Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.107 (DAT)
 1,471 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sodium-dependent transporters for dopamine, norepinephrine, and serotonin that regulate neurotransmission, also translocate the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)). Previous studies implicated residues in transmembrane helix (TMH) XI of DAT as important sites for MPP(+) transport. We examined the importance of TMH XI residues F551 and F556 for MPP(+) translocation by human SERT. Mutations at hSERT F556, but not F551, reduced both 5-HT and MPP(+) transport compared to wild type. However, F556S/hSERT showed a reduction in surface expression explaining the decrease of transport activity for 5-HT, but did not account for the decrease in MPP(+) transport observed. Cysteine mutants at those positions confirmed the accessibility of hSERT/F556 to different methanethiosulfonate (MTS) reagents, suggesting its presence in a hydrophilic environment of the protein. In the presence of MTSET, current induced by 5-HT and MPP(+) was inhibited at the F556C mutant. In agreement with our homology model of SERT, based on the leucine transporter (LeuT(Aa)) from Aquifex aeolicus structure, these results are consistent with the hypothesis that a portion of TMH XI lines the entrance into the substrate permeation pathway.
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PMID:Helix XI contributes to the entrance of the serotonin transporter permeation pathway. 1862 41

X-ray crystallography, structural bioinformatics and computational chemistry have become important techniques in the discovery and development of effective and safe new drugs. From a drug discovery point of view, membrane proteins are among the most interesting molecular targets, but the current knowledge about detailed 3D structures of membrane proteins is sparse. Homology modeling techniques may provide structural knowledge about membrane proteins and their interactions with drugs and other molecules. The neurotransmitter sodium symporters (NSS) are the molecular targets of many pharmacologically active substances, and we have used three different secondary transporters as templates for modeling the NSS proteins DAT, NET and SERT. The first template was based on the electron density projection map of the Escherichia coli Na+/H+ antiporter (NhaA), while later the X-ray structure of Lac Permease (symporter) was used as a template. The helical architectures of these templates have a lot in common, and models based on both could contribute with structural explanations of several experimental studies in spite of low homology with NSS proteins. In 2005 the crystal structure of a bacterial homologue of the human monoamine neurotransmitter transporter Aquifex aeolicus (LeuTAa) was reported. This structure was the first experimental structure of a NSS family member, and represented a breakthrough for homology modeling of pharmacological important NSS proteins. Since then several X-ray structures LeuTAa in complex with pharmacologically important compounds have been published. Homology models of NSS proteins, combined with site-directed mutagenesis data, have identified ligand binding sites and contributed with important knowledge for new drug development.
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PMID:Templates and models of monoamine transporter proteins. 2186 75

Undoped and transition metals (3d TM) doped sodium borophosphate glasses were prepared. UV-visible absorption spectra were measured in the region 200-900nm before and after gamma irradiation. Experimental optical data indicate that the undoped sodium borophosphate glass reveals before irradiation strong and broad UV absorption and no visible bands could be identified. Such UV absorption is related to the presence of unavoidable trace iron impurities within the raw materials used for preparation of this base borophosphate glass. The TMs-doped glasses show absorption bands within the UV and/or visible regions which are characteristic to each respective TM ion in addition to the UV absorption observed from the host base glass. Infrared absorption spectra of the undoped and TMs-doped glasses reveal complex FTIR consisting of extended characteristic vibrational bands which are specific for phosphate groups as a main constituent but with the sharing of some vibrations due to the borate groups. This criterion was investigated and approved using DAT (deconvolution analysis technique). The effects of different TMs ions on the FTIR spectra are very limited due to the low doping level (0.2%) introduced in the glass composition. Gamma irradiation causes minor effect on the FTIR spectra specifically the decrease of intensities of some bands. Such behavior is related to the change of bond angles and/or bond lengths of some structural building units upon gamma irradiation.
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PMID:Optical and infrared absorption spectra of 3d transition metal ions-doped sodium borophosphate glasses and effect of gamma irradiation. 2299 47

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta.
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PMID:p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon. 2437 57

The physiological functions of neurotransmitter:sodium symporters (NSS) in reuptake of neurotransmitters from the synapse into the presynaptic nerve have been shown to be complemented by their involvement, together with non-plasma membrane neurotransmitter transporters, in the reverse transport of substrate (efflux) in response to psychostimulants. Recent experimental evidence implicates highly anionic phosphatidylinositol 4,5-biphosphate (PIP(2)) lipids in such functions of the serotonin (SERT) and dopamine (DAT) transporters. Thus, for both SERT and DAT, neurotransmitter efflux has been shown to be strongly regulated by the presence of PIP(2) lipids in the plasma membrane, and the electrostatic interaction of the N-terminal region of DAT with the negatively charged PIP(2) lipids. We examine the experimentally established phenotypes in a structural context obtained from computational modeling based on recent crystallographic data. The results are shown to set the stage for a mechanistic understanding of physiological actions of neurotransmitter transporters in the NSS family of membrane proteins. This article is part of a Special Issue entitled: Lipid-protein interactions.
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PMID:Functional mechanisms of neurotransmitter transporters regulated by lipid-protein interactions of their terminal loops. 2584 98

Human dopamine (DA) transporter (hDAT) regulates dopaminergic signaling in the central nervous system by maintaining the synaptic concentration of DA at physiological levels, upon reuptake of DA into presynaptic terminals. DA translocation involves the co-transport of two sodium ions and the channeling of a chloride ion, and it is achieved via alternating access between outward-facing (OF) and inward-facing states of DAT. hDAT is a target for addictive drugs, such as cocaine, amphetamine (AMPH), and therapeutic antidepressants. Our recent quantitative systems pharmacology study suggested that orphenadrine (ORPH), an anticholinergic agent and anti-Parkinson drug, might be repurposable as a DAT drug. Previous studies have shown that DAT-substrates like AMPH or -blockers like cocaine modulate the function of DAT in different ways. However, the molecular mechanisms of modulation remained elusive due to the lack of structural data on DAT. The newly resolved DAT structure from Drosophila melanogaster opens the way to a deeper understanding of the mechanism and time evolution of DAT-drug/ligand interactions. Using a combination of homology modeling, docking analysis, molecular dynamics simulations, and molecular biology experiments, we performed a comparative study of the binding properties of DA, AMPH, ORPH, and cocaine and their modulation of hDAT function. Simulations demonstrate that binding DA or AMPH drives a structural transition toward a functional form predisposed to translocate the ligand. In contrast, ORPH appears to inhibit DAT function by arresting it in the OF open conformation. The analysis shows that cocaine and ORPH competitively bind DAT, with the binding pose and affinity dependent on the conformational state of DAT. Further assays show that the effect of ORPH on DAT uptake and endocytosis is comparable to that of cocaine.
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PMID:Insights into the Modulation of Dopamine Transporter Function by Amphetamine, Orphenadrine, and Cocaine Binding. 2610 64

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay (comet assay), p-phenylenediamine dihydrochloride (PPD), o-phenylphenol sodium salt (OPP), and 2,4-diaminotoluene (2,4-DAT), were analyzed in this laboratory as coded test chemicals. Male Sprague-Dawley rats (7-9 weeks of age) were given three oral doses of the test compounds, 24 and 21 h apart and liver and stomach were sampled 3h after the final dose administration. Under the conditions of the test, no increases in DNA damage were observed in liver and stomach with PPD and OPP up to 100 and 1000 mg/kg/day, respectively. 2,4-DAT, a known genotoxic carcinogen, induced a weak but reproducible, dose-related and statistically significant increase in DNA damage in liver cells while no increases were observed in stomach cells.
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PMID:Evaluation of p-phenylenediamine, o-phenylphenol sodium salt, and 2,4-diaminotoluene in the rat comet assay as part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay. 2621 6

Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.
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PMID:Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer. 2822 22

Transporter-mediated uptake determines the peak concentration, duration, and physical spread of released monoamines. Most studies of monoamine clearance focus on the presynaptic uptake1 transporters SERT, NET and DAT. However, recent studies have demonstrated the expression of the uptake2 transporter OCT3 (organic cation transporter 3), throughout the rodent brain. In contrast to NET, DAT and SERT, OCT3 has higher capacity and lower affinity for substrates, is sodium-independent, and is multi-specific, with the capacity to transport norepinephrine, dopamine, serotonin and histamine. OCT3 is insensitive to inhibition by cocaine and antidepressant drugs but is inhibited directly by the glucocorticoid hormone corticosterone. Thus, OCT3 represents a novel, stress hormone-sensitive, monoamine transport mechanism. Incorporating this transporter into current models of monoaminergic neurotransmission requires information on: A) the cellular and subcellular localization of the transporter; B) the effects of OCT3 inhibitors on monoamine clearance; and C) the consequences of decreased OCT3-mediated transport on physiology and/or behavior. This review summarizes studies describing the anatomical distribution of OCT3, its cellular and subcellular localization, its contribution to the regulation of dopaminergic signaling, and its roles in the regulation of behavior. Together, these and other studies suggest that both Uptake1 and Uptake2 transporters play key roles in regulating monoaminergic neurotransmission and the effects of monoamines on behavior.
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PMID:Roles for the uptake2 transporter OCT3 in regulation of dopaminergic neurotransmission and behavior. 3005 94

Neurotransmitter removal from glycine-mediated synapses relies on two sodium-driven high-affinity plasma membrane GlyTs that control neurotransmitter availability. Mostly glial GlyT1 is the main regulator of glycine synaptic levels, whereas neuronal GlyT2 promotes the recycling of synaptic glycine and supplies neurotransmitter for presynaptic vesicle refilling. The GlyTs differ in sodium:glycine symport stoichiometry, showing GlyT1 a 2:1 and GlyT2 a 3:1 sodium:glycine coupling. Sodium binds to the GlyTs at two conserved Na+ sites: Na1 and Na2. The location of GlyT2 Na3 site remains unknown, although Glu650 has been involved in the coordination. Here, we have used comparative MD simulations of a GlyT2 model constructed by homology to the crystalized DAT from Drosophila melanogaster by placing the Na3 ion at two different locations. By combination of in silico and experimental data obtained by biochemical and electrophysiological analysis of GlyTs mutants, we provide evidences suggesting the GlyT2 third sodium ion is held by Glu-250 and Glu-650, within a region with robust allosteric properties involved in cation-specific sensitivity. Substitution of Glu650 in GlyT2 by the corresponding methionine in GlyT1 reduced the charge-to-flux ratio to the level of GlyT1 without producing transport uncoupling. Chloride dependence of glycine transport was almost abolished in this GlyT2 mutant but simultaneous substitution of Glu250 and Glu650 by neutral amino acids rescued chloride sensitivity, suggesting that protonation/deprotonation of Glu250 substitutes chloride function. The differential behavior of equivalent GlyT1 mutations sustains a GlyT2-specific allosteric coupling between the putative Na3 site and the chloride site.
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PMID:Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport. 3031 54


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