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Antibody-Conjugated Single Quantum Dot Tracking of Membrane Neurotransmitter Transporters in Primary Neuronal Cultures.
Bailey DM, Kovtun O, Rosenthal SJ
(2017) Methods Mol Biol 1570: 165-177
MeSH Terms: Algorithms, Animals, Fluorescent Antibody Technique, Immunoconjugates, Models, Theoretical, Molecular Imaging, Neurons, Neurotransmitter Transport Proteins, Primary Cell Culture, Quantum Dots, Rats, Single Molecule Imaging, Statistics as Topic
Show Abstract · Added April 3, 2018
Single particle tracking (SPT) experiments have provided the scientific community with invaluable single-molecule information about the dynamic regulation of individual receptors, transporters, kinases, lipids, and molecular motors. SPT is an alternative to ensemble averaging approaches, where heterogeneous modes of motion might be lost. Quantum dots (QDs) are excellent probes for SPT experiments due to their photostability, high brightness, and size-dependent, narrow emission spectra. In a typical QD-based SPT experiment, QDs are bound to the target of interest and imaged for seconds to minutes via fluorescence video microscopy. Single QD spots in individual frames are then linked to form trajectories that are analyzed to determine their mean square displacement, diffusion coefficient, confinement index, and instantaneous velocity. This chapter describes a generalizable protocol for the single particle tracking of membrane neurotransmitter transporters on cell membranes with either unmodified extracellular antibody probes and secondary antibody-conjugated quantum dots or biotinylated extracellular antibody probes and streptavidin-conjugated quantum dots in primary neuronal cultures. The neuronal cell culture, the biotinylation protocol and the quantum dot labeling procedures, as well as basic data analysis are discussed.
0 Communities
2 Members
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MeSH Terms
The brain in flux: genetic, physiologic, and therapeutic perspectives on transporters in the CNS.
Blakely RD, Ortega A, Robinson MB
(2014) Neurochem Int 73: 1-3
MeSH Terms: Animals, Brain Chemistry, Brain Diseases, Carrier Proteins, Humans, Neurotransmitter Transport Proteins
Added February 12, 2015
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1 Members
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6 MeSH Terms
Conformational dynamics of ligand-dependent alternating access in LeuT.
Kazmier K, Sharma S, Quick M, Islam SM, Roux B, Weinstein H, Javitch JA, McHaourab HS
(2014) Nat Struct Mol Biol 21: 472-9
MeSH Terms: Bacterial Proteins, Ligands, Models, Molecular, Neurotransmitter Transport Proteins, Protein Structure, Tertiary, Spin Labels, Symporters
Show Abstract · Added May 30, 2014
The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial homolog of neurotransmitter/sodium symporters (NSSs) that catalyze reuptake of neurotransmitters at the synapse. Crystal structures of wild-type and mutants of LeuT have been interpreted as conformational states in the coupled transport cycle. However, the mechanistic identities inferred from these structures have not been validated, and the ligand-dependent conformational equilibrium of LeuT has not been defined. Here, we used distance measurements between spin-label pairs to elucidate Na(+)- and leucine-dependent conformational changes on the intracellular and extracellular sides of the transporter. The results identify structural motifs that underlie the isomerization of LeuT between outward-facing, inward-facing and occluded states. The conformational changes reported here present a dynamic picture of the alternating-access mechanism of LeuT and NSSs that is different from the inferences reached from currently available structural models.
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1 Members
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7 MeSH Terms
Pharmacological characterization of designer cathinones in vitro.
Simmler LD, Buser TA, Donzelli M, Schramm Y, Dieu LH, Huwyler J, Chaboz S, Hoener MC, Liechti ME
(2013) Br J Pharmacol 168: 458-70
MeSH Terms: Amphetamines, Blood-Brain Barrier, Cell Line, Designer Drugs, Dopamine, HEK293 Cells, Humans, Illicit Drugs, Norepinephrine, Plasma Membrane Neurotransmitter Transport Proteins, Serotonin
Show Abstract · Added August 3, 2013
BACKGROUND AND PURPOSE - Designer β-keto amphetamines (e.g. cathinones, 'bath salts' and 'research chemicals') have become popular recreational drugs, but their pharmacology is poorly characterized.
EXPERIMENTAL APPROACH - We determined the potencies of cathinones to inhibit DA, NA and 5-HT transport into transporter-transfected HEK 293 cells, DA and 5-HT efflux from monoamine-preloaded cells, and monoamine receptor binding affinity.
KEY RESULTS - Mephedrone, methylone, ethylone, butylone and naphyrone acted as non-selective monoamine uptake inhibitors, similar to cocaine. Mephedrone, methylone, ethylone and butylone also induced the release of 5-HT, similar to 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) and other entactogens. Cathinone, methcathinone and flephedrone, similar to amphetamine and methamphetamine, acted as preferential DA and NA uptake inhibitors and induced the release of DA. Pyrovalerone and 3,4-methylenedioxypyrovalerone (MDPV) were highly potent and selective DA and NA transporter inhibitors but unlike amphetamines did not evoke the release of monoamines. The non-β-keto amphetamines are trace amine-associated receptor 1 ligands, whereas the cathinones are not. All the cathinones showed high blood-brain barrier permeability in an in vitro model; mephedrone and MDPV exhibited particularly high permeability.
CONCLUSIONS AND IMPLICATIONS - Cathinones have considerable pharmacological differences that form the basis of their suggested classification into three groups. The predominant action of all cathinones on the DA transporter is probably associated with a considerable risk of addiction.
© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.
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11 MeSH Terms
Labeling of neuronal receptors and transporters with quantum dots.
Chang JC, Kovtun O, Blakely RD, Rosenthal SJ
(2012) Wiley Interdiscip Rev Nanomed Nanobiotechnol 4: 605-19
MeSH Terms: Animals, Fluorescent Dyes, Humans, Molecular Imaging, Molecular Probes, Neurons, Neurotransmitter Transport Proteins, Protein Transport, Quantum Dots, Receptors, Neurotransmitter
Show Abstract · Added July 10, 2013
The ability to efficiently visualize protein targets in cells is a fundamental goal in biological research. Recently, quantum dots (QDots) have emerged as a powerful class of fluorescent probes for labeling membrane proteins in living cells because of breakthrough advances in QDot surface chemistry and biofunctionalization strategies. This review discusses the increasing use of QDots for fluorescence imaging of neuronal receptors and transporters. The readers are briefly introduced to QDot structure, photophysical properties, and common synthetic routes toward the generation of water-soluble QDots. The following section highlights several reports of QDot application that seek to unravel molecular aspects of neuronal receptor and transporter regulation and trafficking. This article is closed with a prospectus of the future of derivatized QDots in neurobiological and pharmacological research.
Copyright © 2012 Wiley Periodicals, Inc.
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3 Members
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10 MeSH Terms
Vesicular and plasma membrane transporters for neurotransmitters.
Blakely RD, Edwards RH
(2012) Cold Spring Harb Perspect Biol 4:
MeSH Terms: Animals, Cell Membrane, Exocytosis, Humans, Neurotransmitter Agents, Neurotransmitter Transport Proteins, Synaptic Transmission, Synaptic Vesicles
Show Abstract · Added July 10, 2013
The regulated exocytosis that mediates chemical signaling at synapses requires mechanisms to coordinate the immediate response to stimulation with the recycling needed to sustain release. Two general classes of transporter contribute to release, one located on synaptic vesicles that loads them with transmitter, and a second at the plasma membrane that both terminates signaling and serves to recycle transmitter for subsequent rounds of release. Originally identified as the target of psychoactive drugs, these transport systems have important roles in transmitter release, but we are only beginning to understand their contribution to synaptic transmission, plasticity, behavior, and disease. Recent work has started to provide a structural basis for their activity, to characterize their trafficking and potential for regulation. The results indicate that far from the passive target of psychoactive drugs, neurotransmitter transporters undergo regulation that contributes to synaptic plasticity.
1 Communities
1 Members
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8 MeSH Terms
Bound to be different: neurotransmitter transporters meet their bacterial cousins.
Henry LK, Meiler J, Blakely RD
(2007) Mol Interv 7: 306-9
MeSH Terms: Antidepressive Agents, Bacterial Proteins, Binding Sites, Humans, Models, Molecular, Multiprotein Complexes, Neurotransmitter Transport Proteins
Show Abstract · Added July 10, 2013
The neurotransmitter transporters belonging to the solute carrier 6 (SLC6) family, including the gamma-aminobutyric acid (GAT), norepinephrine (NET), serotonin (SERT) and dopamine (DAT) transporters are extremely important drug targets of great clinical relevance. These Na+, Cl(-)-dependent transporters primarily function following neurotransmission to reset neuronal signaling by transporting neurotransmitter out of the synapse and back into the pre-synaptic neuron. Recent studies have tracked down an elusive binding site for Cl(-) that facilitates neurotransmitter transport using structural differences evident with bacterial family members (e.g., the Aquifex aeolicus leucine transporter LeuT Aa) that lack Cl(-) dependence. Additionally, the crystal structures of antidepressant-bound LeuT Aa reveals a surprising mode of drug interaction that may have relevance for medication development. The study of sequence and structural divergence between LeuT Aa and human SLC6 family transporters can thus inform us as to how and why neurotransmitter transporters evolved a reliance on extracellular Cl(-) to propel the transport cycle; what residue changes and helical rearrangements give rise to recognition of different substrates; and how drugs such as antidepressants, cocaine, and amphetamines halt (or reverse) the transport process.
2 Communities
2 Members
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7 MeSH Terms
Microarray analysis reveals distinctive signaling between the bed nucleus of the stria terminalis, nucleus accumbens, and dorsal striatum.
Olsen CM, Huang Y, Goodwin S, Ciobanu DC, Lu L, Sutter TR, Winder DG
(2008) Physiol Genomics 32: 283-98
MeSH Terms: Animals, Corpus Striatum, Databases, Factual, Gene Expression Profiling, Gene Regulatory Networks, Genes, Reporter, Green Fluorescent Proteins, Male, Mice, Mice, Inbred C57BL, Nerve Tissue Proteins, Neural Pathways, Neuronal Plasticity, Neurotransmitter Agents, Neurotransmitter Transport Proteins, Nucleus Accumbens, Oligonucleotide Array Sequence Analysis, Receptors, Neurotransmitter, Septal Nuclei, Signal Transduction, Synaptic Transmission, Transcription, Genetic
Show Abstract · Added May 19, 2014
To identify distinct transcriptional patterns between the major subcortical dopamine targets commonly studied in addiction we studied differences in gene expression between the bed nucleus of the stria terminalis (BNST), nucleus accumbens (NAc), and dorsal striatum (dStr) using microarray analysis. We first tested for differences in expression of genes encoding transcripts for common neurotransmitter systems as well as calcium binding proteins routinely used in neuroanatomical delineation of brain regions. This a priori method revealed differential expression of corticotropin releasing hormone (Crh), the GABA transporter (Slc6a1), and prodynorphin (Pdyn) mRNAs as well as several others. Using a gene ontology tool, functional scoring analysis, and Ingenuity Pathway Analysis, we further identified several physiological pathways that were distinct among these brain regions. These two different analyses both identified calcium signaling, G-coupled protein receptor signaling, and adenylate cyclase-related signaling as significantly different among the BNST, NAc, and dStr. These types of signaling pathways play important roles in, amongst other things, synaptic plasticity. Investigation of differential gene expression revealed several instances that may provide insight into reported differences in synaptic plasticity between these brain regions. The results support other studies suggesting that crucial pathways involved in neurotransmission are distinct among the BNST, NAc, and dStr and provide insight into the potential use of pharmacological agents that may target region-specific signaling pathways. Furthermore, these studies provide a framework for future mouse-mouse comparisons of transcriptional profiles after behavioral/pharmacological manipulation.
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1 Members
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22 MeSH Terms
All aglow about presynaptic receptor regulation of neurotransmitter transporters.
Blakely RD, DeFelice LJ
(2007) Mol Pharmacol 71: 1206-8
MeSH Terms: Extracellular Signal-Regulated MAP Kinases, Humans, Neurotransmitter Transport Proteins, Pyridinium Compounds, Receptors, Presynaptic, Spectrometry, Fluorescence
Show Abstract · Added July 10, 2013
Mounting evidence supports the idea that neurotransmitter transporters are subject to many forms of post-translational regulation typically associated with receptors and ion channels, including receptor and kinase-mediated changes in transporter phosphorylation, cell surface trafficking, and/or catalytic activation. Although hints of this regulation can be achieved with traditional radiolabeled substrate flux techniques, higher resolution methods are needed that can localize transporter function in situ as well as permit real-time monitoring of transport function without confounds associated with coincident receptor activation. The elegant study by Bolan et al. (p. 1222) capitalizes on the fluorescent properties of a recently introduced substrate for the dopamine (DA) transporter (DAT), termed 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+), to illuminate a pertussis toxin-sensitive, extracellular signal-regulated kinase (ERK1/2)-dependent pathway by which presynaptic DA D(2) receptors regulate DATs.
1 Communities
1 Members
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6 MeSH Terms
The functional impact of SLC6 transporter genetic variation.
Hahn MK, Blakely RD
(2007) Annu Rev Pharmacol Toxicol 47: 401-41
MeSH Terms: Attention Deficit Disorder with Hyperactivity, Biological Transport, Depression, Humans, Multigene Family, Mutation, Plasma Membrane Neurotransmitter Transport Proteins, Polymorphism, Genetic
Show Abstract · Added July 10, 2013
Solute carrier 6 (SLC6) is a gene family of ion-coupled plasma membrane cotransporters, including transporters of neurotransmitters, amino acids, and osmolytes that mediate the movement of their substrates into cells to facilitate or regulate synaptic transmission, neurotransmitter recycling, metabolic function, and fluid homeostasis. Polymorphisms in transporter genes may influence expression and activity of transporters and contribute to behavior, traits, and disease. Determining the relationship between the monoamine transporters and complex psychiatric disorders has been a particular challenge that is being met by evolving approaches. Elucidating the functional consequences of and interactions among polymorphic sites is advancing our understanding of this relationship. Examining the influence of environmental influences, especially early-life events, has helped bridge the gap between genotype and phenotype. Refining phenotypes, through assessment of endophenotypes, specific behavioral tasks, medication response, and brain network properties has also improved detection of the impact of genetic variation on complex behavior and disease.
1 Communities
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8 MeSH Terms