Biochemical and Biophysical Research Communications 372 (2008) 656661

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

PI3K signaling supports amphetamine-induced dopamine efux q

Brandon J. Lute a, Habibeh Khoshbouei b, Christine Saunders c, Namita Sen d, Richard Z. Lin e, Jonathan A. Javitch d, Aurelio Galli a,*
a

Department of Molecular Physiology and Biophysics, Center for Molecular Neuroscience, Vanderbilt University, 465 21st Avenue South, Nashville, TN 37232-8548, USA Department of Neurobiology and Neurotoxicology, Department of Biomedical Sciences, Meharry Medical College, Nashville, TN 37208, USA c Department of Pharmacology, Center for Molecular Neuroscience, Vanderbilt University, 465 21st Avenue South, Nashville, TN 37232-8548, USA d Departments of Psychiatry and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA e Departments of Medicine and Physiology and Biophysics, Stony Brook University, Stony Brook, NY, USA
b

a r t i c l e

i n f o

a b s t r a c t
The dopamine (DA) transporter (DAT) is a major molecular target of the psychostimulant amphetamine (AMPH). AMPH, as a result of its ability to reverse DAT-mediated inward transport of DA, induces DA efux thereby increasing extracellular DA levels. This increase is thought to underlie the behavioral effects of AMPH. We have demonstrated previously that insulin, through phosphatidylinositol 3-kinase (PI3K) signaling, regulates DA clearance by ne-tuning DAT plasma membrane expression. PI3K signaling may represent a novel mechanism for regulating DA efux evoked by AMPH, since only active DAT at the plasma membrane can efux DA. Here, we show in both a heterologous expression system and DA neurons that inhibition of PI3K decreases DAT cell surface expression and, as a consequence, AMPH-induced DA efux. 2008 Elsevier Inc. All rights reserved.

Article history: Received 9 May 2008 Available online 27 May 2008

Keywords: Dopamine transporter (DAT) Amphetamine (AMPH) Dopamine (DA) Phosphatidylinositol 3-kinase (PI3K) Insulin Drug abuse Transient current

Dopamine (DA) is involved in the control of locomotion, cognition, and reward [1]. Upon exocytic release of DA into the synapse, the DA transporter (DAT) coordinates the spatial and temporal regulation of dopaminergic neurotransmission by actively clearing DA from the extracellular space. DAT belongs to the Na+/Cl-dependent transporter gene family, which also consists of plasmalemmal carriers for norepinephrine, epinephrine, and serotonin [1]. DAT is the major molecular target for several psychostimulants including amphetamine (AMPH) [2]. AMPH, as a substrate of DAT, elevates extracellular DA levels by stimulating DAT-mediated DA efux thereby inducing its behavioral effects [2]. Importantly, AMPH-induced DA efux depends on the turnover rate of an individual transporter and the number of functional transporters expressed at the plasma membrane. Therefore, regulation of DAT cell surface expression may be a potential therapeutic target for psychostimulant abuse. Recent studies suggest a role for insulin and insulin-like growth factors (e.g., IGF1 and IGF2) in the regulation of DA homeostasis [3]. It is well documented that a high density of insulin and IGF 12 receptors are located in limbic areas and the striatum [4], a
This work was supported in part the Peter F. McManus Charitable Trust Grant (C.S.) and by NIH Grants DA21069 (B.J.L.), DA13975 and MH058921 (A.G.), DK62722 (R.Z.L.), DA12408 (A.G. and J.A.J.), and DA022413 (J.A.J.). * Corresponding author. Fax: +1 615 936 3745. E-mail address: Aurelio.Galli@vanderbilt.edu (A. Galli). 0006-291X/$ - see front matter 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.091
q

brain region in which DAT is abundantly expressed. Insulin receptors and IGF1-2 receptors function as receptor tyrosine kinases (RTKs), which stimulate phosphatidylinositol 3-kinase (PI3K) signaling [5]. Importantly, PI3K signaling regulates DA clearance through a mechanism that seems to rely on DAT trafcking [6]. Consistent with the idea that PI3K signaling regulates DAT cell surface expression, rats made hypoinsulinemic display decreased DA clearance [3]. Furthermore, insulin-depleted, diabetic rodents are resistant to the motor stimulant properties of AMPH [7]. Taken together, these studies suggest that insulin signaling pathways in the brain play an important role in regulating DAT activity, extracellular DA levels, and AMPH action. Still, little is known about how DAT trafcking regulates the ability of AMPH to cause DA efux. To investigate the importance of this relationship, we explore whether perturbations in PI3K signaling regulate the ability of AMPH to induce DA efux by altering DAT plasma membrane expression. Materials and methods Plasmid construction, transfection, and cell culture. We made a hDAT construct labeled with yellow uorescent protein (YFP), YFPDAT, as described previously [8]. This construct was subcloned into either a bicistronic expression vector or a pcDNA5/ FRT/TO-TOPO vector, both of which were modied to express the

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synthetic YFPDAT as described previously [8]. EM4 cells, a HEK293 cell line stably transfected with macrophage scavenger receptor to increase their adherence (R. Horlick; Pharmacopeia), were transfected with the bicistronic YFPDAT vector (i.e., hDAT cells) [8]. FlpIn TREx HEK293 cells were transfected with YFP DAT pcDNA5/FRT/TO-TOPO using lipofectamine (Invitrogen), and a stably transfected pool (i.e., inducible hDAT cells) was selected in 100 lg/mL hygromycin and 15 lg/mL blasticidin. Transgenic mice. Postnatally derived mouse midbrain neuronal cultures were obtained from transgenic mouse strains generated as described by Zhang et al. [9]. In order to create a source of DA neurons that do not express DAT, we developed a line of mice from which midbrain DA neurons carrying the red uorescent protein (RFP) marker on a DAT knockout background were generated. DAT knockout mice provided by Dr. Marc Caron [10]. Electrophysiology. Cells were washed twice with bath solution containing the following (in mM): 130 NaCl, 10 Hepes, 1.5 CaCl2, 0.5 MgSO4, 1.3 KH2PO4, and 34 dextrose adjusted to pH 7.35. The pipette solution contained the following (in mM): 90 KCl, 30 NaCl, 2.0 MgCl26H2O, 10 Hepes, 0.1 CaCl2, 1.1 EGTA, and 30 dextrose (pH 7.35) plus 2 mM DA as described by Khoshbouei et al. [11]. Whole cell currents were low-pass ltered at 5000 Hz. The whole cell and amperometric currents recorded from midbrain dopaminergic neurons were obtained as described previously [12]. We used the external and internal solutions described by Ingram et al. [13], except for isoosmotically replacing 30 mM KCl with NaCl and adding 2 mM DA to the pipette solution. We also added 1 lM sulpiride to the bath solution. The DAT transient charge (Q) movement, in response to a voltage step to 140 mV from a holding potential of 20 mV, was obtained by integrating the relaxation component of the AMPH-induced current (Fig. 2C). Time-dependent changes in Q were used to evaluate DAT cell surface expression as described previously [8]. Amperometry. Amperometric currents were recorded using a second Axopatch 200B amplier connected to a carbon ber as described previously [11] with a low-pass lter set at 10 Hz. The AMPH-induced, DAT-mediated steady-state, transient, and amperometric currents were dened as the current recorded in the presence of AMPH minus the current recorded after the addition of cocaine to the bath with AMPH still present. Data were recorded and analyzed off-line as described previously [11]. Cell surface biotinylation and immunoblotting. Cell surface biotinylation experiments were performed as described previously [14]. Inducible hDAT cells were seeded in 6-well plates (106 cells/well) approximately 48 h prior to the experiment. After 24 h, when the cells had grown to 60% conuence, 1 lg/mL tetracycline was added and experiments were performed at 0, 4, 8, and 24 h, respectively. YFPDAT was detected by anti-GFP primary antibody (Abcam) and antirabbit-HRP secondary antibody (Santa Cruz Biotechnology), with ECL-Plus (Amersham) and uorescence detection and quantitation on a Fluoro-Chem imager. Uptake of [3H]tyramine. Tyramine was used as a radiolabeled substrate as described previously [14]. In contrast to DA, tyramine is not degraded by catechol-O-methyl transferase and therefore avoids potential complications with kinetics of uptake.

Results PI3K signaling pathways, which are stimulated by activation of insulin receptors and other RTKs [5], play a critical role in the maintenance of DA clearance and DAT cell surface expression [15]. By ne-tuning DAT plasma membrane expression, PI3Kdependent signaling pathways may regulate the ability of AMPH to cause DAT-mediated DA efux. To test this hypothesis, we used both hDAT cells [8] and midbrain DA neurons transgenically

labeled with RFP to visualize DAT-expressing cells [12,16]. Importantly, untransfected cells and DA neurons from DAT knockout mice did not produce any measurable DAT-mediated whole cell or amperometric currents (data not shown). The membrane potential of the patched DA neurons was held at 20 mV and stepped, in 20 mV intervals, to voltages between +100 and 100 mV. Fig. 1A shows a representative AMPH-induced, DATmediated electrical current recorded in the whole cell conguration during a voltage step from 20 to +100 mV. The DAT-mediated current was dened as the current recorded in the presence of 10 lM AMPH minus the nonspecic current recorded after the application of 10 lM cocaine with AMPH still present [16]. To test whether PI3K signaling modulates AMPH regulation of DAT function, we measured DAT-mediated electrical currents in the presence of a specic inhibitor of PI3K, LY294002. At +100 mV, bath application of 50 lM LY294002 (AMPH + LY) substantially reduced DAT-mediated ionic currents in DA neurons (Fig. 1A). The rewarding effects of AMPH are attributed to the exocyticindependent rise in extracellular DA levels. We coupled the patchclamp technique with amperometry to measure DAT-mediated ionic currents and DA efux simultaneously as described previously [12]. Briey, we placed a 5 lm carbon ber held at +700 mV %1 lm from the neuronal plasma membrane. The amperometric current is generated by the oxidation of DA by the carbon ber electrode. An upward deection in the amperometric current corresponds to an outward efux of DA. We dened DAT-mediated DA efux by subtracting the nonspecic efux measured after the application of 10 lM cocaine (with AMPH still present) from the oxidation current recorded in the presence of 10 lM AMPH. Fig. 1B shows the AMPH-induced DA efux during a voltage step from 20 mV to +100 mV. Consistent with the whole cell data, DA efux induced by AMPH was inhibited by LY294002 (AMPH + LY) in DA neurons (Fig. 1B). Fig. 1C displays the currentvoltage relationship for the substrate-induced ionic current mediated by DAT. In Fig. 1D, we plot oxidation currents against the test voltage applied to the neuron. AMPH-induced DA efux increased at positive potentials (Fig. 1D). AMPH-induced ionic currents and DA efux were signicantly reduced by LY294002 at positive potentials as shown by the currentvoltage and amperometricvoltage relationships (Fig. 1C and D). In contrast, treatment with LY303511 (50 lM), a structural inactive analog of LY294002, or vehicle (DMSO) did not reduce AMPH-induced ionic currents or DA efux (data not shown). Importantly, we did not observe DAT-mediated currents in DAT knockout mice, demonstrating that the AMPH-induced efux we measure is not due to exocytic release of DA from synaptic vesicles (data not shown). Similar results were obtained in hDAT cells (data not shown). The PI3K-mediated effects we observed may be due to modication of transporter activity on the cell surface, changes in DAT cell surface expression, or both. We rst addressed whether the amount of DAT cell surface expression correlates with DAT activity by virtue of our ability to measure surface DAT with electrophysiological methods. We subcloned DAT into an inducible, tetracycline-based expression vector and stably transfected HEK293 cells (i.e., inducible hDAT cells). Next, we induced DAT expression with 1 lg/mL tetracycline for 0, 4, 8, or 24 h. Fig. 2A displays a representative immunoblot showing biotinylated cell extracts (cell surface) and total extracts (total) after incubation with tetracycline for the indicated time points. The normalized cell surface band densities were plotted against the Vmax for DAT-specic [3H]tyramine, a DAT substrate, uptake (Fig. 2B). These data demonstrate that the maximal rate of transport linearly correlates with DAT cell surface expression. Measurements of time-dependent changes in transient charge (Q) movements have been adopted to evaluate plasma membrane

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60

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* * AMPH
AMPH + LY

40

20

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-120 -80 -40 -20 40 80 120 -120 -80 -40 -0.01 40 80 120

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Fig. 1. PI3K signaling regulates AMPH-induced DA efux in DA neurons. Representative DAT-mediated ionic currents (A) and DA efux (B) recorded from DA neurons at +100 mV in response to either 10 lM AMPH (AMPH) or 10 lM AMPH followed by 50 lM LY294002 (AMPH + LY). Currentvoltage (C) and amperometricvoltage (D) relationships in response to either 10 lM AMPH (open circles) or to 10 lM AMPH followed by 50 lM LY294002 (closed triangles). Currents were collected in a voltage range between +100 and 100 mV from a holding potential of 20 with 20 mV increments. Data points represent means SE (n = 3; one-way ANOVA followed by Bonferronis post hoc test; *p < 0.05 comparing AMPH to AMPH + LY294002 treatment for each voltage).

expression of DAT [8]. Following a voltage jump, hDAT cells display current relaxations that persist for several milliseconds reecting the time required to charge the membrane capacitance. The DAT component of these current relaxations (i.e., DAT-mediated transient current) can be dened by bath application of DA uptake blockers such as mazindol and cocaine [8]. We isolated DAT-mediated transient currents by subtracting the current recorded in the presence of AMPH and cocaine from the current recorded in the presence of AMPH alone. DAT-mediated Q has been shown to be at virtual saturation at 140 mV [8]. In inducible hDAT cells, we recorded DAT-mediated transient currents in response to AMPH by stepping the membrane voltage from 20 to 140 mV (Fig. 2C). We measured DAT-mediated transient currents while varying transporter expression in inducible hDAT cells (1 lg/mL tetracycline for 0, 4, 8, or 24 h) to further characterize the relationship between DAT cell surface expression and DAT-mediated Q. We plotted the normalized band density values from Fig. 2A against DAT Q values. Fig. 2D demonstrates that DAT-mediated Q linearly correlates with DAT cell surface expression. Next, we evaluated whether the reduction in AMPH-induced ionic currents and DA efux caused by LY294002 is promoted by a decrease in DAT cell surface expression. We monitored DAT levels at the plasma membrane by transient current analysis in DA neurons. In these experiments, the DAT-mediated Q in response to a voltage step was obtained by integrating either the on or the off of the relaxation component of the AMPH-induced current.

DAT-mediated transient currents were not seen in DA neurons from DAT knockout mice or untransfected cells (data not shown). Fig. 3A and B shows that treatment with AMPH (10 lM) followed by LY294002 (50 lM) results in a signicant decrease in the DAT-mediated Q with respect to AMPH alone. In response to LY294002, the transient charge decreased both at the on and off of the voltage step with respect to the initial transient current (Fig. 3C and D, respectively). In contrast, treatment with LY303511 (50 lM) or vehicle (DMSO) did not result in any signicant reductions (data not shown). Although not signicantly different, AMPH alone has a slight tendency to decrease the initial DAT-mediated transient currents. Similar results were obtained in hDAT cells (data not shown). These ndings, together with our electrochemical data (Fig. 1), support the hypothesis that the reduction in AMPH-induced DA efux caused by LY294002 is a consequence of reduced DAT plasma membrane levels and are consistent with the previously reported effects of PI3K regulation of transporter trafcking [6,15,17]. In order to further explore this PI3K regulation of DAT cell surface expression, we plotted the time-dependent decrease of DATmediated Q recorded in hDAT cells in response to LY294002 (50 lM) (Fig. 4A). After bath application of LY294002, the AMPHinduced transient current decreased linearly (Fig. 4A). To determine the temporal relationship between the LY294002-induced DAT cell surface redistribution and the reduction of DA efux, we plotted the Q values (see Fig. 4A) against the AMPH-induced DA

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B
[3H]Tyramine Uptake (pmol/min/g protein)
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r = 1.00 p < 0.0001

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Fig. 2. Increased DAT plasma membrane expression correlates with DAT activity and DAT-mediated transient currents. (A) Representative immunoblot showing biotinylated (cell surface) and total extracts (total) after induction of DAT expression with 1 lg/mL tetracycline for the indicated time periods. Varying protein amounts were loaded to avoid signal saturation of the DAT band at 24 h and to detect surface DAT for 0 and 4 h. (B) Displays biotinylated band densities plotted against the Vmax for [3H]tyramine uptake after identical induction times for DAT expression. Biotinylated band densities were normalized to protein loaded and expressed as fold increase with respect to the band density at the 0 h induction time. Data are reported as means SE (n = 4; one-way ANOVA followed by Bonferronis post hoc test; *,#p < 0.01 for the uptake and biotinylation data, respectively, relative to values at the 24 h DAT induction time). (C) Representative DAT-mediated transient current stimulated by 10 lM AMPH. The transient current was recorded from a hDAT cell at the on of a voltage step. Transient charge (Q) was obtained by integrating the DAT-mediated transient current. (D) Inducible hDAT cells were used to correlate the transient charge stimulated by AMPH with DAT cell surface expression measured by biotinylation during parallel induction times. The AMPH-induced DAT Q was normalized to the initial DAT Q in the absence of AMPH. Biotinylated DAT was normalized as in (B). Data are expressed as mean SE; (n = 45; one-way ANOVA followed by Bonferronis post hoc test; *,#p < 0.01 for the transient charge and biotinylation data, respectively, relative to values at the 24 h DAT induction time).

efux recorded immediately after the transient measurements (Fig. 4B). DAT-mediated DA efux was measured at +100 mV. Changes in DAT cell surface expression strongly correlate with changes in AMPH-induced DA efux. These data further support our hypothesis that inhibition of PI3K signaling results in reduced DAT cell surface expression and, as a consequence, attenuates AMPH-induced DA efux. Discussion Insulin/PI3K signaling has been widely acknowledged to regulate dopaminergic neurotransmission, but the underlying molecular mechanism has been under considerable debate. Importantly, PI3K-dependent signaling has been implicated in the regulation of psychostimulant abuse [15]. Here, we demonstrate that pharmacological inhibition of PI3K signaling reduces the ability of AMPH to evoke DAT-mediated ionic currents and DA efux as measured by whole cell patchclamp and amperometry, respectively (Fig. 1). We show for the rst time, in a single DA neuron, that this decrease in AMPH-induced currents and DA efux is associated with a loss of DAT plasma membrane expression (Fig. 3).

Establishing a strong correlation between DAT cell surface expression and transporter function in a single neuron has been restricted by the methodologies employed. Indeed, while effective for measuring the average characteristics from a population of transporters, techniques such as [3H]DA uptake and cell surface biotinylation are restricted by a low time resolution and detection limits. In addition, important cellular aspects driving transporter function (e.g., voltage and ionic gradients) cannot be accurately controlled with these experimental techniques. To circumvent these obstacles, we utilized DAT-mediated transient current analysis, which allows for determination of DAT cell surface expression at the single cell level. Several lines of evidence from these current studies support the conclusion that the cocaine-sensitive transient currents originate from DAT and correlate with DAT cell surface expression: (1) they are absent in untransfected cells and DAT knockout neurons (data not shown), (2) they are blocked by cocaine, a pharmacological inhibitor of DAT activity, and (3) the DAT-mediated Q correlates with cell surface DAT over a wide range of expression levels (Fig. 2). Similar to what we have reported for DAT [8] and what others have shown for the c-aminobutyric acid transporter GAT-1 [18], the DAT-mediated transient currents in DA neurons appear to be capacitive in nature. This is because the

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AMPH

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AMPH

AMPH + LY

Fig. 3. PI3K signaling sustains DAT plasma membrane expression in DA neurons. Representative DAT-mediated transient currents from DA neurons for the on (A) and off (B) of a voltage step to 140 mV from a holding potential of 20 mV. Transient currents were measured in the presence of either 10 lM AMPH (AMPH) or 10 lM AMPH followed by 50 lM LY294002 (AMPH + LY). Quantitation of DAT-mediated transient currents for the on (C) and off (D) of a voltage step to 140 mV from a holding potential of 20 mV in response to either 10 lM AMPH (open bar) or 10 lM AMPH followed by 50 lM LY294002 (closed bar). The AMPH-induced DAT Q was normalized to the transient current recoded before AMPH treatment (QCTR). Data points represent mean SE (n = 3; unpaired t test; *p < 0.05).

A
1.2

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LY
Transient Charge (Q/Qinitial)
r = 0.81 p < 0.0001

1.2 1.0 0.8 0.6 0.4 0.2 0.0 0.0

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r = 0.81 p < 0.0001

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DA Efflux (I/Iinitial)

Fig. 4. PI3K signaling regulates both DAT cell surface expression and AMPH-induced DA efux. (A) Time-dependent decrease of DAT-mediated transient charge in the presence of 10 lM AMPH plus 50 lM LY294002 (n = 5). Time zero is dened as the DAT-mediated Q recorded immediately after bath application of LY294002 (LY). The AMPH-induced transient charge was generated by a voltage jump from 20 to 140 mV and normalized to the Q calculated at time zero for the on of the voltage step. (B) Changes in DAT membrane expression measured by transient charge analysis from (A) are plotted against DAT-mediated, AMPH-induced DA efux recorded during subsequent voltage steps to +100 mV. Data were normalized to time zero dened as the DAT-mediated transient charge movement and DA efux recorded immediately after bath application of LY294002. Each symbol in (A,B) signies individual recordings representative of ve experiments.

on and off charge movements are equal and opposite in sign (Fig. 3). By utilizing transient current analysis we are able to measure LY294002-induced changes in both DAT activity and plasma membrane expression from a single neuron, simultaneously. Here, we show in DA neurons that LY294002, but not the inactive analog LY303511, decreases AMPH-induced DA efux and io-

nic currents. The reduction in AMPH-induced ionic currents and DA efux in response to LY294002 could result either from modication of transporter activity on the cell surface, changes in DAT cell surface expression, or a combination of both. To discriminate between these possibilities, we correlate over time both the AMPH-induced transient currents and DA efux in response to

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voltage steps (Fig. 4). Our data strongly suggest that the reduction in substrate-induced currents and DA efux originate from changes in DAT cell surface expression. This is because: (a) LY294002 does not cause a signicant change in the reversal potential for DAT-mediated ionic currents in either hDAT cells or DA neurons (Fig. 1); and (b) the decrease we measure for AMPH-induced ionic currents and DA efux in response to LY294002 is temporally correlated with a concomitant reduction in DAT cell surface expression as quantied by transient current analysis. Importantly, our data show that LY294002-mediated inhibition of PI3K signaling impairs the ability of AMPH to increase extracellular DA levels. Collectively, these data support the hypothesis that PI3K signaling sustains DAT at the plasma membrane, which is required for AMPH to stimulate DA efux. Therefore, PI3K signaling may provide a new cellular target for the development of novel treatments of AMPH abuse and regulation of dopaminergic tone. Acknowledgment We thank Dr. Marc G. Caron for providing DAT knockout mice. References
[1] B. Giros, M.G. Caron, Molecular characterization of the dopamine transporter, Trends Pharmacol. Sci. 14 (1993) 4349. [2] D. Sulzer, M.S. Sonders, N.W. Poulsen, A. Galli, Mechanisms of neurotransmitter release by amphetamines: a review, Prog. Neurobiol. 75 (2005) 406433. [3] W.A. Owens, R.J. Sevak, R. Galici, X. Chang, M.A. Javors, A. Galli, C.P. France, L.C. Daws, Decits in dopamine clearance and locomotion in hypoinsulinemic rats unmask novel modulation of dopamine transporters by amphetamine, J. Neurochem. 94 (2005) 14021410. [4] D.P. Figlewicz, S.B. Evans, J. Murphy, M. Hoen, D.G. Baskin, Expression of receptors for insulin and leptin in the ventral tegmental area/substantia nigra (VTA/SN) of the rat, Brain Res. 964 (2003) 107115. [5] C. Taha, A. Klip, The insulin signaling pathway, J. Membr. Biol. 169 (1999) 112.

[6] B. Garcia, Y. Wei, J.A. Moron, R.Z. Lin, J.A. Javitch, A. Galli, Akt is essential for insulin modulation of amphetamine- induced human dopamine transporter cell surface redistribution, Mol. Pharmacol. (2005). [7] N. Rowland, J.N. Joyce, L.L. Bellush, Stereotyped behavior and diabetes mellitus in rats: reduced behavioral effects of amphetamine and apomorphine and reduced in vivo brain binding of [3H]spiroperidol, Behav. Neurosci. 99 (1985) 831841. [8] K.M. Kahlig, J.A. Javitch, A. Galli, Amphetamine regulation of dopamine transport. Combined measurements of transporter currents and transporter imaging support the endocytosis of an active carrier, J. Biol. Chem. 279 (2004) 89668975. [9] D.Q. Zhang, J.F. Stone, T. Zhou, H. Ohta, D.G. McMahon, Characterization of genetically labeled catecholamine neurons in the mouse retina, Neuroreport 15 (2004) 17611765. [10] B. Giros, M. Jaber, S.R. Jones, R.M. Wightman, M.G. Caron, Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter, Nature 379 (1996) 606612. [11] H. Khoshbouei, H. Wang, J.D. Lechleiter, J.A. Javitch, A. Galli, Amphetamineinduced dopamine efux. A voltage-sensitive and intracellular Na+ dependent mechanism, J. Biol. Chem. 278 (2003) 1207012077. [12] J.U. Fog, H. Khoshbouei, M. Holy, W.A. Owens, C.B. Vaegter, N. Sen, Y. Nikandrova, E. Bowton, D.G. McMahon, R.J. Colbran, L.C. Daws, H.H. Sitte, J.A. Javitch, A. Galli, U. Gether, Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport, Neuron 51 (2006) 417429. [13] S.L. Ingram, B.M. Prasad, S.G. Amara, Dopamine transporter-mediated conductances increase excitability of midbrain dopamine neurons, Nat. Neurosci. 5 (2002) 971978. [14] H. Khoshbouei, N. Sen, B. Guptaroy, L. Johnson, D. Lund, M.E. Gnegy, A. Galli, J.A. Javitch, N-terminal phosphorylation of the dopamine transporter is required for amphetamine-induced efux, PLoS Biol. 2 (2004) 387393. [15] J.M. Williams, W.A. Owens, G.H. Turner, C. Saunders, C. Dipace, R.D. Blakely, C.P. France, J.C. Gore, L.C. Daws, M.J. Avison, A. Galli, Hypoinsulinemia regulates amphetamine-induced reverse transport of dopamine, PLoS Biol. 5 (2007) 23692378. [16] K.M. Kahlig, F. Binda, H. Khoshbouei, R.D. Blakely, D.G. McMahon, J.A. Javitch, A. Galli, Amphetamine induces dopamine efux through a dopamine transporter channel, Proc. Natl. Acad. Sci. USA 102 (2005) 34953500. [17] L. Carvelli, J.A. Moron, K.M. Kahlig, J.V. Ferrer, N. Sen, J.D. Lechleiter, L.M. LeebLundberg, G. Merrill, E.M. Lafer, L.M. Ballou, T.S. Shippenberg, J.A. Javitch, R.Z. Lin, A. Galli, PI 3-kinase regulation of dopamine uptake, J. Neurochem. 81 (2002) 859869. [18] S. Mager, J. Naeve, M. Quick, C. Labarca, N. Davidson, H.A. Lester, Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes, Neuron 10 (1993) 177188.