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PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein

Nature Chemical Biology volume 10, pages 582–589 (2014)Cite this article

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Abstract

Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux without altering the hDAT physiological function of DA uptake. We evaluated the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present what is to our knowledge the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.

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Figure 1: PIP2 interacts with hDAT.
Figure 2: hDAT-PIP2 electrostatic interactions are mediated by the hDAT N terminus.
Figure 3: Sequestration or depletion of PIP2 inhibits AMPH-induced DA efflux.
Figure 4: The hDAT N-terminal lysine regulates hDAT-PIP2 interaction.
Figure 5: N-terminal Lys3 and Lys5 regulate specific modalities of hDAT function.
Figure 6: Expression of hDAT K/A in Drosophila dopaminergic neurons does not affect circadian locomotor activity yet impairs AMPH-induced locomotion and neuronal DA efflux.

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Acknowledgements

We thank S. Singh (Yale University) for the gift of the dDAT cDNA. Computational resources were provided by Teragrid allocation MCB120008 on the Ranger machine, by the NERSC allocation (repository m1710) on the Carver supercomputer and the David Cofrin Center for Biomedical Information of the Institute for Computational Biomedicine at Weill Cornell Medical College. This work was supported by US National Science Foundation Graduate Research Fellowship DGE0909667 (P.J.H.) and grants F31 DA 035535-01 (P.J.H.), 23658-B11 (H.H.S.), DA035263 (A.G.), P01 DA012408 (A.G., H.W. and J.A.J.) and U54GM087519 (H.W. and J.A.J.).

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Author notes

  1. Peter J Hamilton and Andrea N Belovich: These authors contributed equally to this work.

  2. Heinrich J G Matthies and Aurelio Galli: These authors equally directed this work.

Authors and Affiliations

  1. Vanderbilt Brain Institute, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Peter J Hamilton & Aurelio Galli

  2. Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Andrea N Belovich, Christine Saunders & Aurelio Galli

  3. Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, New York, USA

    George Khelashvili & Harel Weinstein

  4. Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Kevin Erreger, Heinrich J G Matthies & Aurelio Galli

  5. Department of Psychiatry, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Jonathan A Javitch

  6. Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York, USA

    Jonathan A Javitch

  7. Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University Vienna, Vienna, Austria

    Harald H Sitte

  8. HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute of Computational Biomedicine, Weill Cornell Medical College of Cornell University, New York, New York, USA

    Harel Weinstein

  9. Neuroscience Program in Substance Abuse, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Peter J Hamilton, Kevin Erreger, Heinrich J G Matthies & Aurelio Galli

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Contributions

P.J.H., A.N.B., G.K., C.S., K.E. and H.J.G.M. performed experiments. P.J.H., A.N.B., G.K., C.S., K.E., J.A.J., H.H.S., H.W., H.J.G.M. and A.G. designed experiments and analyzed data. G.K. and H.W. generated computational models and analyses. J.A.J. supplied expression vectors and plasmid DNA. P.J.H., A.N.B., H.W., H.J.G.M., G.K. and A.G. wrote the manuscript. All of the authors participated in the discussion of results and contributed to the preparation of the manuscript.

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Correspondence to Heinrich J G Matthies or Aurelio Galli.

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Hamilton, P., Belovich, A., Khelashvili, G. et al. PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein. Nat Chem Biol 10, 582–589 (2014). https://doi.org/10.1038/nchembio.1545

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