Palmitoylation mechanisms in dopamine transporter regulation

Abstract

The neurotransmitter dopamine (DA) plays a key role in several biological processes including reward, mood, motor activity and attention. Synaptic DA homeostasis is controlled by the dopamine transporter (DAT) which transports extracellular DA into the presynaptic neuron after release and regulates its availability to receptors. Many neurological disorders such as schizophrenia, bipolar disorder, Parkinson disease and attention-deficit hyperactivity disorder are associated with imbalances in DA homeostasis that may be related to DAT dysfunction. DAT is also a target of psychostimulant and therapeutic drugs that inhibit DA reuptake and lead to elevated dopaminergic neurotransmission. We have recently demonstrated the acute and chronic modulation of DA reuptake activity and DAT stability through S-palmitoylation, the linkage of a 16-carbon palmitate group to cysteine via a thioester bond. This review summarizes the properties and regulation of DAT palmitoylation and describes how it serves to affect various transporter functions. Better understanding of the role of palmitoylation in regulation of DAT function may lead to identification of therapeutic targets for modulation of DA homeostasis in the treatment of dopaminergic disorders.

Keywords: 2 Bromopalmitate; Acyl protein thioesterase; Palmitoyl acyl transferase; Phosphorylation; Posttranslational modification; Protein trafficking.

Fig. 1

Schematic diagram of the expanded membrane topology of DAT with 12 TMDs and intracellularly facing N- and C-termini. Vertical stacks of amino acids (blue, black and white) represent α-helical membrane spanning domains arranged in two antiparallel aligned pentahelical bundles (TMDs 1–5 and 6–10) with TMDs 11 and 12 located peripheral to these bundles. The DA binding site is positioned within the inner core (black) with critical residues contained within unwound segments found in the middle of TMD1 and TMD6 (white). The known sites of glycosylation (black branched sticks), phosphorylation within the PKC-domain containing Ser7 (blue circles) and palmitoylation (orange circle with red appendage) are shown. Potential additional sites of palmitoylation are shown as large orange circles. The C-terminal helix (mauve, FREKLAYA) is also shown with Ras-like GTPase Rin 1 bound (Rin, mauve) and red dashed lines depicting hydrogen bonding between Arg 587 and Ser 581.

Fig. 2

Illustration of the alternating access mechanism of DA transport. Substrate (S) and ions (Na⁺ and Cl⁻) access the binding site of the outward facing transporter via the open outer gate, the outer gate closes yielding the occluded state, which then transitions to the inward facing state releasing substrate and ions to the cytosol. The transporter then rectifies to the outward facing structure. Black sticks represent the gating network of amino acid side chains.

Fig. 3

Functional consequences of DAT palmitoylation. The cycle of DAT palmitoylation and depalmitoylation by palmitoyl acyltransferases (green, PAT / DHHC) and acyl protein thioesterases (yellow, APT), respectively, is shown. Identified palmitoylation site Cys580 (red, PAL) and the reciprocally regulated Ser 7 phosphorylation site (dark blue, P) are also shown. DAT destined for degradation is directed toward the lysosome via an unknown sorting mechanism which could include depalmitoylation by lysosomal localized palmitoyl-protein thioesterase (PPT, pink).