Tag: DAT: Dopamine Transporter

What is a Dopamine Transporter?

Published on by Andrew Marshall6 Comments

Introduction

The dopamine transporter (DAT) also (sodium-dependent dopamine transporter) is a membrane-spanning protein coded for in the human by the SLC6A3 gene, (also known as DAT1), that pumps the neurotransmitter dopamine out of the synaptic cleft back into cytosol. In the cytosol, other transporters sequester the dopamine into vesicles for storage and later release. Dopamine reuptake via DAT provides the primary mechanism through which dopamine is cleared from synapses, although there may be an exception in the prefrontal cortex, where evidence points to a possibly larger role of the norepinephrine transporter.

DAT is implicated in a number of dopamine-related disorders, including attention deficit hyperactivity disorder, bipolar disorder, clinical depression, eating disorders, and substance use disorders. 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 type of genetic polymorphism, known as a variable number tandem repeat, in the SLC6A3 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-favourable 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.

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-labelled 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 build-up 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 depolarises.

DAT–Cav Coupling

Preliminary evidence suggests that the dopamine transporter couples to L-type voltage-gated calcium channels (particularly Cav1.2 and Cav1.3), which are expressed in virtually all dopamine neurons. As a result of DAT–Cav coupling, DAT substrates that produce depolarising currents through the transporter are able to open calcium channels that are coupled to the transporter, resulting in a calcium influx in dopamine neurons. This calcium influx is believed to induce CAMKII-mediated phosphorylation of the dopamine transporter as a downstream effect; since DAT phosphorylation by CAMKII results in dopamine efflux in vivo, activation of transporter-coupled calcium channels is a potential mechanism by which certain drugs (e.g. amphetamine) trigger neurotransmitter release.

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 characterisation 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. The exact structure of the Drosophila melanogaster dopamine transporter (dDAT) 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 the 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 substantia nigra pars compacta.

DAT in the mesocortical pathway, labelled 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 colocalisation 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. TAAR1 is a presynaptic intracellular receptor that is also colocalised with DAT and which has the opposite effect of the D2 autoreceptor when activated; i.e. it internalises dopamine transporters and induces efflux through reversed transporter function via PKA and PKC signalling.

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 is localised to axonal and dendritic (i.e. pre- and post-synaptic) plasma membranes.

Within the perikarya of pars compacta neurons, DAT was localised 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 gene for DAT, known as DAT1, 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) and another in the 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 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. MAPK, CAMKII, PKA, and PKC can modulate the rate at which the transporter moves dopamine or cause the internalisation of DAT. Co-localised TAAR1 is an important regulator of the dopamine transporter that, when activated, phosphorylates DAT through protein kinase A (PKA) and protein kinase C (PKC) signalling. Phosphorylation by either protein kinase can result in DAT internalisation (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) contains a high affinity extracellular zinc binding site which, upon zinc binding, inhibits dopamine reuptake and amplifies amphetamine-induced dopamine efflux in vitro. In contrast, the human serotonin transporter (hSERT) and human norepinephrine transporter (hNET) do not contain zinc binding sites. Zinc supplementation may reduce the minimum effective dose of amphetamine when it is used for the treatment of attention deficit hyperactivity disorder.

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. An allele of the DAT gene with normal protein levels is associated with non-smoking behaviour 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.

Mutations in DAT have been shown to cause dopamine transporter deficiency syndrome, an autosomal recessive movement disorder characterised by progressively worsening dystonia and parkinsonism.

Pharmacology

The dopamine transporter is the target of substrates, dopamine releasers, transport inhibitors and allosteric modulators.

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 the firing rate of the postsynaptic neuron and triggers protein kinase A and protein kinase C signalling, 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. Amphetamine also produces dopamine efflux through a second TAAR1-independent mechanism involving CAMKIIα-mediated phosphorylation of the transporter, which putatively arises from the activation of DAT-coupled L-type calcium channels by amphetamine.

The dopaminergic mechanisms of each drug are believed to underlie the pleasurable feelings elicited by these substances.

Interactions

Dopamine transporter has been shown to interact with:

  • Alpha-synuclein
  • PICK1
  • TGFB1I1

Apart from these innate protein-protein interactions, recent studies demonstrated that viral proteins such as HIV-1 Tat protein interacts with the DAT and this binding may alter the dopamine homeostasis in HIV positive individuals which is a contributing factor for the HIV-associated neurocognitive disorders.

This page is based on the copyrighted Wikipedia article < https://en.wikipedia.org/wiki/Dopamine_transporter >; it is used under the Creative Commons Attribution-ShareAlike 3.0 Unported License (CC-BY-SA). You may redistribute it, verbatim or modified, providing that you comply with the terms of the CC-BY-SA.

What is a Serotonin-Dopamine Reuptake Inhibitor?

Published on by Andrew Marshall2 Comments

Introduction

A serotonin–dopamine reuptake inhibitor (SDRI) is a type of drug which acts as a reuptake inhibitor of the monoamine neurotransmitters serotonin and dopamine by blocking the actions of the serotonin transporter (SERT) and dopamine transporter (DAT), respectively. This in turn leads to increased extracellular concentrations of serotonin and dopamine, and, therefore, an increase in serotonergic and dopaminergic neurotransmission.

A closely related type of drug is a serotonin–dopamine releasing agent (SDRA).

Comparison to SNDRIs

Relative to serotonin–norepinephrine–dopamine reuptake inhibitors (SNDRIs), which also inhibit the reuptake of norepinephrine in addition to serotonin and dopamine, SDRIs might be expected to have a reduced incidence of certain side effects, namely insomnia, appetite loss, anxiety, and heart rate and blood pressure changes.

Examples of SDRIs

Unlike the case of other combination monoamine reuptake inhibitors such as serotonin–norepinephrine reuptake inhibitors (SNRIs) and norepinephrine–dopamine reuptake inhibitors (NDRIs), on account of the very similar chemical structures of their substrates, it is exceptionally difficult to tease apart affinity for the DAT from the norepinephrine transporter (NET) and inhibit the reuptake of dopamine alone. As a result, selective dopamine reuptake inhibitors (DRIs) are rare, and comparably, SDRIs are even more so.

Pharmaceutical Drugs

Medifoxamine (Cledial, Gerdaxyl) is an antidepressant that appears to act as an SDRI as well as a 5-HT2 receptor antagonist. Sibutramine (Reductil, Meridia, Siredia, Sibutrex) is a withdrawn anorectic that itself as a molecule in vitro is an SNDRI but preferentially an SDRI, with 18.3- and 5.8-fold preference for inhibiting the reuptake of serotonin and dopamine over norepinephrine, respectively. However, the metabolites of sibutramine are substantially more potent and possess different ratios of monoamine reuptake inhibition in comparison, and sibutramine appears to be acting in vivo mainly as a prodrug to them; accordingly, it was found to act as an SNRI (73% and 54% for norepinephrine and serotonin reuptake inhibition, respectively) in human volunteers with only very weak inhibition of dopamine reuptake (16%).

Sertraline

Sertraline (Zoloft) is a selective serotonin reuptake inhibitor (SSRI), but, uniquely among most antidepressants, it shows relatively high (nanomolar) affinity for the DAT as well. As such, it has been suggested that clinically it may weakly inhibit the reuptake of dopamine, particularly at high dosages. For this reason, sertraline has sometimes been described as an SDRI. This is relevant as dopamine is thought to be involved in the pathophysiology of depression, and increased dopaminergic signaling by sertraline in addition to serotonin may have additional benefits against depression.

Tatsumi et al. (1997) found Ki values of sertraline at the SERT, DAT, and NET of 0.29, 25, and 420 nM, respectively. The selectivity of sertraline for the SERT over the DAT was 86-fold. In any case, of the wide assortment of antidepressants assessed in the study, sertraline showed the highest affinity of them all for the DAT, even higher than the norepinephrine–dopamine reuptake inhibitors (NDRIs) nomifensine (Ki = 56 nM) and bupropion (Ki = 520 nM). Sertraline is also said to have similar affinity for the DAT as the NDRI methylphenidate. It is notable that tametraline (CP-24,441), a very close analogue of sertraline and the compound from which sertraline was originally derived, is an NDRI that was never marketed.

Single doses of 50 to 200 mg sertraline have been found to result in peak plasma concentrations of 20 to 55 ng/mL (65–180 nM), while chronic treatment with 200 mg/day sertraline, the maximum recommended dosage, has been found to result in maximal plasma levels of 118 to 166 ng/mL (385–542 nM). However, sertraline is highly protein-bound in plasma, with a bound fraction of 98.5%. Hence, only 1.5% is free and theoretically bioactive. Based on this percentage, free concentrations of sertraline would be 2.49 ng/mL (8.13 nM) at the very most, which is only about one-third of the Ki value that Tatsumi et al. found with sertraline at the DAT. A very high dosage of sertraline of 400 mg/day has been found to produce peak plasma concentrations of about 250 ng/mL (816 nM). This can be estimated to result in a free concentration of 3.75 ng/mL (12.2 nM), which is still only about half of the Ki of sertraline for the DAT.

As such, it seems unlikely that sertraline would produce much inhibition of dopamine reuptake even at clinically used dosages well in excess of the recommended maximum clinical dosage. This is in accordance with its 86-fold selectivity for the SERT over the DAT and hence the fact that nearly 100-fold higher levels of sertraline would be necessary to also inhibit dopamine reuptake. In accordance, while sertraline has very low abuse potential and may even be aversive at clinical dosages, a case report of sertraline abuse described dopaminergic-like effects such as euphoria, mental overactivity, and hallucinations only at a dosage 56 times the normal maximum and 224 times the normal minimum. For these reasons, significant inhibition of dopamine reuptake by sertraline at clinical dosages is controversial, and occupation by sertraline of the DAT is thought by many experts to not be clinically relevant.

Research Chemicals

Two SDRIs that are known in research at present are RTI-83 and UWA-101, though other related compounds are also known. Based on its chemical structure, UWA-101 may actually also possess some activity as a releasing agent, and if so, unlike RTI-83, it would not be an SDRI in the purest sense and would also be an SDRA. Manning et al. presented two high-affinity MAT-ligands with good binding selectivity for SERT and DAT, namely the 4-indolyl and 1-naphthyl arylalkylamines ent-16b (Ki 0.82, 3.8, 4840 nM for SERT, DAT, NET) and ent-13b respectively. AN-788 (NSD-788) is another SDRI, and has been under development for the treatment of depressive and anxiety disorders.

This page is based on the copyrighted Wikipedia article < https://en.wikipedia.org/wiki/Serotonin%E2%80%93dopamine_reuptake_inhibitor >; it is used under the Creative Commons Attribution-ShareAlike 3.0 Unported License (CC-BY-SA). You may redistribute it, verbatim or modified, providing that you comply with the terms of the CC-BY-SA.

What is Dopaminergic?

Published on by Andrew Marshall3 Comments

Introduction

Dopaminergic means “related to dopamine” (literally, “working on dopamine”), dopamine being a common neurotransmitter. Dopaminergic substances or actions increase dopamine-related activity in the brain.

Outline

Dopaminergic brain pathways facilitate dopamine-related activity. For example, certain proteins such as the dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), and dopamine receptors can be classified as dopaminergic, and neurons that synthesize or contain dopamine and synapses with dopamine receptors in them may also be labelled as dopaminergic.

Enzymes that regulate the biosynthesis or metabolism of dopamine such as aromatic L-amino acid decarboxylase or DOPA decarboxylase, monoamine oxidase (MAO), and catechol O-methyl transferase (COMT) may be referred to as dopaminergic as well. Also, any endogenous or exogenous chemical substance that acts to affect dopamine receptors or dopamine release through indirect actions (for example, on neurons that synapse onto neurons that release dopamine or express dopamine receptors) can also be said to have dopaminergic effects, two prominent examples being opioids, which enhance dopamine release indirectly in the reward pathways, and some substituted amphetamines, which enhance dopamine release directly by binding to and inhibiting VMAT2.

This page is based on the copyrighted Wikipedia article < https://en.wikipedia.org/wiki/Dopaminergic >; it is used under the Creative Commons Attribution-ShareAlike 3.0 Unported License (CC-BY-SA). You may redistribute it, verbatim or modified, providing that you comply with the terms of the CC-BY-SA.

What is a Norepinephrine-Dopamine Reuptake Inhibitor?

Published on by Andrew Marshall5 Comments

Introduction

A norepinephrine–dopamine reuptake inhibitor (NDRI) is a drug used for the treatment of clinical depression, attention deficit hyperactivity disorder (ADHD), narcolepsy, and the management of Parkinson’s disease. The drug acts as a reuptake inhibitor for the neurotransmitters norepinephrine and dopamine by blocking the action of the norepinephrine transporter (NET) and the dopamine transporter (DAT), respectively. This in turn leads to increased extracellular concentrations of both norepinephrine and dopamine and, therefore, an increase in adrenergic and dopaminergic neurotransmission.

A closely related type of drug is a norepinephrine–dopamine releasing agent (NDRA).

List of NDRIs

The section only lists compounds that are selective for NET and DAT relative to the serotonin transporter (SERT). For a list of compounds that inhibit reuptake at all three transporters, see serotonin–norepinephrine–dopamine reuptake inhibitor.

Many NDRIs exist, including the following:

  • Amineptine (Survector, Maneon, Directim)
  • Bupropion (Wellbutrin, Zyban)
  • Desoxypipradrol (2-DPMP)
  • Dexmethylphenidate (Focalin)
  • Difemetorex (Cleofil)
  • Diphenylprolinol (D2PM)
  • Ethylphenidate
  • Fencamfamine (Glucoenergan, Reactivan)
  • Fencamine (Altimina, Sicoclor)
  • Lefetamine (Santenol)
  • Methylenedioxypyrovalerone (MDPV)
  • Methylphenidate (Ritalin, Concerta, Metadate, Methylin)
  • Nomifensine (Merital)
  • O-2172
  • Phenylpiracetam (Phenotropil, Carphedon)
  • Pipradrol (Meretran)
  • Prolintane (Promotil, Katovit)
  • Pyrovalerone (Centroton, Thymergix)
  • Solriamfetol (Sunosi)
  • Tametraline (CP-24,411)
  • WY-46824

Amphetamine and many of its immediate derivatives (i.e., the substituted amphetamines) are also both non-competitive and competitive inhibitors of the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT) proteins. Amphetamine itself has comparatively low affinity for SERT relative to DAT and NET. Consequently, amphetamine is usually classified as an NDRI instead of an SNDRI. However, the substituted amphetamines have a very diverse effects profile, and many of them have significant inhibiting effects on the SERT.

Amphetamine and many of the other substituted amphetamines are inhibitors of VMAT2 and potent agonists of the trace amine-associated receptor 1 (TAAR1); agonism of TAAR1 triggers phosphorylation events that result in both non-competitive reuptake inhibition and reversed transport direction of monoamine transporter proteins. As a result, monoamines flow out of the cell and into the synaptic cleft. Thus, amphetamine and its derivatives have a pharmacological profile that is much different than classical NDRIs, but analogous to trace amines.

This page is based on the copyrighted Wikipedia article < https://en.wikipedia.org/wiki/Norepinephrine-dopamine_reuptake_inhibitor >; it is used under the Creative Commons Attribution-ShareAlike 3.0 Unported License (CC-BY-SA). You may redistribute it, verbatim or modified, providing that you comply with the terms of the CC-BY-SA.

Schizophrenia & the Dopamine Transporter

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Research Paper Title

Altered levels of dopamine transporter in the frontal pole and dorsal striatum in schizophrenia.

Background

The dopamine hypothesis proposes that there is a hypodopaminergic state in the prefrontal cortex and a hyperdopaminergic state in the striatum of patients with schizophrenia.

Evidence suggests the hyperdopaminergic state in the striatum is due to synaptic dopamine elevation, particularly in the dorsal striatum.

However, the molecular mechanisms causing disrupted dopaminergic function in schizophrenia remains unclear.

The researchers postulated that the dopamine transporter (DAT), which regulates intra-synaptic dopamine concentrations by transporting dopamine from the synaptic cleft into the pre-synaptic neuron, could be involved in dopaminergic dysfunction in schizophrenia.

Methods

Therefore, they measured levels of DAT in the cortex and striatum from patients with schizophrenia and controls using postmortem human brain tissue. Levels of desmethylimipramine-insensitive mazindol-sensitive [3H]mazindol binding to DAT were measured using in situ radioligand binding and autoradiography in gray matter from Brodmann’s area (BA) 10, BA 17, the dorsal striatum, and nucleus accumbens from 15 patients with schizophrenia and 15 controls.

Results

Levels of desmethylimipramine-insensitive mazindol-sensitive [3H]mazindol binding were significantly higher in BA 10 from patients with schizophrenia (p = 0.004) and significantly lower in the dorsal striatum (dorsal putamen p = 0.005; dorsal caudate p = 0.007) from those with the disorder.

There were no differences in levels of desmethylimipramine-insensitive [3H]mazindol binding in BA 17 or nucleus accumbens.

Conclusions

These data raise the possibility that high levels of DAT in BA 10 could be contributing to lower synaptic cortical dopamine, whereas lower levels of DAT could be contributing to a hyperdopaminergic state in the dorsal striatum.

Reference

Sekiguchi, H., Pavey, G. & Dean, B. (2019) Altered levels of dopamine transporter in the frontal pole and dorsal striatum in schizophrenia. NPJ Schizophrenia. 5(1), pp.20. doi: 10.1038/s41537-019-0087-7.