Dopamine transporter

The dopamine transporter (also dopamine active transporter, DAT, SLC6A3) is a membrane spanning protein that binds the neurotransmitter dopamine and moves it from the synapse into a neuron. When DAT binds to dopamine, sodium and cloride ions on the outside of the cell, it undergoes a conformational change that releases both into the cell. An electrochemical gradient forces sodium into the cell and pulls dopamine along. DAT is present in the peri-synaptic area of dopaminergic neurons in areas of the brain where dopamine signaling is common. Because DAT terminates the dopamine signal, it is implicated in a number of dopamine related disorders including: Attention Deficit hyperactivity disorder, Bipolar Disorder, Depression, and Alcoholism. The gene that encodes the DAT protein is located on 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.

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.

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.

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. Chlorine 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 membrane potential and the sodium gradient, activity-induced changes in membrane polarity can dramatically influence transport rates. Additionally, 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 transmembrane domains (TMD) with a large extracellular loop between the third and fourth TMDs. Further characterization of this protein used proteases, which digest proteins into smaller fragments, and glycosylation, which only occurs on extracellular loops, and largely verified the initial predictions of membrane topology.

Location and distribution
Regional distribution of DAT has been found in areas of the brain with established dopaminergic circuitry including: mesostriatal, mesolimbic, and mesocortical pathways. 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.

Genetics & Regulation
The gene for DAT is located on chromosome 5p15. The protein encoding region of the gene is over 64 kb long and is comprised of 15 coding segments or exons. This gene has a variable number tandem repeat (VNTR) at the 3’ end. 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.

Nurr1, a 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 transcription factor Sp-1.

While transcription factors control which cells express DAT, functional regulation of this protein is largely accomplished by kinases. Both MAPK and PKC 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. These characteristics have striking similarities to the symptoms of ADHD.

Differences in the functional 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 alcoholism, although this is a point of controversy. Interestingly, an allele of the DAT gene with normal protein levels is associated with non-smoking behavior and ease of quitting.

Reduced levels of DAT in the brain are also associated with several different disorders, including depression and alcohol abuse. Decreasing levels of DAT expression are also associated with aging, and likely underlie a compensatory mechanism for the decreases in dopamine release as a person ages.

Pharmacology
DAT is also the target of several “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. 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.

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.