This gene is a member of the family of human serotonin receptors, which are G protein-coupled receptors that stimulate cAMP production in response to serotonin (5-hydroxytryptamine). The gene product is a glycosylated transmembrane protein that functions in both the peripheral and central nervous system to modulate the release of various neurotransmitters. Multiple transcript variants encoding proteins with distinct C-terminal sequences have been described, but the full-length nature of some transcript variants has not been determined.
Location
The receptor is located in the alimentary tract, urinary bladder, heart and adrenal gland as well as the central nervous system (CNS). In the CNS the receptor appears in the putamen, caudate nucleus, nucleus accumbens, globus pallidus, and substantia nigra, and to a lesser extent in the neocortex, raphe, pontine nuclei, and some areas of the thalamus. It has not been found in the cerebellum.
Isoforms
Internalisation is isoform-specific.
Ligands
Several drugs that act as 5-HT4 selective agonists have recently been introduced into use in both scientific research and clinical medicine. Some drugs that act as 5-HT4 agonists are also active as 5-HT3 antagonists, such as mosapride, metoclopramide, renzapride, and zacopride, and so these compounds cannot be considered highly selective. Research in this area is ongoing. Amongst these agonists prucalopride has >150-fold higher affinity for 5-HT4 receptors than for other receptors.
SB-207,145 radiolabelled with carbon-11 is used as a radioligand for 5-HT4 in positron emission tomography pig and human studies.
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The 5-HT3 receptor belongs to the Cys-loop superfamily of ligand-gated ion channels (LGICs) and therefore differs structurally and functionally from all other 5-HT receptors (5-hydroxytryptamine, or serotonin receptors) which are G protein-coupled receptors. This ion channel is cation-selective and mediates neuronal depolarisation and excitation within the central and peripheral nervous systems.
As with other ligand gated ion channels, the 5-HT3 receptor consists of five subunits arranged around a central ion conducting pore, which is permeable to sodium (Na), potassium (K), and calcium (Ca) ions. Binding of the neurotransmitter 5-hydroxytryptamine (serotonin) to the 5-HT3 receptor opens the channel, which, in turn, leads to an excitatory response in neurons. The rapidly activating, desensitising, inward current is predominantly carried by sodium and potassium ions. 5-HT3 receptors have a negligible permeability to anions. They are most closely related by homology to the nicotinic acetylcholine receptor.
Brief History
Identification of the 5-HT3 receptor did not take place until 1986, lacking selective pharmacological tools. However, with the discovery that the 5-HT3 receptor plays a prominent role in chemotherapy- and radiotherapy-induced vomiting, and the concomitant development of selective 5-HT3 receptor antagonists to suppress these side effects aroused intense interest from the pharmaceutical industry and therefore the identification of 5-HT3 receptors in cell lines and native tissues quickly followed.
Structure
The 5-HT3 receptor differs markedly in structure and mechanism from the other 5-HT receptor subtypes, which are all G-protein-coupled. A functional channel may be composed of five identical 5-HT3A subunits (homopentameric) or a mixture of 5-HT3A and one of the other four 5-HT3B, 5-HT3C, 5-HT3D, or 5-HT3E subunits (heteropentameric). It appears that only the 5-HT3A subunits form functional homopentameric channels. All other subunit subtypes must heteropentamerise with 5-HT3A subunits to form functional channels. Additionally, there has not currently been any pharmacological difference found between the heteromeric 5-HT3AC, 5-HT3AD, 5-HT3AE, and the homomeric 5-HT3A receptor. N-terminal glycosylation of receptor subunits is critical for subunit assembly and plasma membrane trafficking.
The subunits surround a central ion channel in a pseudo-symmetric manner. Each subunit comprises an extracellular N-terminal domain which comprises the orthosteric ligand-binding site; a transmembrane domain consisting of four interconnected alpha helices (M1-M4), with the extracellular M2-M3 loop involved in the gating mechanism; a large cytoplasmic domain between M3 and M4 involved in receptor trafficking and regulation; and a short extracellular C-terminus. Whereas extracellular domain is the site of action of agonists and competitive antagonists, the transmembrane domain contains the central ion pore, receptor gate, and principle selectivity filter that allows ions to cross the cell membrane.
Human and Mouse Genes
The genes encoding human 5-HT3 receptors are located on chromosomes 11 (HTR3A, HTR3B) and 3 (HTR3C, HTR3D, HTR3E), so it appears that they have arisen from gene duplications. The genes HTR3A and HTR3B encode the 5-HT3A and 5-HT3B subunits and HTR3C, HTR3D and HTR3E encode the 5-HT3C, 5-HT3D and 5-HT3E subunits. HTR3C and HTR3E do not seem to form functional homomeric channels, but when co-expressed with HTR3A they form heteromeric complex with decreased or increased 5-HT efficacies. The pathophysiological role for these additional subunits has yet to be identified.
The human 5-HT3A receptor gene is similar in structure to the mouse gene which has 9 exons and is spread over ~13 kb. Four of its introns are exactly in the same position as the introns in the homologous α7-acetylcholine receptor gene, clearly showing their evolutionary relationship.
Expression: The 5-HT3C, 5-HT3D and 5-HT3E genes tend to show peripherally restricted pattern of expression, with high levels in the gut. In human duodenum and stomach, for example, 5-HT3C and 5-HT3E mRNA might be greater than for 5-HT3A and 5-HT3B.
Polymorphism: In patients treated with chemotherapeutic drugs, certain polymorphism of the HTR3B gene could predict successful antiemetic treatment. This could indicate that the 5-HTR3B receptor subunit could be used as biomarker of antiemetic drug efficacy.
Tissue Distribution
The 5-HT3 receptor is expressed throughout the central and peripheral nervous systems and mediates a variety of physiological functions. On a cellular level, it has been shown that postsynaptic 5-HT3 receptors mediate fast excitatory synaptic transmission in rat neocortical interneurons, amygdala, and hippocampus, and in ferret visual cortex. 5-HT3 receptors are also present on presynaptic nerve terminals. There is some evidence for a role in modulation of neurotransmitter release, but evidence is inconclusive.
Effects
When the receptor is activated to open the ion channel by agonists, the following effects are observed:
Central nervous system (CNS): nausea and vomiting centre in brain stem, anxiety, as well as anticonvulsant and pro-nociceptive activity.
Peripheral nervous system (PNS): neuronal excitation (in autonomic, nociceptive neurons), emesis.
Agonists
Agonists for the receptor include:
Cereulide
2-methyl-5-HT
Alpha-Methyltryptamine
Bufotenin
Chlorophenylbiguanide
Ethanol
Ibogaine
Phenylbiguanide
Quipazine
RS-56812: Potent and selective 5-HT3 partial agonist, 1000x selectivity over other serotonin receptors
SR-57227
Varenicline
YM-31636
S 21007(SAR c.f. CGS-12066A)
Antagonists
Antagonists for the receptor (sorted by their respective therapeutic application) include:
These agents are not agonists at the receptor, but increase the affinity or efficacy of the receptors for an agonist:
Indole Derivatives
5-chloroindole
Small Organic Anaesthetics
Ethanol
Chloroform
Halothane
Isoflurane
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The 5-HT1 receptors are a subfamily of the 5-HT serotonin receptors that bind to the endogenous neurotransmitter serotonin (also known as 5-hydroxytryptamine, or 5-HT). The 5-HT1 subfamily consists of five G protein-coupled receptors (GPCRs) that share 40% to 63% overall sequence homology, including 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F. Receptors of the 5-HT1 type, specifically, the 5-HT1A and 5-HT1D receptor subtypes, are present on the cell bodies. Receptors of the 5-HT1 type, specifically, the 5-HT1B and 5-HT1D receptor subtypes, are also present on the nerve terminals. These receptors are broadly distributed throughout the brain and are recognised to play a significant part in regulating synaptic levels of 5-HT.
The receptor subfamily is coupled to Gi/Go and mediate inhibitory neurotransmission by inhibiting the function of adenylate cyclase and modulating downstream ionic effects. This R-coupling to Gi/Go proteins leads to a reduction in local concentrations of cAMP, proving that 5-HT1 are primarily inhibitory. There is no 5-HT1C receptor, as it was reclassified as the 5-HT2C receptor.
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The 5-HT2 receptors are a subfamily of 5-HT receptors that bind the endogenous neurotransmitter serotonin (5-hydroxytryptamine, 5-HT).
Outline
The 5-HT2 subfamily consists of three G protein-coupled receptors (GPCRs) which are coupled to Gq/G11 and mediate excitatory neurotransmission, including:
5-HT2A
5-HT2B
5-HT2C
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5-HT receptors, 5-hydroxytryptamine receptors, or serotonin receptors, are a group of G protein-coupled receptor and ligand-gated ion channels found in the central and peripheral nervous systems. They mediate both excitatory and inhibitory neurotransmission. The serotonin receptors are activated by the neurotransmitter serotonin, which acts as their natural ligand.
The serotonin receptors modulate the release of many neurotransmitters, including glutamate, GABA, dopamine, epinephrine/norepinephrine, and acetylcholine, as well as many hormones, including oxytocin, prolactin, vasopressin, cortisol, corticotropin, and substance P, among others. Serotonin receptors influence various biological and neurological processes such as aggression, anxiety, appetite, cognition, learning, memory, mood, nausea, sleep, and thermoregulation. They are the target of a variety of pharmaceutical and recreational drugs, including many antidepressants, antipsychotics, anorectics, antiemetics, gastroprokinetic agents, antimigraine agents, hallucinogens, and entactogens.
Serotonin receptors are found in almost all animals and are even known to regulate longevity and behavioural ageing in the primitive nematode, Caenorhabditis elegans.
Classification
5-hydroxytryptamine receptors or 5-HT receptors, or serotonin receptors are found in the central and peripheral nervous systems. They can be divided into 7 families of G protein-coupled receptors which activate an intracellular second messenger cascade to produce an excitatory or inhibitory response. The exception to this is the 5-HT3 receptor which is a ligand-gated ion channel. In 2014, a novel 5-HT receptor was isolated from the small white butterfly, Pieris rapae, and named pr5-HT8. It does not occur in mammals and shares relatively low similarity to the known 5-HT receptor classes.
The 7 general serotonin receptor classes include a total of 14 known serotonin receptors. The 15th receptor 5-HT1P has been distinguished on the basis of functional and radioligand binding studies, its existence has never been definitely affirmed or refuted.
5-HT1A
5-HT1B
5-HT1D
5-HT1E
5-HT1F
5-HT1P
5-HT2A
5-HT2B
5-HT2C
5-HT3
5-HT4
5-HT5A
5-HT5B
5-HT6
5-HT7
Note that there is no 5-HT1C receptor since, after the receptor was cloned and further characterised, it was found to have more in common with the 5-HT2 family of receptors and was redesignated as the 5-HT2C receptor.
Very nonselective agonists of 5-HT receptor subtypes include ergotamine (an antimigraine), which activates 5-HT1A, 5-HT1D, 5-HT1B, D2 and norepinephrine receptors. LSD (a psychedelic) is a 5-HT1A, 5-HT2A, 5-HT2C, 5-HT5A and 5-HT6 agonist.
Expression Patterns
The genes coding for serotonin receptors are expressed across the mammalian brain. Genes coding for different receptors types follow different developmental curves. Specifically, there is a developmental increase of HTR5A expression in several subregions of the human cortex, paralleled by a decreased expression of HTR1A from the embryonic period to the post-natal one.
5-HT1-Like
A number of receptors were classed as “5-HT1-like” – by 1998 it was being argued that, since these receptors were “a heterogeneous population of 5-HT1B, 5-HT1D and 5-HT7” receptors the classification was redundant.
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5-HTTLPR (serotonin-transporter-linked promoter region) is a degenerate repeat (redundancy in the genetic code) polymorphic region in SLC6A4, the gene that codes for the serotonin transporter.
Since the polymorphism was identified in the middle of the 1990s, it has been extensively investigated, e.g., in connection with neuropsychiatric disorders. A 2006 scientific article stated that “over 300 behavioural, psychiatric, pharmacogenetic and other medical genetics papers” had analysed the polymorphism. While often discussed as an example of gene-environment interaction, this contention is contested.
Alleles
The polymorphism occurs in the promoter region of the gene. Researchers commonly report it with two variations in humans: A short (“s”) and a long (“l”), but it can be subdivided further. The short (s)- and long (l)- alleles have been thought to be related to stress and psychiatric disorders. In connection with the region are two single nucleotide polymorphisms (SNP): rs25531 and rs25532.
One study published in 2000 found 14 allelic variants (14-A, 14-B, 14-C, 14-D, 15, 16-A, 16-B, 16-C, 16-D, 16-E, 16-F, 19, 20 and 22) in a group of around 200 Japanese and Europeans. The difference between 16-A and 16-D is the rs25531 SNP. It is also the difference between 14-A and 14-D.
Some studies have found that long allele results in higher serotonin transporter mRNA transcription in human cell lines. The higher level may be due to the A-allele of rs25531, such that subjects with the long-rs25531(A) allelic combination (sometimes written LA) have higher levels while long-rs25531(G) carriers have levels more similar to short-allele carriers. Newer studies examining the effects of genotype may compare the LA/LA genotype against all other genotypes. The allele frequency of this polymorphism seems to vary considerably across populations, with a higher frequency of the long allele in Europe and lower frequency in Asia. It is argued that the population variation in the allele frequency is more likely due to neutral evolutionary processes than natural selection.
Neuropsychiatric Disorders
In the 1990s it has been speculated that the polymorphism might be related to affective disorders, and an initial study found such a link. However, another large European study found no such link. A decade later two studies found that 5-HTT polymorphism influences depressive responses to life stress; an example of gene-environment interaction (GxE) not considered in the previous studies. However, a 2017 meta-analysis found no such association. Earlier, two 2009 meta-analyses found no overall GxE effect, while a 2011 meta-analysis, demonstrated a positive result. In turn, the 2011 meta-analysis has been criticised as being overly inclusive (e.g. including hip fractures as outcomes), for deeming a study supportive of the GxE interaction which is actually in the opposite direction, and because of substantial evidence of publication bias and data mining in the literature. This criticism points out that if the original finding were real, and not the result of publication bias, we would expect that those replication studies which are closest in design to the original are the most likely to replicate—instead we find the opposite. This suggests that authors may be data dredging for measures and analytic strategies which yield the results they want.
Treatment Response
With the results from one study the polymorphism was thought to be related to treatment response so that long-allele patients respond better to antidepressants. Another antidepressant treatment response study did, however, rather point to the rs25531 SNP, and a large study by the group of investigators found a “lack of association between response to an SSRI and variation at the SLC6A4 locus”.
Amygdala
The 5-HTTLPR has been thought to predispose individuals to affective disorders such as anxiety and depression. There have been some studies that test whether this association is due to the effects of variation in 5-HTTLPR on the reactivity of the human amygdala. In order to test this, researchers gathered a group of subjects and administered a harm avoidance (HA) subset of the Tridimensional Personality Questionnaire as an initial mood and personality assessment. Subjects also had their DNA isolated and analysed in order to be genotyped. Next, the amygdala was then engaged by having the subject match fearful facial expressions during an fMRI scan (by the 3-T GE Signa scanner). The results of the study showed that there was bilateral activity in the amygdala for every subject when processing the fearful images, as expected. However, the activity in the right amygdala was much higher for subjects with the s-allele, which shows that the 5-HTTLPR has an effect on amygdala activity. There did not seem to be the same effect on the left amygdala.
Insomnia
There has been speculation that the 5-HTTLPR gene is associated with insomnia and sleep quality. Primary insomnia is one of the most common sleep disorders and is defined as having trouble falling or staying asleep, enough to cause distress in one’s life. Serotonin (5-HT) has been associated with the regulation of sleep for a very long time now. The 5-HT transporter (5-HTT) is the main regulator of serotonin and serotonergic energy and is therefore targeted by many antidepressants. There also have been several family and twin studies that suggest that insomnia is heavily genetically influenced. Many of these studies have found that there is a genetic and environment dual-factor that influences insomnia. It has been hypothesized that the short 5-HTTLPR genotype is related to poor sleep quality and, therefore, also primary insomnia. It is important to note that research studies have found that this variation does not cause insomnia, but rather may predispose an individual to experience worse quality of sleep when faced with a stressful life event.
Brummett
The effect that the 5-HTTLPR gene had on sleep quality was tested by Brummett in a study conducted at Duke University Medical Centre from 2001 to 2004. The sleep quality of 344 participants was measured using The Pittsburgh Sleep Quality Index. The study found that caregivers with the homozygous s-allele had poorer sleep quality, which shows that the stress of caregiving combined with the allele gave way to worse sleep quality. Although the study found that the 5-HTTLPR genotype did not directly affect sleep quality, the 5-HTTLPR polymorphism’s effect on sleep quality was magnified by one’s environmental stress. It supports the notion that the 5-HTTLPR s-allele is what leads to hyperarousal when exposed to stress; hyperarousability is commonly associated with insomnia.
Deuschle
However, in a 2007 study conducted by a sleep laboratory in Germany, it was found that the 5-HTTLPR gene did have a strong association with both insomnia and depression both in participants with and without lifetime affective disorders. This study included 157 insomnia patients and a control group of 836 individuals that had no psychiatric disorders. The subjects were then genotyped through polymerase chain reaction (PCR) techniques. The researchers found that the s-allele was greater represented in the vast majority of patients with insomnia compared to those who had no disorder. This shows that there is an association between the 5-HTTPLR genotype and primary insomnia. However, it is important to consider the fact that there was a very limited number of subjects with insomnia tested in this study.
Personality Traits
5-HTTLPR may be related to personality traits: Two 2004 meta-analyses found 26 research studies investigating the polymorphism in relation to anxiety-related traits. The initial and classic 1996 study found s-allele carriers to on average have slightly higher neuroticism score with the NEO PI-R personality questionnaire, and this result was replicated by the group with new data. Some other studies have, however, failed to find this association, nor with peer-rated neuroticism, and a 2006 review noted the “erratic success in replication” of the first finding. A meta-analysis published in 2004 stated that the lack of replicability was “largely due to small sample size and the use of different inventories”. They found that neuroticism as measured with the NEO-family of personality inventories had quite significant association with 5-HTTLPR while the trait harm avoidance from the Temperament and Character Inventory family did not have any significant association. A similar conclusion was reached in an updated 2008 meta-analysis.] However, based on over 4000 subjects, the largest study that used the NEO PI-R found no association between variants of the serotonin transporter gene (including 5-HTTLPR) and neuroticism, or its facets (Anxiety, Angry-Hostility, Depression, Self-Consciousness, Impulsiveness, and Vulnerability).
In a study published in 2009, authors found that individuals homozygous for the long allele of 5-HTTLPR paid more attention on average to positive affective pictures while selectively avoiding negative affective pictures presented alongside the positive pictures compared to their heterozygous and short-allele-homozygous peers. This biased attention of positive emotional stimuli suggests they may tend to be more optimistic. Other research indicates carriers of the short 5-HTTLPR allele have difficulty disengaging attention from emotional stimuli compared to long allele homozygotes. Another study published in 2009 using an eye tracking assessment of information processing found that short 5-HTTLPR allele carriers displayed an eye gaze bias to view positive scenes and avoid negative scenes, while long allele homozygotes viewed the emotion scenes in a more even-handed fashion. This research suggests that short 5-HTTLPR allele carriers may be more sensitive to emotional information in the environment than long allele homozygotes.
Another research group have given evidence for a modest association between shyness and the long form in grade school children. This is, however, just a single report and the link is not investigated as intensively as for the anxiety-related traits.
Neuroimaging
Molecular neuroimaging studies have examined the association between genotype and serotonin transporter binding with positron emission tomography (PET) and SPECT brain scanners. Such studies use a radioligand that binds—preferably selectively—to the serotonin transporter so an image can be formed that quantifies the distribution of the serotonin transporter in the brain. One study could see no difference in serotonin transporter availability between long/long and short/short homozygotes subjects among 96 subjects scanned with SPECT using the iodine-123 β-CIT radioligand. Using the PET radioligand carbon-11-labeled McN 5652 another research team could neither find any difference in serotonin transporter binding between genotype groups. Newer studies have used the radioligand carbon-11-labeled DASB with one study finding higher serotonin transporter binding in the putamen of LA homozygotes compared to other genotypes. Another study with similar radioligand and genotype comparison found higher binding in the midbrain.
Associations between the polymorphism and the grey matter in parts of the anterior cingulate brain region have also been reported based on magnetic resonance imaging (MRI) brain scanning and voxel-based morphometry analysis. 5-HTTLPR short allele–driven amygdala hyperreactivity was confirmed in a large (by MRI study standards) cohort of healthy subjects with no history of psychiatric illness or treatment. Brain blood flow measurements with positron emission tomography brain scanners can show genotype-related changes. The glucose metabolism in the brain has also been investigated with respect to the polymorphism, and the functional magnetic resonance imaging (fMRI) brain scans have also been correlated to the polymorphism.
The amygdala brain structure has, especially, been the focus of the functional neuroimaging studies.
Electrophysiology
The relationship between the Event Related Potentials P3a and P3b and the genetic variants of 5-HTTLPR were investigated using an auditory oddball paradigm and revealed short allele homozygotes mimicked those of COMT met/met homozygotes with an enhancement of the frontal, but not parietal P3a and P3b. This suggests a frontal-cortical dopaminergic and serotoninergic mechanism in bottom-up attentional capture.
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A transport protein (variously referred to as a transmembrane pump, transporter, escort protein, acid transport protein, cation transport protein, or anion transport protein) is a protein that serves the function of moving other materials within an organism. Transport proteins are vital to the growth and life of all living things. There are several different kinds of transport proteins.
Outline
Carrier proteins are proteins involved in the movement of ions, small molecules, or macromolecules, such as another protein, across a biological membrane. Carrier proteins are integral membrane proteins; that is, they exist within and span the membrane across which they transport substances. The proteins may assist in the movement of substances by facilitated diffusion (i.e., passive transport) or active transport. These mechanisms of movement are known as carrier-mediated transport. Each carrier protein is designed to recognise only one substance or one group of very similar substances. Research suggests that potassium, calcium and sodium channels can function as oxygen sensors in mammals and plants, and has correlated defects in specific carrier proteins with specific diseases. A membrane transport protein (or simply transporter) is a membrane protein that acts as such a carrier.
A vesicular transport protein is a transmembrane or membrane associated protein. It regulates or facilitates the movement by vesicles of the contents of the cell.
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Reuptake is the reabsorption of a neurotransmitter by a neurotransmitter transporter located along the plasma membrane of an axon terminal (i.e., the pre-synaptic neuron at a synapse) or glial cell after it has performed its function of transmitting a neural impulse.
Reuptake is necessary for normal synaptic physiology because it allows for the recycling of neurotransmitters and regulates the level of neurotransmitter present in the synapse, thereby controlling how long a signal resulting from neurotransmitter release lasts. Because neurotransmitters are too large and hydrophilic to diffuse through the membrane, specific transport proteins are necessary for the reabsorption of neurotransmitters. Much research, both biochemical and structural, has been performed to obtain clues about the mechanism of reuptake.
Protein Structure
The first primary sequence of a reuptake protein was published in 1990. The technique for protein sequence determination relied upon the purification, sequencing, and cloning of the transporter protein in question, or expression cloning strategies in which transport function was used as an assay for cDNA species coding for that transporter. After separation, it was realised that there were many similarities between the two DNA sequences. Further exploration in the field of reuptake proteins found that many of the transporters associated with important neurotransmitters within the body were also very similar in sequence to the GABA and norepinephrine transporters. The members of this new family include transporters for dopamine, norepinephrine, serotonin, glycine, proline and GABA. They were called Na+/Cl− dependent neurotransmitter transporters. Sodium and chloride ion dependence will be discussed later in the mechanism of action. Using the commonalities among sequences and hydropathy plot analyses, it was predicted that there are 12 hydrophobic membrane spanning regions in the ‘Classical’ transporter family. In addition to this, the N– and C-termini exist in the intracellular space. These proteins also all have an extended extracellular loop between the third and fourth transmembrane sequences. Site-directed chemical labelling experiments verified the predicted topological organisation of the serotonin transporter.
In addition to neurotransmitter transporters, many other proteins in both animals and prokaryotes were found with similar sequences, indicating a larger family of Neurotransmitter:Sodium Symporters (NSS). One of these proteins, LeuT, from Aquifex aeolicus, was crystallised by Yamashita et al. with very high resolution, revealing a molecule of leucine and two Na+ ions bound near the centre of the protein. They found that the transmembrane (TM) helices 1 and 6 contained unwound segments in the middle of the membrane. Along with these two helices, TM helices 3 and 8 and the areas surrounding the unwound sections of 1 and 6 formed the substrate and sodium ion binding sites. The crystal structure revealed pseudo-symmetry in LeuT, in which the structure of TM helices 1-5 is reflected in the structure of helices 6–10.
There is an extracellular cavity in the protein, into which protrudes a helical hairpin formed by extracellular loop EL4. In TM1, an aspartate distinguishes monoamine NSS transporters from amino acid transporters which contain a glycine at the same position. External and internal “gates” were assigned to pairs of negatively and positively charged residues in the extracellular cavity and near the cytoplasmic ends of TM helices 1 and 8.
Mechanism of Action
The classic transporter proteins use transmembrane ion gradients and electrical potential to transport neurotransmitter across the membrane of the presynaptic neuron. Typical neurotransmitter sodium symport (NSS) transporters, which are Na+ and Cl− ion dependent, take advantage of both Na+ and Cl− gradients, inwardly directed across the membrane. The ions flow down their concentration gradients, in many cases leading to transmembrane charge movement that is enhanced by the membrane potential. These forces pull the neurotransmitter substrate into the cell, even against its own concentration gradient. At a molecular level, Na+ ions stabilise amino acid binding at the substrate site and also hold the transporter in an outward-open conformation that allows substrate binding. The role of the Cl− ion in the symport mechanism has been proposed to be for stabilising the charge of the symported Na+.
After ion and substrate binding have taken place, some conformational change must occur. From the conformational differences between the structure of TMs 1-5 and that of TMs 6–10, and from the identification of a substrate permeation pathway between the binding site of SERT and the cytoplasm, a mechanism for conformational change was proposed in which a four-helix bundle composed of TMs 1, 2, 6 and 7 changes its orientation within the rest of the protein. A structure of LeuT in the inward-open conformation subsequently demonstrated that the major component of the conformational change represents movement of the bundle relative to the rest of the protein.
Mechanism of Reuptake Inhibition
The main objective of a reuptake inhibitor is to substantially decrease the rate by which neurotransmitters are reabsorbed into the presynaptic neuron, increasing the concentration of neurotransmitter in the synapse. This increases neurotransmitter binding to pre- and postsynaptic neurotransmitter receptors. Depending on the neuronal system in question, a reuptake inhibitor can have drastic effects on cognition and behaviour. Non-competitive inhibition of the bacterial homologue LeuT by tricyclic antidepressants resulted from binding of these inhibitors in the extracellular permeation pathway. However, the competitive nature of serotonin transport inhibition by antidepressants suggests that in neurotransmitter transporters, they bind in a site overlapping the substrate site.
Human Systems
Refer to Pharmacology of Antidepressants.
Horschitz et al.[12] examined reuptake inhibitor selectivity among the rat serotonin reuptake protein (SERT) expressed in human embryonic kidney cells (HEK-SERT). They presented SERT with varying doses of either citalopram (an SSRI) or desipramine (an inhibitor of norepinephrine reuptake protein, NET). By examining the dose-response curves (using a normal medium as control), they were able to quantify that citalopram acted on SERT as an SSRI, and that desipramine had no effect on SERT. In a separate experiment, Horschitz et al. exposed HEK-SERT with citalopram on a long-term basis. They noticed that long-term exposure led to a down-regulation of binding sites. These results suggest some mechanism for long-term changes in the pre-synaptic neuron after drug therapy. Horschitz et al. found that after removing citalopram from the system, normal levels of SERT binding site expression returned.
Depression has been suggested to be a result of a decrease of serotonin found in the synapse, although this hypothesis has been challenged since as early as the 1980s. It was initially supported by the successful reduction of depressive symptoms after administration of tricyclic antidepressants (such as desipramine) and SSRIs. Tricyclic antidepressants inhibit the reuptake of both serotonin and norepinephrine by acting upon both the SERT and NET. SSRIs selectively inhibit the reuptake of serotonin by acting upon SERT. The net result is an increased amount of serotonin in the synapse, thus increasing the probability that serotonin will interact with a serotonin receptor of the postsynaptic neuron. There are additional mechanisms by which serotonin autoreceptor desensitisation must occur, but the net result is the same. This increases serotonin signaling, which according to the hypothesis is believed to elevate mood and thus relieve depressive symptoms. This proposal for the antidepressant mechanism of serotonin reuptake inhibitors does not account for the time course of the therapeutic effect, which takes weeks to months, while transporter inhibition is essentially immediate.
The net effect of amphetamine (AMPH) use is an increase of dopamine, norepinephrine and serotonin in the synapse. It has been shown that AMPH acts upon trace amine-associated receptor 1 (TAAR1) to induce efflux and reuptake inhibition in the serotonin, norepinephrine, and dopamine transporters. This effect requires the transporter and TAAR1 to be co-localised (occur together) within the same neuron.
Neuroprotective Role
Astrocytes seem to utilise reuptake mechanisms for a neuroprotective role. Astrocytes use excitatory amino acid transporter 2 (EAAT2, aka GLT-1) to remove glutamate from the synapse. EAAT2 knockout mice were more prone to lethal and spontaneous seizures and acute brain injuries among the cortex. These effects could be linked to increased concentrations of glutamate in the brains of EAAT2 knockout mice, analysed post-mortem.
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The serotonin transporter (SERT or 5-HTT) also known as the sodium-dependent serotonin transporter and solute carrier family 6 menmber 4 is a protein that in humans is encoded by the SLC6A4 gene. SERT is a type of monoamine transporter protein that transports the neurotransmitter serotonin from the synaptic cleft back to the presynaptic neuron, in a process known as serotonin reuptake.
This transport of serotonin by the SERT protein terminates the action of serotonin and recycles it in a sodium-dependent manner. Many antidepressant medications of the SSRI and tricyclic antidepressant classes work by binding to SERT and thus reducing serotonin reuptake. It is a member of the sodium:neurotransmitter symporter family. A repeat length polymorphism in the promoter of this gene has been shown to affect the rate of serotonin uptake and may play a role in sudden infant death syndrome, aggressive behaviour in Alzheimer disease patients, post-traumatic stress disorder and depression-susceptibility in people experiencing emotional trauma.
Mechanism of Action
Serotonin-reuptake transporters are dependent on both the concentration of potassium ion in the cytoplasm and the concentrations of sodium and chloride ions in the extracellular fluid. In order to function properly the serotonin transporter requires the membrane potential created by the sodium-potassium adenosine triphosphatase.
The serotonin transporter first binds a sodium ion, followed by the serotonin, and then a chloride ion; it is then allowed, thanks to the membrane potential, to flip inside the cell freeing all the elements previously bound. Right after the release of the serotonin in the cytoplasm a potassium ion binds to the transporter which is now able to flip back out returning to its active state.
Function
The serotonin transporter removes serotonin from the synaptic cleft back into the synaptic boutons. Thus, it terminates the effects of serotonin and simultaneously enables its reuse by the presynaptic neuron.
Neurons communicate by using chemical messengers like serotonin between cells. The transporter protein, by recycling serotonin, regulates its concentration in a gap, or synapse, and thus its effects on a receiving neuron’s receptors.
Medical studies have shown that changes in serotonin transporter metabolism appear to be associated with many different phenomena, including alcoholism, clinical depression, obsessive–compulsive disorder (OCD), romantic love, hypertension and generalized social phobia.
The serotonin transporter is also present in platelets; there, serotonin functions as a vasoconstrictive substance. It also serves as a signalling molecule to induce platelet aggregation.
Pharmacology
In 1995 and 1996, scientists in Europe had identified the polymorphism 5-HTTLPR, a serotonin-transporter in the gene SLC6A4. In December 1996, a group of researchers led by D.A. Collier of the Institute of Psychiatry, Psychology and Neuroscience, published their findings in Molecular Psychiatry, that, “5-HTTLPR-dependent variation in functional 5-HTT expression is a potential genetic susceptibility factor for affective disorders.”
SERT spans the plasma membrane 12 times. It belongs to the NE, DA, SERT monoamine transporter family. Transporters are important sites for agents that treat psychiatric disorders. Drugs that reduce the binding of serotonin to transporters (serotonin reuptake inhibitors, or SRIs) are used to treat mental disorders. The selective serotonin reuptake inhibitor (SSRI) fluoxetine and the tricyclic antidepressant (TCA) clomipramine are examples of serotonin reuptake inhibitors.
Following the elucidation of structures of the homologous bacterial transporter, LeuT, co-crystallised with tricyclic antidepressants in the vestibule leading from the extracellular space to the central substrate site it was inferred that this binding site did also represent the binding site relevant for antidepressant binding in SERT. However, studies on SERT showed that tricyclic antidepressants and selective serotonin reuptake inhibitors bind to the central binding site overlapping the substrate binding site. The Drosophila dopamine transporter, which displays a pharmacology similar to SERT, was crystallised with tricyclic antidepressants and confirmed the earlier finding that the substrate binding site is also the antidepressant binding site.
Ligands
DASB, also known as 3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile, is a compound that binds to the serotonin transporter.
compound 4b: Ki = 17 pM; 710-fold and 11,100-fold selective over DAT and NET
compound (+)-12a: Ki = 180 pM at hSERT; >1000-fold selective over hDAT, hNET, 5-HT1A, and 5-HT6. Isosteres
The gene that encodes the serotonin transporter is called solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 (SLC6A4, refer to Solute carrier family). In humans the gene is found on chromosome 17 on location 17q11.1–q12.
Mutations associated with the gene may result in changes in serotonin transporter function, and experiments with mice have identified more than 50 different phenotypic changes as a result of genetic variation. These phenotypic changes may, e.g., be increased anxiety and gut dysfunction. Some of the human genetic variations associated with the gene are:
Length variation in the serotonin-transporter-gene-linked polymorphic region (5-HTTLPR)
rs25531 — a single nucleotide polymorphism (SNP) in the 5-HTTLPR
rs25532 — another SNP in the 5-HTTLPR
STin2 — a variable number of tandem repeats (VNTR) in the functional intron 2
G56A on the second exon
I425V on the ninth exon
Length Variation in 5-HTTLPR
Refer to 5-HTTLPR.
According to a 1996 article in The Journal of Neurochemistry, the promoter region of the SLC6A4 gene contains a polymorphism with “short” and “long” repeats in a region: 5-HTT-linked polymorphic region (5-HTTLPR or SERTPR). The short variation has 14 repeats of a sequence while the long variation has 16 repeats. A second 1996 article stated that the short variation leads to less transcription for SLC6A4, and it has been found that it can partly account for anxiety-related personality traits. This polymorphism has been extensively investigated in over 300 scientific studies (as of 2006). The 5-HTTLPR polymorphism may be subdivided further: One study published in 2000 found 14 allelic variants (14-A, 14-B, 14-C, 14-D, 15, 16-A, 16-B, 16-C, 16-D, 16-E, 16-F, 19, 20 and 22) in a group of around 200 Japanese and Caucasian people.
In addition to altering the expression of SERT protein and concentrations of extracellular serotonin in the brain, the 5-HTTLPR variation is associated with changes in brain structure. One 2005 study found less grey matter in perigenual anterior cingulate cortex and amygdala for short allele carriers of the 5-HTTLPR polymorphism compared to subjects with the long/long genotype.
In contrast, a 2008 meta-analysis found no significant overall association between the 5-HTTLPR polymorphism and autism. A hypothesized gene–environment interaction between the short/short allele of the 5-HTTLPR and life stress as predictor for major depression has suffered a similar fate: after an influential initial report in 2003 there were mixed results in replication in 2008, and a 2009 meta-analysis was negative.
rs25532
rs25532 is a SNP (C>T) close to the site of 5-HTTLPR. It has been examined in connection with obsessive compulsive disorder (OCD).
I425V
I425V is a rare mutation on the ninth exon. In 2003, researchers from Japan and the US reported that they had found this genetic variation in unrelated families with OCD, and have found that it leads to faulty transporter function and regulation. A second variant in the same gene of some patients with this mutation suggests a genetic “double hit”, resulting in greater biochemical effects and more severe symptoms.
VNTR in STin2
Another noncoding polymorphism is a VNTR in the second intron (STin2). In a 2005 study, it was found with three alleles: 9, 10 and 12 repeats. A meta-analysis has found that the 12 repeat allele of the STin2 VNTR polymorphism had some minor (with odds ratio 1.24), but statistically significant, association with schizophrenia. A 2008 meta-analysis found no significant overall association between the STin2 VNTR polymorphism and autism. Furthermore, a 2003 meta-analysis of affective disorders, major depressive disorder and bipolar disorder, found a minor association to the intron 2 VNTR polymorphism, but the results of the meta-analysis were dependent upon a large effect from one individual study.
The polymorphism has also been related to personality traits with a 2008 Russian study finding individuals with the STin2.10 allele having lower neuroticism scores as measured with the Eysenck Personality Inventory.
Neuroimaging
The distribution of the serotonin transporter in the brain may be imaged with positron emission tomography using radioligands called DASB and DAPP; the first such studies on the human brain were reported in 2000. DASB and DAPP are not the only radioligands for the serotonin transporter. There are numerous others, with the most popular probably being the β-CIT radioligand with an iodine-123 isotope that is used for brain scanning with single-photon emission computed tomography (SPECT) according to a 1993 article in the Journal of Neural Transmission. The radioligands were used in 2006 to examine whether variables such as age, gender or genotype are associated with differential serotonin transporter binding. Healthy subjects that have a high score of neuroticism—a personality trait in the Revised NEO Personality Inventory—were found to have more serotonin transporter binding in the thalamus in 2007.
Neuroimaging and Genetics
Studies on the serotonin transporter have combined neuroimaging and genetics methods, e.g., a voxel-based morphometry study found less grey matter in perigenual anterior cingulate cortex and amygdala for short allele carriers of the 5-HTTLPR polymorphism compared to subjects with the long/long genotype.
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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.
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