Garcia-Olivares J et al. (2013), Inhibition of dopamine transporter activity by ...

XB-ART-50514

PLoS One 2013 Jan 01;83:e59788. doi: 10.1371/journal.pone.0059788.

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Inhibition of dopamine transporter activity by G protein βγ subunits.

Garcia-Olivares J , Torres-Salazar D , Owens WA , Baust T , Siderovski DP , Amara SG , Zhu J , Daws LC , Torres GE .

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Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson's disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.

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Species referenced: Xenopus laevis
Genes referenced: slc1a3 slc6a3

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	Figure 2. Direct interaction between Gβγ subunits and the carboxy terminus of DAT.(A) Schematic representation of DAT depicting cytoplasmic segments used in GST pull-down assays. (B) The carboxy terminus of DAT (GST-DATc) precipitated Gβγ subunits from mouse striata. (C) Immuno-far-western assay showing a direct interaction between GST-DATc and purified Gβγ from bovine brain. RP = Ponceau Red.
	Figure 3. Activation of G proteins with GTP-γ-S inhibits DAT function in heterologous systems.(A) [3H]-DA uptake assays in Xenopus laevis oocytes expressing DAT after intracellular injection with water (white bar, n = 8), 10 µM GTP-γ-S (black bar, n = 8), or 10 µM GDP-β-S (gray bar, n = 8). (B) Specific [3H]-DA uptake and kinetic analysis for HEK293-DAT permeabilized with streptolysin-O (SLO) and dialyzed with 50 µM GTP-γ-S (white circles, n = 3). or control buffer (black circles, n = 3). **p<0.01.
	Figure 4. Overexpression of Gβγ subunits results in decreased DAT uptake activity in HEK-293-DAT cells.(A, B) Specific [3H]-DA uptake and kinetic analysis for HEK293-DAT control cells (white bar, n = 6) or transfected with Gβ1γ2 (n = 3), Gαi2 (n = 3), or Gαi2Gβ1γ2 (n = 3). (C) Overexpression of Gβ4 (n = 5) or Gβ5 (n = 7) (gray bars), decreased [3H]-DA uptake in HEK293-DAT cells when compared to control (white bar, n = 19). Overexpression of the Gβγ scavengers (black bars), Gαt (n = 6), or GRK2ct (n = 4) increased [3H]-DA uptake. (D, E) Overexpression of Gβ1γ2 (n = 4), Gβ4 (n = 5), or Gβ5 (n = 7) in HEK-DAT cells did not alter the plasma membrane levels of DAT as measured by biotinylation. T = total fraction, B = biotinylated fractions, **p<0.01.
	Figure 5. Activation of Gβγ with mSIRK resulted in an inhibition of DAT activity in heterologous systems.(A) Oocytes expressing DAT (white bars) or EAAT1 (gray bars) were incubated for 30 min with 0.5% DMSO (DAT/n = 3; EAAT1/n = 25), 5 µM mSIRK (DAT/n = 20, EAAT1/n = 23), or 25 µM mSIRK (DAT/n = 15, EAAT1/n = 15) before uptake assays with [H3]-DA or [3H]-Glutamate, respectively. (B) Dose-dependent inhibition of [3H]-DA uptake in HEK293-DAT cells by mSIRK (n = 4, white circle). (C) 5 min pre-incubation of HEK293-DAT cells with 10 µM mSIRK produced a reduction of Vmax with no changes in Km (n = 3). (D) In HEK293-DAT, 10 µM mSIRK reduced uptake (control, black bar, n = 32; mSIRK, white bar, n = 15). The inhibitory effect of 10 µM mSIRK was attenuated in HEK-DAT cells expressing GRK2ct (control, n = 16; mSIRK, n = 16). *p<0.01 or p<0.05.
	Figure 6. Activation of Gβγ subunits reduces DAT activity in brain synaptosomes(A) mSIRK reduces [3H]-DA uptake in rat striatal synaptosomes. Samples were pre-incubated for 10 min with various concentrations of mSIRK (open circles) or scb-mSIRK (filled circles) at 34°C, followed by the addition of [3H]-DA (final concentration, 0.1 µM) for 8 min, n = 3. Nonspecific [3H]-DA uptake was determined in the presence of 10 µM nomifensine. (B) Kinetic analysis of the synaptosomal [3H]-DA uptake was determined in the absence (vehicle) or presence of 5 µM mSIRK, n = 3. **p<0.01.
	Figure 7. Activation of Gβγ subunits reduces DAT activity in vivo.(A) Clearance of exogenously applied DA from the striatum of anesthetized mice. Once a baseline for DA clearance was established, mSIRK or scr-mSIRK was applied, and then 5 min later, DA was applied and again, 10, 20, 30 and 40 min after the peptides were introduced (arrows). The amplitude of the DA signal augment as observed in a representative traces of DA signal after intrastriatal injection of mSIRK (upper panel) or scb-mSIRK (bottom panel). (B) Clearance time (T80) of exogenous DA was increased in the presence of mSIRK treatment. *p<0.01, p<0.05.
	Figure 1. Co-immunoprecipitation of DAT and Gβγ subunits.(A, B) Immunoprecipitation of DAT with two DAT antibodies directed against different DAT epitopes results in the co-precipitation of Gβ subunits from mouse striatum. Control experiments include immunoprecipitations with nonspecific rat or rabbit IgGs or immunoprecipitations from DAT knockout striatum or mouse cerebellum. (C, D) Immunoprecipitation of DAT resulted in the co-immunoprecipitation of Gβ subunits from MN9D-DAT cells or HEK293-DAT cells. Control experiments include immunoprecipitations from MN9D or HEK293 mock-transfected cells. Three different DAT antibodies were used; DAT1 (Mab369, Millipore), DAT2 (H-80, Santa Cruz), and DAT3 (c-20, Santa Cruz). Immunoblotting was performed with a Gβ pan-antibody (T-20, Santa Cruz). Cereb = cerebellum, St = striatum, DAT-KO = DAT knockout.

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