Dopamine transporter

The dopamine transporter (also dopamine active transporter, DAT, SLC6A3) is a membrane-spanning protein that pumps the is_associated_with::neurotransmitter is_associated_with::dopamine out of the synapse back into is_associated_with::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 is_associated_with::synapses, although there may be an exception in the prefrontal cortex, where evidence points to a possibly larger role of the is_associated_with::norepinephrine transporter. DAT is implicated in a number of dopamine-related disorders, including attention deficit hyperactivity disorder, is_associated_with::bipolar disorder, is_associated_with::clinical depression, and is_associated_with::alcoholism. The is_associated_with::gene that encodes the DAT protein is located on human chromosome 5, consists of 15 coding is_associated_with::exons, and is roughly 64 kbp long. Evidence for the associations between DAT and dopamine related disorders has come from a type of is_associated_with::genetic polymorphism, known as a is_associated_with::VNTR, in the DAT gene (is_associated_with::DAT1), which influences the amount of protein expressed.

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

Mechanism
DAT is a is_associated_with::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 is_associated_with::co-transport of two Na+ is_associated_with::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.

In the most widely accepted model for is_associated_with::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.

Studies using is_associated_with::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.

Because of the tight coupling of the is_associated_with::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.

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 is_associated_with::transmembrane domains (TMD) with a large extracellular loop between the third and fourth TMDs. Further characterization of this protein used is_associated_with::proteases, which digest proteins into smaller fragments, and glycosylation, which occurs only on extracellular loops, and largely verified the initial predictions of membrane topology. The exact structure of the transporter was elucidated in 2013 by X-ray crystallography.

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. The nuclei that make up these pathways have distinct patterns of expression. Gene expression patterns in the adult mouse show high expression in the is_associated_with::substantia nigra pars compacta.

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

Staining in the is_associated_with::striatum and is_associated_with::nucleus accumbens of the mesolimbic pathway was dense and heterogeneous. In the striatum, DAT is localized in the plasma membrane of axon terminals. Double is_associated_with::immunocytochemistry demonstrated DAT colocalization with two other markers of is_associated_with::nigrostriatal terminals, is_associated_with::tyrosine hydroxylase and D2 dopamine receptors. The latter was thus demonstrated to be an is_associated_with::autoreceptor on cells that release dopamine. is_associated_with::TAAR1 is a presynaptic intracellular receptor that is also colocalized with DAT and which has the opposite effect of the D2 autoreceptor when activated; i.e., it internalizes dopamine transporters and induces efflux through reversed transporter function via PKA and PKC signaling.

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 is_associated_with::substantia nigra, DAT appears is localized to axonal and dendritic (i.e., pre- and post-synaptic) is_associated_with::plasma membranes.

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

Genetics and regulation
The is_associated_with::gene for DAT, known as is_associated_with::DAT1, is located on chromosome 5p15. The protein encoding region of the gene is over 64 kb long and comprises 15 coding segments or is_associated_with::exons. This gene has a is_associated_with::variable number tandem repeat (VNTR) at the 3’ end (is_associated_with::rs28363170) and another in the is_associated_with::intron 8 region. Differences in the VNTR have been shown to affect the basal level of expression of the transporter; consequently, researchers have looked for associations with dopamine related disorders.

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

While transcription factors control which cells express DAT, functional regulation of this protein is largely accomplished by is_associated_with::kinases. is_associated_with::MAPK, PKA, and PKC can modulate the rate at which the transporter moves dopamine or cause the internalization of DAT. Co-localized is_associated_with::TAAR1 is an important regulator of the dopamine transporter that, when activated, phosphorylates DAT through is_associated_with::protein kinase A (PKA) and is_associated_with::protein kinase C (PKC) signaling. is_associated_with::Phosphorylation by either protein kinase can result in DAT internalization (non-competitive reuptake inhibition), but PKC-mediated phosphorylation alone induces reverse transporter function (dopamine efflux). Dopamine autoreceptors also regulate DAT by directly opposing the effect of TAAR1 activation. The human dopamine transporter, hDAT, has a high affinity zinc binding site that, upon binding Zn2+, facilitates is_associated_with::amphetamine induced efflux and inhibits reuptake of hDAT substrates.

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. These characteristics have striking similarities to the symptoms of is_associated_with::ADHD.

Differences in the functional is_associated_with::VNTR have been identified as risk factors for bipolar disorder and ADHD. Data has emerged that suggests there is also an association with stronger withdrawal symptoms from is_associated_with::alcoholism, although this is a point of controversy. Interestingly, an is_associated_with::allele of the DAT gene with normal protein levels is associated with non-smoking behavior and ease of quitting. 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.

Increased activity of DAT is associated with several different disorders, including is_associated_with::clinical depression.

Pharmacology
DAT is also the target of several "DAT-releasers" and “DAT-blockers” including is_associated_with::amphetamine and is_associated_with::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. In contrast, amphetamine enters the presynaptic neuron directly through the neuronal membrane or through DAT, competing for reuptake with dopamine. Once inside, it binds to TAAR1 or enters synaptic vesicles through VMAT2. When amphetamine binds to TAAR1, it reduces is_associated_with::dopamine receptor firing rate and triggers is_associated_with::protein kinase A and is_associated_with::protein kinase C signaling, resulting in DAT phosphorylation. Phosphorylated DAT then either operates in reverse or withdraws into the presynaptic neuron and ceases transport. When amphetamine enters the synaptic vesicles through VMAT2, dopamine is released into the cytosol.

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.

Ligands







 * is_associated_with::LBT-999
 * RTI-470

Interactions
Dopamine transporter has been shown to interact with:
 * is_associated_with::Alpha-synuclein,
 * is_associated_with::PICK1, and
 * is_associated_with::TGFB1I1.