Dopamine transporter
Encyclopedia
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 synapse
s except in the prefrontal cortex, where dopamine uptake via the norepinephrine transporter
plays that role.
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.
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.
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+ ion
s 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 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. 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 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.
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 occurs only on extracellular loops, and largely verified the initial predictions of membrane topology.
, 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.
for DAT is located on chromosome 5p15.
The protein encoding region of the gene is over 64 kb long and comprises 15 coding segments or exons.
This gene has a variable number tandem repeat
(VNTR) at the 3’ end (rs28363170
).
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.
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. 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 clinical depression
. 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.
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.
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.
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.
with TGFB1I1
, PICK1
and Alpha-synuclein
.
Dopamine
Dopamine is a catecholamine neurotransmitter present in a wide variety of animals, including both vertebrates and invertebrates. In the brain, this substituted phenethylamine functions as a neurotransmitter, activating the five known types of dopamine receptors—D1, D2, D3, D4, and D5—and their...
transporter
Active transport
Active transport is the movement of a substance against its concentration gradient . In all cells, this is usually concerned with accumulating high concentrations of molecules that the cell needs, such as ions, glucose, and amino acids. If the process uses chemical energy, such as from adenosine...
(also dopamine active transporter, DAT, SLC6A3) is a membrane-spanning protein that pumps the neurotransmitter
Neurotransmitter
Neurotransmitters are endogenous chemicals that transmit signals from a neuron to a target cell across a synapse. Neurotransmitters are packaged into synaptic vesicles clustered beneath the membrane on the presynaptic side of a synapse, and are released into the synaptic cleft, where they bind to...
dopamine
Dopamine
Dopamine is a catecholamine neurotransmitter present in a wide variety of animals, including both vertebrates and invertebrates. In the brain, this substituted phenethylamine functions as a neurotransmitter, activating the five known types of dopamine receptors—D1, D2, D3, D4, and D5—and their...
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 synapse
Synapse
In the nervous system, a synapse is a structure that permits a neuron to pass an electrical or chemical signal to another cell...
s except in the prefrontal cortex, where dopamine uptake via the norepinephrine transporter
Norepinephrine transporter
The norepinephrine transporter , also known as solute carrier family 6 member 2 , is a protein that in humans is encoded by the SLC6A2 gene....
plays that role.
DAT is thought to be implicated in a number of dopamine-related disorders, including attention deficit hyperactivity disorder, bipolar disorder
Bipolar disorder
Bipolar disorder or bipolar affective disorder, historically known as manic–depressive disorder, is a psychiatric diagnosis that describes a category of mood disorders defined by the presence of one or more episodes of abnormally elevated energy levels, cognition, and mood with or without one or...
, clinical depression
Clinical depression
Major depressive disorder is a mental disorder characterized by an all-encompassing low mood accompanied by low self-esteem, and by loss of interest or pleasure in normally enjoyable activities...
, and alcoholism
Alcoholism
Alcoholism is a broad term for problems with alcohol, and is generally used to mean compulsive and uncontrolled consumption of alcoholic beverages, usually to the detriment of the drinker's health, personal relationships, and social standing...
. The gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
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 proteinIntegral membrane protein
An integral membrane protein is a protein molecule that is permanently attached to the biological membrane. Proteins that cross the membrane are surrounded by "annular" lipids, which are defined as lipids that are in direct contact with a membrane protein...
that removes dopamine from the synaptic cleft and deposits it into surrounding cells, thus terminating the signal of the neurotransmitter. Dopamine
Dopamine
Dopamine is a catecholamine neurotransmitter present in a wide variety of animals, including both vertebrates and invertebrates. In the brain, this substituted phenethylamine functions as a neurotransmitter, activating the five known types of dopamine receptors—D1, D2, D3, D4, and D5—and their...
underlies several aspects of cognition, including reward, and DAT facilitates regulation of that signal.
Mechanism
DAT is a symporterSymporter
A cotransporter is an integral membrane protein that is involved in secondary active transport. It works by binding to two molecules or ions at a time and using the gradient of one solute's concentration to force the other molecule or ion against its gradient....
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
Co-transport
Co-transport, also known as coupled transport or secondary active transport, refers to the simultaneous or sequential passive transfer of molecules or ions across biological membranes in a fixed ratio...
of two Na+ ion
Ion
An ion is an atom or molecule in which the total number of electrons is not equal to the total number of protons, giving it a net positive or negative electrical charge. The name was given by physicist Michael Faraday for the substances that allow a current to pass between electrodes in a...
s 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 monoamine transporter
Monoamine transporter
Monoamine transporters are protein structures that function as integral plasma membrane transporters to regulate concentrations of extracellular monoamine neurotransmitters. Three major classes of MATs are responsible for the reuptake of their associated amine neurotransmitters...
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
Electrophysiology
Electrophysiology is the study of the electrical properties of biological cells and tissues. It involves measurements of voltage change or electric current on a wide variety of scales from single ion channel proteins to whole organs like the heart...
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 membrane potential
Membrane potential
Membrane potential is the difference in electrical potential between the interior and exterior of a biological cell. All animal cells are surrounded by a plasma membrane composed of a lipid bilayer with a variety of types of proteins embedded in it...
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 analysisHydrophilicity plot
A hydrophilicity plot is a quantitative analysis of the degree of hydrophobicity or hydrophilicity of amino acids of a protein. It is used to characterize or identify possible structure or domains of a protein....
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 occurs only 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: nigrostriatalNigrostriatal pathway
The nigrostriatal pathway is a neural pathway that connects the substantia nigra with the striatum. It is one of the four major dopamine pathways in the brain, and is particularly involved in the production of movement, as part of a system called the basal ganglia motor loop.Loss of dopamine...
, mesolimbic
Mesolimbic pathway
The mesolimbic pathway is one of the dopaminergic pathways in the brain. The pathway begins in the ventral tegmental area of the midbrain and connects to the limbic system via the nucleus accumbens, the amygdala, and the hippocampus as well as to the medial prefrontal cortex...
, and mesocortical
Mesocortical pathway
The mesocortical pathway is a neural pathway that connects the ventral tegmentum to the cerebral cortex, particularly the frontal lobes. It is one of the four major dopamine pathways in the brain...
pathways. The nuclei
Nucleus (neuroanatomy)
In neuroanatomy, a nucleus is a brain structure consisting of a relatively compact cluster of neurons. It is one of the two most common forms of nerve cell organization, the other being layered structures such as the cerebral cortex or cerebellar cortex. In anatomical sections, a nucleus shows up...
that make up these pathways have distinct patterns of expression.
DAT in the mesocortical pathway
Mesocortical pathway
The mesocortical pathway is a neural pathway that connects the ventral tegmentum to the cerebral cortex, particularly the frontal lobes. It is one of the four major dopamine pathways in the brain...
, labeled with radioactive antibodies, was found to be enriched in dendrites and cell bodies of neurons in the substantia nigra
Substantia nigra
The substantia nigra is a brain structure located in the mesencephalon that plays an important role in reward, addiction, and movement. Substantia nigra is Latin for "black substance", as parts of the substantia nigra appear darker than neighboring areas due to high levels of melanin in...
pars compacta and ventral tegmental area. This pattern makes sense for a protein that regulates dopamine levels in the synapse.
Staining in the striatum
Striatum
The striatum, also known as the neostriatum or striate nucleus, is a subcortical part of the forebrain. It is the major input station of the basal ganglia system. The striatum, in turn, gets input from the cerebral cortex...
and nucleus accumbens
Nucleus accumbens
The nucleus accumbens , also known as the accumbens nucleus or as the nucleus accumbens septi , is a collection of neurons and forms the main part of the ventral striatum...
of the mesolimbic pathway was dense and heterogeneous. In the striatum, DAT is localized in the plasma membrane of axon terminals. Double immunocytochemistry
Immunocytochemistry
Immunocytochemistry is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes. These bound antibodies can then be detected using several different methods. ICC allows researchers to evaluate whether or not cells in a...
demonstrated DAT colocalization with two other markers of nigrostriatal terminals, tyrosine hydroxylase
Tyrosine hydroxylase
Tyrosine hydroxylase or tyrosine 3-monooxygenase is the enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to dihydroxyphenylalanine . It does so using tetrahydrobiopterin as a coenzyme. DOPA is a precursor for dopamine, which, in turn, is a precursor for norepinephrine ...
and D2 dopamine receptors. The latter was thus demonstrated to be an autoreceptor
Autoreceptor
An autoreceptor is a receptor located on presynaptic nerve cell membranes and serves as a part of a feedback loop in signal transduction. It is sensitive only to those neurotransmitters or hormones that are released by the neuron in whose membrane the autoreceptor sits.Canonically, a presynaptic...
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
Substantia nigra
The substantia nigra is a brain structure located in the mesencephalon that plays an important role in reward, addiction, and movement. Substantia nigra is Latin for "black substance", as parts of the substantia nigra appear darker than neighboring areas due to high levels of melanin in...
, 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
Pars compacta
-Anatomy:In humans, the nerve cell bodies of the pars compacta are coloured black by the pigment neuromelanin. The degree of pigmentation increases with age. This pigmentation is visible as a distinctive black stripe in brain sections and is the origin of the name given to this area. The neurons...
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 and regulation
The geneGene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
for DAT is located on chromosome 5p15.
The protein encoding region of the gene is over 64 kb long and comprises 15 coding segments or exons.
This gene has a variable number tandem repeat
Variable number tandem repeat
A Variable Number Tandem Repeat is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length between individuals. Each variant acts as an inherited allele, allowing them to be used for...
(VNTR) at the 3’ end (rs28363170
Rs28363170
In genetics, rs28363170 is a genetic variation at SLC6A3, the gene that encodes the dopamine transporter. It is polymorphism as a 40 base pairs VNTR in the 3' untranslated region.It is an deletion/insertion polymorphism ....
).
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
Nuclear receptor related 1 protein
The Nuclear receptor related 1 protein also known as NR4A2 is a protein that in humans is encoded by the NR4A2 gene. NURR1 is a member of the nuclear receptor family of intracellular transcription factors.NURR1 plays a key role in the maintenance of the dopaminergic system of the brain...
, a nuclear receptor
Nuclear receptor
In the field of molecular biology, nuclear receptors are a class of proteins found within cells that are responsible for sensing steroid and thyroid hormones and certain other molecules...
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
Transcription factor
In molecular biology and genetics, a transcription factor is a protein that binds to specific DNA sequences, thereby controlling the flow of genetic information from DNA to mRNA...
Sp-1.
While transcription factors control which cells express DAT, functional regulation of this protein is largely accomplished by kinases. Both MAPK and PKC
Protein kinase C
Protein kinase C also known as PKC is a family of enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues on these proteins. PKC enzymes in turn are activated by signals such as increases in...
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
Withdrawal
Withdrawal can refer to any sort of separation, but is most commonly used to describe the group of symptoms that occurs upon the abrupt discontinuation/separation or a decrease in dosage of the intake of medications, recreational drugs, and alcohol...
symptoms from alcoholism
Alcoholism
Alcoholism is a broad term for problems with alcohol, and is generally used to mean compulsive and uncontrolled consumption of alcoholic beverages, usually to the detriment of the drinker's health, personal relationships, and social standing...
, although this is a point of controversy. Interestingly, an allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
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 clinical depression
Clinical depression
Major depressive disorder is a mental disorder characterized by an all-encompassing low mood accompanied by low self-esteem, and by loss of interest or pleasure in normally enjoyable activities...
. 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.
Pharmacology
Cocaine
Cocaine is a crystalline tropane alkaloid that is obtained from the leaves of the coca plant. The name comes from "coca" in addition to the alkaloid suffix -ine, forming cocaine. It is a stimulant of the central nervous system, an appetite suppressant, and a topical anesthetic...
. 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.
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.
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
- LBT-999
- RTI-470
Interactions
Dopamine transporter has been shown to interactProtein-protein interaction
Protein–protein interactions occur when two or more proteins bind together, often to carry out their biological function. Many of the most important molecular processes in the cell such as DNA replication are carried out by large molecular machines that are built from a large number of protein...
with TGFB1I1
TGFB1I1
Transforming growth factor beta-1-induced transcript 1 protein is a protein that in humans is encoded by the TGFB1I1 gene.-Interactions:TGFB1I1 has been shown to interact with PTPN12, PTK2, Androgen receptor, PTK2B, Dopamine transporter and Hsp27....
, PICK1
PICK1
PRKCA-binding protein is a protein that in humans is encoded by the PICK1 gene.-Interactions:PICK1 has been shown to interact with HER2/neu, ACCN2, Metabotropic glutamate receptor 7, BNC1, Metabotropic glutamate receptor 3, GRIA4, Dopamine transporter, GRIK1, GRIK2, GRIK3, GRIA2 and GRIA3.-Further...
and Alpha-synuclein
Alpha-synuclein
Alpha-synuclein is a protein that, in humans, is encoded by the SNCA gene. An alpha-synuclein fragment, known as the non-Abeta component of Alzheimer's disease amyloid, originally found in an amyloid-enriched fraction, is shown to be a fragment of its precursor protein, NACP, by cloning of the...
.