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. 2008 Oct;74(4):1101-8.
doi: 10.1124/mol.108.048447. Epub 2008 Jul 10.

Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux

Francesca Binda  1 , Concetta Dipace, Erica Bowton, Sabrina D Robertson, Brandon J Lute, Jacob U Fog, Minjia Zhang, Namita Sen, Roger J Colbran, Margaret E Gnegy, Ulrik Gether, Jonathan A Javitch, Kevin Erreger, Aurelio Galli
Affiliations

Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux

Francesca Binda et al. Mol Pharmacol. 2008 Oct.

Abstract

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells. By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At -60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 microM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at -60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.

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Figures

Figure 1
Figure 1. SYN1A directly interacts with hDAT
A) GST-pull-down experiments confirm a direct interaction between SYN1A and hDAT and demonstrate that the hDAT N-terminus mediates this interaction. Purified SYN1AΔTM was incubated with GST alone or with GST-hDAT N-terminal fusion proteins comprised of the indicated residue numbers. The samples were then immunoblotted for SYN1A. Each of the three fusion proteins tested showed a signal for SYN1A and showed no signal for GST alone. The experiment displayed is representative of 5 experiments. B) An SDS-PAGE gel of the GST fusion proteins used in A stained with GelCode Blue demonstrating fusion proteins of the correct size with minimal degradation.
Figure 2
Figure 2. AMPH promotes DAT/SYN1A association
A) hDAT cells transfected with SYN1A or mouse synaptosomes were treated with 10 µM AMPH for 5 min at 37°C. DAT proteins were immunoprecipitated and immunoblotted for SYN1A. B) Surface fractions were isolated by biotinylation. DAT proteins were immunoprecipitated and immunoblotted for SYN1A. C) The immunoprecipitated band densities were quantified, normalized to the corresponding density of total SYN1A (not shown) and expressed as a percentage of vehicle control. (n=3–7; *p<0.05, Student’s t-test).
Figure 3
Figure 3. Overexpression of SYN1A potentiates AMPH-induced efflux from hDAT cells
hDAT cells transiently transfected with either empty vector or SYN1A were incubated with 15µM DA for 30 min at 37°C to load the cells with DA. Cells were placed in a perfusion apparatus with a flow rate of 0.4 mL/min. DA efflux was measured in effluent fractions collected every 2 min. A 2 min application of 10µM AMPH was given at the time indicated. Results are calculated as the fractional release of total cellular DA per 2 min fraction in vector-transfected (○) or SYN1A-transfected (▪) cells. Total cellular DA (in nmol/mg protein) at the begining of the experiment in the vector- and SYN1A-transfected cells was 13.1 ± 2.0 and 9.7 ± 1.2, respectively (p >0.05, Student’s t-test). In a two-way ANOVA, p = 0.002 for the vector, p < 0.0001 for the time and the interaction between vector and time. (n=3; * p<0.01 post-hoc Bonferroni test).
Figure 4
Figure 4. SYN1A supports AMPH-induced efflux from hDAT cells and from DA neurons at physiological voltage
A). hDAT cells were voltage clamped in the whole-cell configuration at −60 mV. The solution in the patch electrode contained either GST alone or a GST- SYN1AΔTM fusion protein (“GST-SYN1A”). A carbon fiber amperometric electrode was placed onto the cell to record oxidation currents due to DA efflux from the cell. AMPH (10 µM) was added to the bath at the time indicated. Cocaine (COC; 10 µM) was added to the bath to establish the baseline (dashed line) for DAT-specific DA efflux. B) Cultured mouse midbrain DA neurons were voltage clamped in the whole-cell configuration at −60 mV. AMPH (10 µM) and COC (10 µM) were added to the bath at the time indicated. C) AMPH induced a significant change in DA efflux from hDAT cells in the presence of GST-SYN1A but not in the GST control (n=4–6; *p<0.05; paired Student’s t test). D) AMPH induces a significant change in DA efflux from DA neurons in the presence of GST-SYN1A but not in the GST control (n=3; *p<0.05; paired Student’s t-test).
Figure 5
Figure 5. CAMKII is required for AMPH-induced SYN1A/DAT interaction and DA efflux
A) hDAT cells transfected with SYN1A were treated with 10 µM AMPH for 5 min at 37°C. FLAG-tagged hDAT proteins were immunoprecipitated from the biotinylated fraction and immunoblotted for SYN1A. AMPH-induced DAT/SYN1A interaction at the plasma membrane is blocked by pre-incubation (30 min) with 5 µM KN93 or 5 µM CaMKIINTIDE. B) The immunoprecipitated band densities were quantified, normalized to the density of the corresponding total SYN1A, and expressed as AMPH band density normalized to the corresponding control without AMPH (n=4–7; *p<0.05, paired Student’s t-test). C) hDAT cells were voltage clamped in the whole-cell configuration at −60 mV. The patch electrode contained either GST or GST- SYN1AΔTM. A carbon fiber amperometric electrode was juxtaposed to the plasma membrane to record oxidation currents due to DA efflux from the cell. Efflux before and after exposure to AMPH was measured as in Fig. 4A relative to the efflux following COC application. Pre-incubation (5 min) with 5 µM KN93 prevented exogenous SYN1A from supporting AMPH-induced DA efflux at −60 mV.
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