics of hTPC2 and of the PI(3,5)P2-insensitive TPC1 from the model plant Arabidopsis thaliana. Combination of simulations with functional analysis of channel mutants revealed the presence of an hTPC2-specific lipid-binding pocket mutually formed by two channel regions exposed to the cytosolic side of the membrane. We showed that PI(3,5)P2 is simultaneously stabilized by positively charged amino acids (K203, K204, and K207) in the linker between transmembrane helices S4 and S5 and by S322 in the cytosolic extension of S6. We suggest that PI(3,5)P2 cross links two parts of the channel, enabling their coordinated movement during channel gating.

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Abbreviations

PI(3,5)P2 :

Phosphatidylinositol-(3,5)-bisphosphate

POPC:

1-Palmitoyl-2-oleoyl phosphatidylcholine

MD:

Molecular dynamics

CG:

Coarse-grained

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Acknowledgements

This work was supported by the Research Training Group 1962 (to RAB and PD) from the Deutsche Forschungsgemeinschaft and by 2015795S5W funding to AC from the Italian Ministry of Education, University and Research. We would like to thank Joachim Scholz-Starke and Margherita Festa (Genova) for supervision of AK during part of his experiments.

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    Authors and Affiliations

    1. Computational Biology, Department of Biology, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

      Sonja A. Kirsch & Rainer A. Böckmann

    2. Molecular Plant Physiology, Department of Biology, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany

      Andreas Kugemann & Petra Dietrich

    3. Institute of Biophysics, National Research Council, Genoa, Italy

      Armando Carpaneto

    4. Department of Earth, Environment and Life Sciences-DISTAV, University of Genoa, Genoa, Italy

      Armando Carpaneto

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    1. Sonja A. Kirsch
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    4. Rainer A. Böckmann
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    5. Petra Dietrich
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    Contributions

    RAB and PD designed and directed research of the simulations and functional analysis, respectively. SAK set up the homology model, and performed CG and atomistic simulations and the related analysis. AK performed cloning, site-directed mutagenesis, functional expression and patch-clamp analysis in plant vacuoles and analyzed the data; AC hosted AK during a laboratory stay to conduct part of the experiments and helped analyzing the data. SAK, PD, RAB, AK, and AC wrote the manuscript.

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    Correspondence to Rainer A. Böckmann or Petra Dietrich.

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    Sonja A. Kirsch and Andreas Kugemann contributed equally.

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    Kirsch, S.A., Kugemann, A., Carpaneto, A. et al. Phosphatidylinositol-3,5-bisphosphate lipid-binding-induced activation of the human two-pore channel 2. Cell. Mol. Life Sci. 75, 3803–3815 (2018). https://doi.org/10.1007/s00018-018-2829-5

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