Dopamine transporter

The dopamine transporter (also dopamine active transporter, DAT, SLC6A3) is a membrane-spanning protein that pumps the neurotransmitter dopamine out of the synapse back into cytosol, from which other transporters sequester DA and NE into vesicles for later storage and release. Dopamine reuptake via DAT provides the primary mechanism through which dopamine is cleared from synapses except in the prefrontal cortex, where dopamine uptake via the norepinephrine transporter plays that role.^[1][2]

DAT is thought to be implicated in a number of dopamine-related disorders, including attention deficit hyperactivity disorder, bipolar disorder, clinical depression, and alcoholism. The gene that encodes the DAT protein is located on human chromosome 5, consists of 15 coding exons, and is roughly 64 kbp long. Evidence for the associations between DAT and dopamine related disorders has come from a genetic polymorphism in the DAT gene, which influences the amount of protein expressed.

Function

DAT is an integral membrane protein that removes dopamine from the synaptic cleft and deposits it into surrounding cells, thus terminating the signal of the neurotransmitter. Dopamine underlies several aspects of cognition, including reward, and DAT facilitates regulation of that signal.^[3]

Mechanism

DAT is a symporter that moves dopamine across the cell membrane by coupling the movement to the energetically-favorable movement of sodium ions moving from high to low concentration into the cell. DAT function requires the sequential binding and co-transport of two Na+ ions and one Cl- ion with the dopamine substrate. The driving force for DAT-mediated dopamine reuptake is the ion concentration gradient generated by the plasma membrane Na+/K+ ATPase.^[4]

In the most widely-accepted model for monoamine transporter function, sodium ions must bind to the extracellular domain of the transporter before dopamine can bind. Once dopamine binds, the protein undergoes a conformational change, which allows both sodium and dopamine to unbind on the intracellular side of the membrane.^[5]

Studies using electrophysiology and radioactive-labeled dopamine have confirmed that the dopamine transporter is similar to other monoamine transporters in that one molecule of neurotransmitter can be transported across the membrane with one or two sodium ions. Chloride ions are also needed to prevent a buildup of positive charge. These studies have also shown that transport rate and direction is totally dependent on the sodium gradient.^[6]

Because of the tight coupling of the membrane potential and the sodium gradient, activity-induced changes in membrane polarity can dramatically influence transport rates. In addition, the transporter may contribute to dopamine release when the neuron depolarizes.^[6]

Protein Structure

The initial determination of the membrane topology of DAT was based upon hydrophobic sequence analysis and sequence similarities with the GABA transporter. These methods predicted twelve transmembrane domains (TMD) with a large extracellular loop between the third and fourth TMDs.^[7] Further characterization of this protein used proteases, which digest proteins into smaller fragments, and glycosylation, which occurs only on extracellular loops, and largely verified the initial predictions of membrane topology.^[8]

Location and distribution

Regional distribution of DAT has been found in areas of the brain with established dopaminergic circuitry including: nigrostriatal, mesolimbic, and mesocortical pathways.^[9] The nuclei that make up these pathways have distinct patterns of expression.

DAT in the mesocortical pathway, labeled with radioactive antibodies, was found to be enriched in dendrites and cell bodies of neurons in the substantia nigra pars compacta and ventral tegmental area. This pattern makes sense for a protein that regulates dopamine levels in the synapse.

Staining in the striatum and nucleus accumbens of the mesolimbic pathway was dense and heterogeneous. In the striatum, DAT is localized in the plasma membrane of axon terminals. Double immunocytochemistry demonstrated DAT colocalization with two other markers of nigrostriatal terminals, tyrosine hydroxylase and D2 dopamine receptors. The latter was thus demonstrated to be an autoreceptor on cells that release dopamine.

Surprisingly, DAT was not identified within any synaptic active zones. These results suggest that striatal dopamine reuptake may occur outside of synaptic specializations once dopamine diffuses from the synaptic cleft.

In the substantia nigra, DAT appears to be specifically transported into dendrites, where it can be found in smooth endoplasmic reticulum, plasma membrane, and pre- and postsynaptic active zones. These localizations suggest that DAT modulates the intracellular and extracellular dopamine levels of nigral dendrites.

Within the perikarya of pars compacta neurons, DAT was localized primarily to rough and smooth endoplasmic reticulum, Golgi complex, and multivesicular bodies, identifying probable sites of synthesis, modification, transport, and degradation.^[10]

Genetics and regulation

The gene for DAT is located on chromosome 5p15.^[11] The protein encoding region of the gene is over 64 kb long and comprises 15 coding segments or exons.^[12] This gene has a variable number tandem repeat (VNTR) at the 3’ end (rs28363170).^[13] Differences in the VNTR have been shown to affect the basal level of expression of the transporter; consequentially, researchers have looked for associations with dopamine related disorders.^[14]

Nurr1, a nuclear receptor that regulates many dopamine related genes, can bind the promoter region of this gene and induce expression.^[15] This promoter may also be the target of the transcription factor Sp-1.

While transcription factors control which cells express DAT, functional regulation of this protein is largely accomplished by kinases. Both MAPK^[16] and PKC^[17] can modulate the rate at which the transporter moves dopamine or cause the internalization of DAT.

Biological role and disorders

The rate at which DAT removes dopamine from the synapse can have a profound effect on the amount of dopamine in the cell. This is best evidenced by the severe cognitive deficits, motor abnormalities, and hyperactivity of mice with no dopamine transporters.^[18] These characteristics have striking similarities to the symptoms of ADHD.

Differences in the functional VNTR have been identified as risk factors for bipolar disorder^[19] and ADHD.^[20] Data has emerged that suggests there is also an association with stronger withdrawal symptoms from alcoholism, although this is a point of controversy.^[21][22] Interestingly, an allele of the DAT gene with normal protein levels is associated with non-smoking behavior and ease of quitting.^[23] Additionally, male adolescents particularly those in high-risk families (ones marked by a disengaged mother and absence of maternal affection) who carry the 10-allele VNTR repeat show a statistically significant affinity for antisocial peers.^[24]

Increased activity of DAT is associated with several different disorders, including clinical depression.^[25] Decreasing levels of DAT expression are associated with aging, and likely underlie a compensatory mechanism for the decreases in dopamine release as a person ages.^[26]

Pharmacology

DAT is also the target of several "DAT-releasers" & “DAT-blockers” including amphetamines and cocaine. These chemicals inhibit the action of DAT and, to a lesser extent, the other monoamine transporters, but their effects are mediated by separate mechanisms.

Cocaine blocks DAT by binding directly to the transporter and reducing the rate of transport.^[7] In contrast, amphetamines trigger a signal cascade thought to involve PKC or MAPK that leads to the internalization of DAT molecules, which are normally expressed on the neuron’s surface.^[27]

Amphetamine on DAT also has a direct effect in the increased levels of secreted dopamine. Lipophilic AMPH diffuses into the cytoplasm and into the dopamine secretory vesicles disrupting the proton gradient established across the vesicle wall. This induces a leaky channel and DA diffuses out into the cytoplasm. Additionally, AMPH causes a reversal of normal DA flow at the DAT. Instead of DA reuptake, in the presence of AMPH, a reversal in the mechanism of DAT occurs causing an outflow of dopamine released into the cytoplasm into the synaptic space changing it from a symporter to an antiporter-like functionality.^{[28][29][30][31]}

Both of these mechanisms result in less removal of dopamine from the synapse and increased signaling, which is thought to underlie the pleasurable feelings elicited by these substances.^[3]

Ligands

• LBT-999^{[32][33][34][35][36]}
• RTI-470^[37]

Interactions

Dopamine transporter has been shown to interact with TGFB1I1,^[38] PICK1^[39] and Alpha-synuclein.^[40][41]

See also

• Norepinephrine transporter
• Serotonin transporter
• Glutamate transporter

References

External links

• Dopamine transporter-related Associations, Experiments, Publications and Clinical Trials
• MeSH Dopamine+Transporter