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ELife Jan 2023The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - that is, the retrieval of serotonin...
The serotonin transporter (SERT/SLC6A4) is arguably the most extensively studied solute carrier (SLC). During its eponymous action - that is, the retrieval of serotonin from the extracellular space - SERT undergoes a conformational cycle. Typical inhibitors (antidepressant drugs and cocaine), partial and full substrates (amphetamines and their derivatives), and atypical inhibitors (ibogaine analogues) bind preferentially to different states in this cycle. This results in competitive or non-competitive transport inhibition. Here, we explored the action of -formyl-1,3-bis (3,4-methylenedioxyphenyl)-prop-2-yl-amine (ECSI#6) on SERT: inhibition of serotonin uptake by ECSI#6 was enhanced with increasing serotonin concentration. Conversely, the K for serotonin was lowered by augmenting ECSI#6. ECSI#6 bound with low affinity to the outward-facing state of SERT but with increased affinity to a potassium-bound state. Electrophysiological recordings showed that ECSI#6 preferentially interacted with the inward-facing state. Kinetic modeling recapitulated the experimental data and verified that uncompetitive inhibition arose from preferential binding of ECSI#6 to the K-bound, inward-facing conformation of SERT. This binding mode predicted a pharmacochaperoning action of ECSI#6, which was confirmed by examining its effect on the folding-deficient mutant SERT-PGAA: preincubation of HEK293 cells with ECSI#6 restored export of SERT-PGAA from the endoplasmic reticulum and substrate transport. Similarly, in transgenic flies, the administration of ECSI#6 promoted the delivery of SERT-PGAA to the presynaptic specialization of serotonergic neurons. To the best of our knowledge, ECSI#6 is the first example of an uncompetitive SLC inhibitor. Pharmacochaperones endowed with the binding mode of ECSI#6 are attractive, because they can rescue misfolded transporters at concentrations, which cause modest transport inhibition.
Topics: Humans; Serotonin Plasma Membrane Transport Proteins; Serotonin; HEK293 Cells; Ion Transport
PubMed: 36648438
DOI: 10.7554/eLife.82641 -
Pharmacological Research Feb 2019A variety of human and animal studies support the hypothesis that serotonin (5-hydroxytryptamine or 5-HT) system dysfunction is a contributing factor to the development... (Review)
Review
A variety of human and animal studies support the hypothesis that serotonin (5-hydroxytryptamine or 5-HT) system dysfunction is a contributing factor to the development of autism in some patients. However, many questions remain about how developmental manipulation of various components that influence 5-HT signaling (5-HT synthesis, transport, metabolism) persistently impair social behaviors. This review will summarize key aspects of central 5-HT function important for normal brain development, and review evidence implicating perinatal disruptions in 5-HT signaling in the pathophysiology of autism spectrum disorder. We discuss the importance, and relative dearth, of studies that explore the possible correlation to autism in the interactions between important intrinsic and extrinsic factors that may disrupt 5-HT homeostasis during development. In particular, we focus on exposure to 5-HT transport altering mechanisms such as selective serotonin-reuptake inhibitors or genetic polymorphisms in primary or auxiliary transporters of 5-HT, and how they relate to neurological stores of serotonin and its precursors. A deeper understanding of the many mechanisms by which 5-HT signaling can be disrupted, alone and in concert, may contribute to an improved understanding of the etiologies and heterogeneous nature of this disorder. We postulate that extreme bidirectional perturbations of these factors during development likely compound or synergize to facilitate enduring neurochemical changes resulting in insufficient or excessive 5-HT signaling, that could underlie the persistent behavioral characteristics of autism spectrum disorder.
Topics: Animals; Autism Spectrum Disorder; Humans; Serotonin; Serotonin Plasma Membrane Transport Proteins; Selective Serotonin Reuptake Inhibitors
PubMed: 30009933
DOI: 10.1016/j.phrs.2018.07.010 -
ELife Jul 2020Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of...
Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation, leading to questions about relationships of these structures to the bonafide conformation and inhibitor binding poses of wild-type transporter. To address these concerns, we determined the structures of ∆N72/∆C13 and ts2-inactive SERT bound to paroxetine analogues using single-particle cryo-EM and x-ray crystallography, respectively. We synthesized enantiopure analogues of paroxetine containing either bromine or iodine instead of fluorine. We exploited the anomalous scattering of bromine and iodine to define the pose of these inhibitors and investigated inhibitor binding to Asn177 mutants of ts2-active SERT. These studies provide mutually consistent insights into how paroxetine and its analogues bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.
Topics: Cryoelectron Microscopy; Crystallography, X-Ray; Humans; Molecular Structure; Paroxetine; Protein Structure, Tertiary; Serotonin; Serotonin Plasma Membrane Transport Proteins; Selective Serotonin Reuptake Inhibitors
PubMed: 32618269
DOI: 10.7554/eLife.56427 -
The Journal of General Physiology Nov 2019Serotonin (5-hydroxytryptamine [5-HT]) is accumulated within nerve endings by the serotonin transporter (SERT), which terminates its extracellular action and provides... (Review)
Review
Serotonin (5-hydroxytryptamine [5-HT]) is accumulated within nerve endings by the serotonin transporter (SERT), which terminates its extracellular action and provides cytoplasmic 5-HT for refilling of synaptic vesicles. SERT is the target for many antidepressant medications as well as psychostimulants such as cocaine and ecstasy (3,4-methylenedioxymethamphetamine). SERT belongs to the SLC6 family of ion-coupled transporters and is structurally related to several other transporter families. SERT was studied in the 1970s and 1980s using membrane vesicles isolated from blood platelets. These studies led to a proposed stoichiometry of transport that has been challenged by high-resolution structures of SERT and its homologues and by studies of SERT electrophysiology. Here, we review the original evidence alongside more recent structural and electrophysiological evidence. A self-consistent picture emerges with surprising insights into the ion fluxes that accompany 5-HT transport.
Topics: Animals; Biological Transport; Humans; Serotonin; Serotonin Plasma Membrane Transport Proteins; Synaptic Transmission
PubMed: 31570504
DOI: 10.1085/jgp.201812066 -
Biochimica Et Biophysica Acta.... Jul 2021Structure and function analysis of human membrane proteins in lipid bilayer environments is acutely lacking despite the fundame1ntal cellular importance of these...
Structure and function analysis of human membrane proteins in lipid bilayer environments is acutely lacking despite the fundame1ntal cellular importance of these proteins and their dominance of drug targets. An underlying reason is that detailed study usually requires a potentially destabilising detergent purification of the proteins from their host membranes prior to subsequent reconstitution in a membrane mimic; a situation that is exacerbated for human membrane proteins due to the inherent difficulties in overexpressing suitable quantities of the proteins. We advance the promising styrene maleic acid polymer (SMA) extraction approach to introduce a detergent-free method of obtaining stable, functional human membrane transporters in bilayer nanodiscs directly from yeast cells. We purify the human serotonin transporter (hSERT) following overexpression in Pichia pastoris using diisobutylene maleic acid (DIBMA) as a superior method to traditional detergents or the more established styrene maleic acid polymer. hSERT plays a pivotal role in neurotransmitter regulation being responsible for the transport of the neurotransmitter 5-hydroxytryptamine (5-HT or serotonin). It is representative of the neurotransmitter sodium symporter (NSS) family, whose importance is underscored by the numerous diseases attributed to their malfunction. We gain insight into hSERT activity through an in vitro transport assay and find that DIBMA extraction improves the thermostability and activity of hSERT over the conventional detergent method.
Topics: Alkenes; Humans; Maleates; Polymers; Protein Stability; Recombinant Proteins; Serotonin; Serotonin Plasma Membrane Transport Proteins; Temperature
PubMed: 33744253
DOI: 10.1016/j.bbamem.2021.183602 -
Frontiers in Immunology 2023Serotonin is involved in leukocyte recruitment during inflammation. Deficiency of the serotonin transporter (SERT) is associated with metabolic changes in humans and...
INTRODUCTION
Serotonin is involved in leukocyte recruitment during inflammation. Deficiency of the serotonin transporter (SERT) is associated with metabolic changes in humans and mice. A possible link and interaction between the inflammatory effects of serotonin and metabolic derangements in SERT-deficient mice has not been investigated so far.
METHODS
SERT-deficient ( ) and wild type (WT) mice were fed a high-fat diet, starting at 8 weeks of age. Metabolic phenotyping (metabolic caging, glucose and insulin tolerance testing, body and organ weight measurements, qPCR, histology) and assessment of adipose tissue inflammation (flow cytometry, histology, qPCR) were carried out at the end of the 19-week high-fat diet feeding period. In parallel, and WT mice received a control diet and were analyzed either at the time point equivalent to high-fat diet feeding or as early as 8-11 weeks of age for baseline characterization.
RESULTS
After 19 weeks of high-fat diet, and WT mice displayed similar whole-body and fat pad weights despite increased relative weight gain due to lower starting body weight in . In obese animals insulin resistance and liver steatosis were enhanced as compared to WT animals. Leukocyte accumulation and mRNA expression of cytokine signaling mediators were increased in epididymal adipose tissue of obese mice. These effects were associated with higher adipose tissue mRNA expression of the chemokine monocyte chemoattractant protein 1 and presence of monocytosis in blood with an increased proportion of pro-inflammatory Ly6C+ monocytes. By contrast, mice fed a control diet did not display adipose tissue inflammation.
DISCUSSION
Our observations suggest that SERT deficiency in mice is associated with inflammatory processes that manifest as increased adipose tissue inflammation upon chronic high-fat diet feeding due to enhanced leukocyte recruitment.
Topics: Humans; Animals; Mice; Diet, High-Fat; Serotonin Plasma Membrane Transport Proteins; Serotonin; Inflammation; Obesity; Adipose Tissue; Weight Gain; RNA, Messenger
PubMed: 37520561
DOI: 10.3389/fimmu.2023.1184010 -
International Journal of Environmental... Mar 2022The current systematic review examines whether there is an association between the genetic 5-HTTPLR polymorphism and parenting, and the mechanisms by which this... (Review)
Review
The current systematic review examines whether there is an association between the genetic 5-HTTPLR polymorphism and parenting, and the mechanisms by which this association operates. The literature was searched in various databases such as PubMed, Scopus, and ScienceDirect. In line with our inclusion criteria, nine articles were eligible out of 22. Most of the studies analysed in this review found an association between 5HTTLPR and parenting. Four studies found a direct association between 5-HTTLPR and parenting with conflicting findings: two studies found that mothers carrying the short variant were more sensitive to their infants, while two studies found that parents carrying the S allele were less sensitive. In addition, several studies found strong interaction between genetic and environmental factors, such as childhood stress and disruptive child behaviour, quality of early care experiences, poor parenting environment, and quality of the environment. Only one study found an association between children's 5HTTLPR and parenting. Parenting can be described as a highly complex construct influenced by multiple factors, including the environment, as well as parent and child characteristics. According to the studies, maternal 5-HTTLPR polymorphism is most likely to be associated with sensitive parenting.
Topics: Child; Female; Humans; Infant; Mothers; Parenting; Polymorphism, Genetic; Problem Behavior; Serotonin Plasma Membrane Transport Proteins
PubMed: 35409736
DOI: 10.3390/ijerph19074052 -
International Journal of Molecular... Sep 2022Conformational changes are fundamental events in the transport mechanism. The serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin after its...
Conformational changes are fundamental events in the transport mechanism. The serotonin transporter (SERT) catalyzes reuptake of the neurotransmitter serotonin after its release by serotonergic neurons and is the molecular target for antidepressant drugs and psychostimulants. Despite significant progress in characterizing the structure-function relationship of SERT, its conformational mechanism has not been fully understood. We present here a cell-based method for determining conformational changes in SERT with its fluorescent substrates by fluorescence imaging analysis. This method fluorometrically measures accessibility of strategically positioned cysteine residues in the substrate permeation pathway to calculate the rate constants of reactivity with MTS reagents in live or permeabilized cells. We validated this method by investigating ligand and ion-induced conformational changes in both the extracellular and cytoplasmic pathways of SERT. Furthermore, we applied this method for examining the influence of Cl binding and vilazodone inhibition on SERT conformation. Our results showed that Cl ion, in the presence of Na, facilitates the conformational conversion from outward to inward open states, and that vilazodone binding stabilizes SERT in an outward open and inward-closed conformation. The present work provided insights into the conformational mechanism of SERT and also indicated that the cell-based fluorometric method is robust, straightforward to perform, and potentially applicable to any monoamine transporters in exploring the transport mechanism and mechanism of action of therapeutic agents for the treatment of several psychiatric disorders.
Topics: Cysteine; Humans; Ligands; Neurotransmitter Agents; Protein Conformation; Serotonin; Serotonin Plasma Membrane Transport Proteins; Vilazodone Hydrochloride
PubMed: 36142837
DOI: 10.3390/ijms231810919 -
Advances in Neurobiology 2023Monoamine transporters (MATs) are targets of a wide range of compounds that have been developed as therapeutic treatments for various neuropsychiatric and... (Review)
Review
Monoamine transporters (MATs) are targets of a wide range of compounds that have been developed as therapeutic treatments for various neuropsychiatric and neurodegenerative disorders such as depression, ADHD, neuropathic pain, anxiety disorders, stimulant use disorders, epilepsy, and Parkinson's disease. The MAT family is comprised of three main members - the dopamine transporter (DAT), the norepinephrine transporter (NET), and the serotonin transporter (SERT). These transporters are through reuptake responsible for the clearance of their respective monoamine substrates from the extracellular space. The determination of X-ray crystal structures of MATs and their homologues bound with various substrates and ligands has resulted in a surge of structure-function-based studies of MATs to understand the molecular basis of transport function and the mechanism of various ligands that ultimately result in their behavioral effects. This review focusses on recent examples of ligand-based structure-activity relationship studies trying to overcome some of the challenges associated with previously developed MAT inhibitors. These studies have led to the discovery of unique and novel structurally diverse MAT ligands including allosteric modulators. These novel molecular scaffolds serve as leads for designing more effective therapeutic interventions by modulating the activities of MATs and ultimately their associated neurotransmission and behavioral effects.
Topics: Humans; Biological Transport; Ligands; Serotonin Plasma Membrane Transport Proteins; Vesicular Monoamine Transport Proteins; Mental Disorders; Drug Discovery
PubMed: 36928847
DOI: 10.1007/978-3-031-21054-9_4 -
Translational Psychiatry Jan 2021Understanding the common dimension of mental disorders (such as anxiety, depression, and drug addiction) might contribute to the construction of biological frameworks...
Understanding the common dimension of mental disorders (such as anxiety, depression, and drug addiction) might contribute to the construction of biological frameworks (Research Domain Criteria, RDoC) for novel ways of treatment. One common dimension at the behavioral level observed across these disorders is a generalization. Testing generalization in serotonin transporter (5-HTT) knockout (KO) rats, an animal model showing depression/anxiety-like behaviors and drug addiction-like behaviors, could therefore provide more insights into this framework. We tested the outcome and stimulus generalization in wild-type (WT) and 5-HTT KO rats. Using a newly established touchscreen-based task, subjects directly responded to visual stimuli (Gabor patch images). We measured the response time and outcome in a precise manner. We found that 5-HTT KO rats processed visual information faster than WT rats during outcome generalization. Interestingly, during stimulus generalization, WT rats gradually responded faster to the stimuli as the sessions progressed, while 5-HTT KO rats responded faster than WT in the initial sessions and did not change significantly as the sessions progressed. This observation suggests that KO rats, compared to WT rats, may be less able to update changes in information. Taken together, KO 5-HTT modulates information processing when the environment changes.
Topics: Animals; Anxiety; Cognition; Disease Models, Animal; Generalization, Stimulus; Mice; Mice, Knockout; Rats; Serotonin Plasma Membrane Transport Proteins
PubMed: 33414390
DOI: 10.1038/s41398-020-01162-0