PIP2 regulates psychostimulant behaviors through its interaction with a membrane protein

By Peter J Hamilton, Andrea N Belovich, George Khelashvili, Christine Saunders, Kevin Erreger, Jonathan Javitch, Harald H Sitte, Harel Weinstein, Heinrich J G Matthies & Aurelio Galli.

Published in Nature Chemical Biology 2014 Jun 1 [Epub ahead of print] PMID: 24880859. Link to publication page.

MPSDC Project: The Transport Cycle in Neurotransmitter Uptake Systems

Figure 1. PIP₂ interacts with hDAT. (a) hDAT and PIP₂ colocalize at the plasma membrane. In hDAT-expressing cells, GFP-hDAT (green) colocalizes (yellow) at the plasma membrane with the PIP₂ sensor PHPLC_δ-mRFP (red; representative image from three live experiments with of 10−15 cells imaged per experiment). Scale bar, 5 μm. (b) PIP₂ associates with endogenous mouse DAT in striatal tissue. In striatal lysate, DAT was detected in PIP₂ immunoprecipitates (IP) using an anti-DAT antibody (+). DAT immunoreactivity was absent in the beads fraction (−) and in PIP₂ immunoprecipitates from DAT KO animals (endogenous mouse IgG is observed; representative of n = 3). WB, western blot; WT, wild type; MW, molecular weight. The full blot is shown in Supplementary Figure 7.

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.

$Figure 2. hDAT-PIP2 electrostatic interactions are mediated by the hDAT N terminus. (a) hDAT N terminus directly binds PIP2. GST-64 hDAT is enriched in liposome pulldowns with liposomes containing PIP2 (+) as compared to liposomes lacking PIP2 (−) (**P ≤ 0.01 by one-way ANOVA followed by Bonferroni post hoc test; n = 3; mean ± s.e.m.). Pulldown of GST control was not altered by the presence (+) of absence (−) of PIP2 in the liposomes (P ≥ 0.05 by one-way ANOVA followed by Bonferroni post hoc test; n = 3; mean ± s.e.m.). Preincubation of liposome containing PIP2 with 3 μM of pal-HRQKHFEKRR (+) inhibited the pulldown of GST-64 hDAT with liposomes containing PIP2 (**P ≤ 0.01 by one-way ANOVA followed by Bonferroni post hoc test; n = 3). MW, molecular weight. The full blot is shown in Supplementary Figure 8. (b) Electrostatic potential isosurfaces (+1kT/e (+ charge)) shown as blue wireframes and (−1kT/e (− charge)) as red wireframes, calculated for the predicted structure of the wild-type N terminus. (c) View from the intracellular side of the N terminus (model) adsorbing on the lipid membrane. For clarity, the orientation of the N terminus in c was obtained by a 180° rotation of the N terminus configuration shown in b. The level of the PIP2 segregation by the N terminus is expressed as the ratio of local and ambient lipid fraction values and illustrated in color code (cold colors represent an enrichment of PIP2). The positive residues in the N terminus (yellow) that attract PIP2 electrostatically are highlighted. The simulations predict the local PIP2 concentration to be 2.5 times larger than the PIP2 ambient concentration.$
Figure 2. hDAT-PIP₂ electrostatic interactions are mediated by the hDAT N terminus.

$Figure 4. The hDAT N-terminal lysine regulates hDAT-PIP2 interaction. (a) Mutating hDAT N-terminal residues Lys3 and Lys5 to uncharged alanine (hDAT K/A) disrupts PIP2 segregation to the N terminus. The view is from the intracellular side of the N terminus of hDAT K/A adsorbing on the lipid membrane. The level of PIP2 segregation by the N terminus residues is expressed as the ratio of local and ambient lipid fraction values and is illustrated in color code, with cold colors representing an enrichment of PIP2. (b) N-terminal lysine residues mediate the hDAT-PIP2 interaction. Top, PIP2 immunoprecipitates (IP) from hDAT and hDAT K/A cells were immunoblotted for DAT (top lane). The beads fraction supports absence of nonspecific binding (middle lane). The bottom lane shows the total amount of DAT proteins. IB, immunoblot; MW, molecular weight. The full blot is shown in Supplementary Figure 9. Right, quantification of PIP2 pulldown band intensities normalized to the respective total DAT and expressed as a percentage hDAT (***P ≤ 0.001 by Student's t-test; n = 4; mean ± s.e.m.). Inset, representative blot for GST-64 hDAT and GST-64 hDAT K/A in pulldowns with liposomes containing PIP2. The full blot is shown in Supplementary Figure 9.$
Figure 4. The hDAT N-terminal lysine regulates hDAT-PIP₂ interaction.