Dopamine Proteins

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 Dopamine Proteins Background

The neurotransmitter Dopamine (DA) regulates cognition, voluntary movement, motivation, and reward. As illustrated in Figure 1, DA is synthesized in the cell bodies of dopaminergic neurons located in the substantia nigra (SN) and the ventral tegmental area (VTA) of the brain. Upon depolarization of the presynaptic terminal, voltage-gated calcium channels (CaVs) activate and allow calcium (Ca2+) to enter and to interact with proteins required for vesicular fusion and exocytotic DA release. DA signaling is a balance of neurotransmitter release during tonic and phasic activity of dopaminergic neurons and its clearance from the cleft. The primary mechanism of clearance is reuptake through the dopamine transporter (DAT, SLC6A), and to a lesser extend degradation by enzymes and diffusion away from the synapse. The plasma membrane protein dopamine transporter (DAT) regulates DA signal transduction and homeostasis by transporting the released synaptic neurotransmitter into the presynaptic neuron using Na+ and Cl- electrochemical gradients. Within dopaminergic neurons in the central nervous system (CNS) another transporter, the vesicular monoamine transporter 2 (VMAT2), uses a proton gradient to recycle and concentrate cytosolic DA back in synaptic vesicles for subsequent release. DATs are strategically located in the soma, dendrites, and perisynaptic areas of dopaminergic neurons. Their proper function and location are crucial for dopaminergic signaling.

Dopaminergic synapse

Figure 1 Dopaminergic synapse. DAT is located at the perisynaptic area of dopaminergic terminal. It transports released synaptic DA back in the presynaptic cell to be repackaged in vesicles for subsequent release. AMPH and COC interact directly with DAT. AMPH acts as substrate and is transported, while COC binds the transporter and arrests its function. Figure adapted from.

Dysfunction in transporter activity or expression levels that affect DA concentration in different locations of the brain may lead to neurological, psychiatric, and neuroendocrine disorders. For example, augmentation of DA in the prefrontal cortex (PFC) is linked to attention deficit hyperactivity disorder (ADHD) and schizophrenia, while decreased DA levels in the striatum are associated with Parkinson’s and Huntington’s disease. Increased levels of DA in the nucleus accumbens (NAcc) has been implicated in drug addiction. Albeit through different mechanisms, all classes of abused drugs increase extracellular levels of DA particularly in NAcc. The released DA activate DA receptors which signal higher brain regions, e.g., the prefrontal cortex, to translate motivational input into behavioral output. The importance of DAT in the psychostimulant dependence is confirmed in animals with disrupted mesolimbic dopaminergic system (via knock-outs or lesions) or dopamine receptor antagonists, where abuse related effects of psychostimulants can be eliminated. Moreover, in DAT knock-in mice enhanced psychostimulant reinforcement is observed.


DAT function

DAT is the primary mediator of synaptic DA clearance. The transport process is Na+ and Cl- dependent and it is driven by Na+ electrochemical gradient. Transporter function is classically described as following the alternating access model (AAM) originally defined in 1966 by Jardetzky for ATPase pumps and since adopted for co-transporters. Flux studies revealed Michaelis-Menten type relationship between transport and concentration of DA, Na+, and Cl-. Independent studies from Krueger and Schenk’s laboratory found Na+ concentration dependence on uptake was sigmoidal while Cl- concentration dependence was rectangular hyperbola, hence assigning a transport stoichiometry of 1DA: 2Na+: 1Cl- per transport cycle. Using radiolabeled ligand uptake and irreversible binding assays, transporter turnover rate was calculated to be one full cycle every two seconds; therefore, using the proposed stoichiometry and because DA is a monovalent cation at physiological pH, one positive charge enters the cell every second through DAT. Thus, the transporter is predicted to be electrogenic and the current generated by 1 million transporters acting simultaneously would be 0.16 pA. Nevertheless, numerous reports exist of the dopamine transporter eliciting much larger currents, well beyond the prediction of AAM. In addition, Sonders et. al. report a variable number of charges moving though DAT at different membrane potentials, thus contradicting the fixed stoichiometry model and proposing transport-uncoupled DAT current. The unaccounted for DAT currents in addition to the fast reuptake of DA required at the synapse suggest for an alternative and/or combined ion channel/transporter mechanism of action. DATgenerated currents through the ion channel mode are sufficient to affect membrane potential and excitability. Because a number of psychostimulants interact with DAT to produce or alter its ionic currents. The mechanisms by which STPs control DAT activity and modulate membrane potential is of particular interest.


DAT regulation

DAT activity is contingent upon its presence at the cell membrane. DAT is synthesized in the endoplasmic reticulum (ER), transported to the Golgi for N-glycosylation at the 2nd extracellular loop and trafficked to the plasma membrane. Different signaling pathways have shown effect on transporter cell surface expression both constitutively and upon stimulation. DAT has several consensus sites for posttranslational modifications at its cytosolic amino (N) and carboxyl (C) terminus. On the N terminus a cluster of serine residues are involved in PKC phosphorylation, which is important in DA efflux. Deletions and mutations of these residues on DAT results in reduced phosphorylation and internalization of the protein upon PKC activation. There is evidence for Ca2+/Calmodulin-dependent protein kinase II interactions with the C-terminus of DAT that regulate DA efflux. Residues 587-596 in DAT’s carboxyl terminus play a role in its constitutive protein internalization. More recent studies show that DAT is regulated by PIP2 suggesting the importance of membrane lipid composition in transporter function. DAT substrates like AMPH and METH cause DAT down-regulation, while DAT reuptake inhibitors or blockers cause transporter up-regulation as determined by immunofluorescence confocal microscopy in mammalian expression system. The reduction and rise of DAT at the plasma membrane can be further monitored using voltage clamp techniques where current amplitude and kinetics can be measured for functional transporters. Moreover, DAT regulation by psychostimulants was confirmed by postmortem binding studies in cocaine overdose victims, showing higher number DAT binding sites. The numerous DAT regulation mechanisms contribute to an already complex picture of DAT function and are an active area of research aiming at understanding drug dependence.