Syntaxin 1A Interaction with the Dopamine Transporter Promotes Amphetamine-Induced Dopamine Efflux

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 Ϫ60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 M) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at Ϫ60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca 2ϩ /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.

Membrane-permeable C-terminal Dopamine Transporter Peptides Attenuate Amphetamine-evoked Dopamine Release*

“…Inhibition of CaMKII␣ activity using a membrane-permeable CaMKII␣ inhibitor decreased the interaction between DAT and syntaxin1A. Importantly, similar effects were obtained by inhibiting CaMKII␣ with KN-93, and it was hypothesized that AMPH-mediated activation of CaMKII␣ increases DAT-syntaxin1A interactions resulting in reversed operation of DAT (45). This is in accordance with our data showing attenuated DA release upon TAT peptides abolishing DAT C-terminal interactions, and it emphasizes the importance of CaMKII␣ for AMPH-induced DA efflux.…”

SLC6 Neurotransmitter Transporters: Structure, Function, and Regulation

“…Compelling evidence suggests that the N termini of SLC6 NTTs interact directly with syntaxin1A, possibly through an interaction with the SNARE motif H3 (Deken et al, 2000;Quick, 2003;Lee et al, 2004). For DAT, the interaction site is contained within the first 33 residues of the N terminus (Binda et al, 2008), whereas residues 30 to 54 of the GAT1 N-terminal domain were shown to interact directly with syntaxin1A (Deken et al, 2000) (Fig. 10).…”

The rare DAT coding variant Val559 perturbs DA neuron function, changes behavior, and alters in vivo responses to psychostimulants

“…AMPH is structurally similar to DA and, as a result, is transported by DAT, competing with DA during the reuptake process. AMPH also induces DAT-mediated nonvesicular release, also termed "DA efflux," a process that involves the actions of intracellular signaling proteins such as CamKIIα (12)(13)(14)(15)(16) and PKCβ (17)(18)(19), alterations in interactions with DAT-associated proteins and phospholipids (12,14,20), and changes in DAT phosphorylation (12,16,(21)(22)(23)) that biases the transporter toward an efflux-competent mechanism. Despite their mechanistic differences, MPH and AMPH both rapidly elevate DA in the CNS and are components of the most frequently prescribed medications for ADHD, Ritalin and Adderall, respectively.…”