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Regulation of dopamine transporter function by the PDZ domain protein PICK1

Final Report Summary - PICK1 AND DAT (Regulation of dopamine transporter function by the PDZ domain protein PICK1)

Protein-protein interactions modulate the activity of the dopamine transporter (DAT) by influencing different conductive states of the transport protein. This type of regulation might be a novel way of modulating the activity of dopaminergic neurons because DAT-mediated conductances were shown to be strong enough to alter neuronal excitability in primary culture. It was accordingly the aim of this project to investigate the modulation of DAT-mediated transport associated current in response to amphetamine (AMPH) when the transporter was in a complex with the SNARE protein syntaxin 1A (Syn1A). Moreover, our goal was to analyze the DAT/Syn1A complex in relation to the scaffolding protein PICK1 (protein interacting with C kinase 1). We first tested the effect of intracellular dopamine (DA) on DAT/Syn1A complex-mediated current. Bath application of AMPH elicited an inward current at negative potentials that was enhanced by intracellular DA in a concentration dependent way. Increasing the DA internal concentration from 1 to 2 mM doubled the DAT-specific current recorded in response to AMPH at - 100 mV with a 30 mM Na+ internal solution. When expressed in complex with Syn1A, the DAT mediated the release of DA from the cells at the physiological potential of - 60 mV as was shown by amperometry experiments in HEK 293 cells. In the same experimental conditions, we were able to identify a DAT-mediated current that likely is associated to the efflux of DA because of its dependence on the intracellular concentration of DA.

A possible role of PICK1 in DAT/Syn1A complex regulation was also investigated. Endogenous expression of PICK1 was verified by western blot that revealed strong expression in CAD cells. To highlight the role played by PICK1, we tested the effect of siRNA mediated knock-down of PICK1 on the DAT/Syn1A-mediated DA efflux current. The siRNA treated cells revealed a DAT/Syn1A-associated DA efflux current that exceeded the one characterizing the DAT/Syn1A complex likely bound to PICK1. Knocking down of PICK1 caused a 3-fold increase in the DA efflux current elicited by AMPH at all potentials tested. A stronger DA efflux current was also associated to the DAT+Ala mutant. As for the wild type (WT) transporter, increasing the intracellular DA concentration positively regulated the AMPH-induced current associated to the transporter/Syn1A complex. The lost ability of the +Ala mutant to bind the scaffolding protein is reflected by the stronger DA efflux current when compared to the WT. According to our data, PICK1 negatively regulates the activity of the transporter when bound to Syn1A. Previous work carried on in the lab showed a negative impact of phosphorylation on Syn1A binding to the DAT. The DAT complex composition could influence the degree of phosphorylation by regulating the transporter accessibility to different kinases. Due to its ability to bind PKC, PICK1 could facilitate a PKC-dependent phosphorylation of the transporter that might result in a decreased interaction of Syn1A with DAT. Release of Syn1A from the complex should result in a diminished DA efflux in response to AMPH. The DAT/Syn1A-mediated DA efflux current was measured in presence of the PKC inhibitor Go6983. As expected, blocking the kinase enhances the DA efflux current associated to the complex supporting the negative regulation proposed for this enzyme.

Our data demonstrate for the first time how multiple binding partners of DAT can cooperate to modulate the activity of the transporter in response to AMPH. More experiments are needed to gain a full description of the proposed model. Nevertheless, the work has opened the way to a new research line that has the potential to provide a novel description of DAT activity as result of multiple interactions of the transporters with its binding partners.