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1.
Sci Rep ; 6: 31385, 2016 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-27514281

RESUMO

Phendimetrazine (PDM) is a clinically available anorectic and a candidate pharmacotherapy for cocaine addiction. PDM has been hypothesized to function as a prodrug that requires metabolism to the amphetamine-like monoamine transporter substrate phenmetrazine (PM) to produce its pharmacological effects; however, whether PDM functions as an inactive prodrug or has pharmacological activity on its own remains unclear. The study aim was to determine PDM pharmacological mechanisms using electrophysiological, neurochemical, and behavioral procedures. PDM blocked the endogenous basal hDAT (human dopamine transporter) current in voltage-clamped (-60 mV) oocytes consistent with a DAT inhibitor profile, whereas its metabolite PM induced an inward hDAT current consistent with a DAT substrate profile. PDM also attenuated the PM-induced inward current during co-application, providing further evidence that PDM functions as a DAT inhibitor. PDM increased nucleus accumbens dopamine levels and facilitated electrical brain stimulation reinforcement within 10 min in rats, providing in vivo evidence supporting PDM pharmacological activity. These results demonstrate that PDM functions as a DAT inhibitor that may also interact with the pharmacological effects of its metabolite PM. Overall, these results suggest a novel mechanism for PDM therapeutic effects via initial PDM DAT inhibition followed by PM DAT substrate-induced dopamine release.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Morfolinas/administração & dosagem , Oócitos/efeitos dos fármacos , Fenmetrazina/administração & dosagem , Animais , Dopamina/metabolismo , Regulação para Baixo , Masculino , Estrutura Molecular , Morfolinas/química , Morfolinas/farmacologia , Núcleo Accumbens/metabolismo , Oócitos/metabolismo , Fenmetrazina/química , Fenmetrazina/farmacologia , Ratos , Xenopus
2.
Mol Pharmacol ; 86(1): 76-85, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24753048

RESUMO

The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr62 revealed T62D-human (h)DAT partitions in a predominately efflux-preferring conformation. Compared with wild-type (WT), T62D-hDAT exhibits reduced [(3)H]DA uptake and enhanced baseline DA efflux, whereas T62A-hDAT and WT-hDAT function in an influx-preferring conformation. We now interrogate the basis of the mutants' altered function with respect to membrane conductance and Na(+) sensitivity. The hDAT constructs were expressed in Xenopus oocytes to investigate if heightened membrane potential would explain the efflux characteristics of T62D-hDAT. In the absence of substrate, all constructs displayed identical resting membrane potentials. Substrate-induced inward currents were present in oocytes expressing WT- and T62A-hDAT but not T62D-hDAT, suggesting equal bidirectional ion flow through T62D-hDAT. Utilization of the fluorescent DAT substrate ASP(+) [4-(4-(dimethylamino)styryl)-N-methylpyridinium] revealed that T62D-hDAT accumulates substrate in human embryonic kidney (HEK)-293 cells when the substrate is not subject to efflux. Extracellular sodium (Na(+) e) replacement was used to evaluate sodium gradient requirements for DAT transport functions. The EC50 for Na(+) e stimulation of [(3)H]DA uptake was identical in all constructs expressed in HEK-293 cells. As expected, decreasing [Na(+)]e stimulated [(3)H]DA efflux in WT- and T62A-hDAT cells. Conversely, the elevated [(3)H]DA efflux in T62D-hDAT cells was independent of Na(+) e and commensurate with [(3)H]DA efflux attained in WT-hDAT cells, either by removal of Na(+) e or by application of amphetamine. We conclude that T62D-hDAT represents an efflux-willing, Na(+)-primed orientation-possibly representing an experimental model of the conformational impact of amphetamine exposure to hDAT.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação/genética , Sódio/metabolismo , Treonina/genética , Animais , Transporte Biológico/genética , Linhagem Celular , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Potenciais da Membrana/genética , Proteínas de Membrana Transportadoras/genética , Oócitos/metabolismo , Treonina/metabolismo , Xenopus
3.
J Am Chem Soc ; 133(44): 17528-31, 2011 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-21970724

RESUMO

The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) protein plays a central role in terminating 5-HT neurotransmission and is the most important therapeutic target for the treatment of major depression and anxiety disorders. We report an innovative, versatile, and target-selective quantum dot (QD) labeling approach for SERT in single Xenopus oocytes that can be adopted as a drug-screening platform. Our labeling approach employs a custom-made, QD-tagged indoleamine derivative ligand, IDT318, that is structurally similar to 5-HT and accesses the primary binding site with enhanced human SERT selectivity. Incubating QD-labeled oocytes with paroxetine (Paxil), a high-affinity SERT-specific inhibitor, showed a concentration- and time-dependent decrease in QD fluorescence, demonstrating the utility of our approach for the identification of SERT modulators. Furthermore, with the development of ligands aimed at other pharmacologically relevant targets, our approach may potentially form the basis for a multitarget drug discovery platform.


Assuntos
Aminas/química , Antidepressivos/farmacologia , Descoberta de Drogas , Fluorescência , Paroxetina/farmacologia , Pontos Quânticos , Animais , Antidepressivos/química , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Ligantes , Oócitos/química , Oócitos/efeitos dos fármacos , Paroxetina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Fatores de Tempo , Xenopus
4.
J Biol Chem ; 286(35): 30823-30836, 2011 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-21730057

RESUMO

Na(+)- and Cl(-)-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl(-) coordination of human serotonin transport have been identified, the role of Cl(-) in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl(-) binding to concentrative neurotransmitter uptake.


Assuntos
Asparagina/química , Cloretos/química , Neurotransmissores/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Animais , Cisteína/química , Eletrofisiologia/métodos , Células HeLa , Humanos , Íons , Mutagênese Sítio-Dirigida , Norepinefrina/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp , Plasmídeos/metabolismo , Ratos , Serotonina/metabolismo , Xenopus laevis
5.
Proc Natl Acad Sci U S A ; 105(37): 14192-7, 2008 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-18768815

RESUMO

The Caenorhabditis elegans (C. elegans) dopamine (DA) transporter (DAT-1) regulates DA signaling through efficient DA reuptake following synaptic release. In addition to its DA transport function, DAT-1 generates detectable DA-gated currents that may influence neuronal excitability. Previously, we provided evidence that single Cl-channel events underlie DAT-1 currents. In these studies, we identified a distinct population of altered DAT-1 currents arising from DAT-1 transgenic constructs bearing an N-terminal GFP fusion. The presence of these channels suggested disruption of an endogenous regulatory mechanism that modulates occupancy of DAT-1 channel states. A leading candidate for such a regulator is the SNARE protein syntaxin 1A (Syn1A), previously found to interact with homologous transporters through N-terminal interactions. Here we establish that UNC-64 (C. elegans Syn1A homologue) associates with DAT-1 and suppresses transporter channel properties. In contrast, GFP::DAT-1 is unable to form stable transporter/UNC-64 complexes that limit channel states. Although DAT-1 and GFP::DAT-1 expressing DA neurons exhibit comparable DA uptake, GFP::DAT-1 animals exhibit swimming-induced paralysis (SWIP), a phenotype associated with excess synaptic DA release and spillover. We propose that loss of UNC-64/DAT-1 interactions leads to enhanced synaptic DA release, providing a novel mechanism for DA neuron sensitization that may be relevant to mechanisms of DA-associated disorders.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Transmissão Sináptica , Sintaxina 1/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Eletrofisiologia , Genes Reporter/genética , Técnicas de Patch-Clamp , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Natação , Sintaxina 1/genética
6.
Mol Pharmacol ; 71(5): 1206-8, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17329498

RESUMO

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.


Assuntos
Proteínas de Transporte de Neurotransmissores/metabolismo , Receptores Pré-Sinápticos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Compostos de Piridínio/metabolismo , Espectrometria de Fluorescência
7.
Annu Rev Physiol ; 69: 87-112, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17059369

RESUMO

This review investigates some key aspects of transport mechanisms and recent advances in our understanding of this ubiquitous cellular process. The prevailing model of cotransport is the alternating access model, which suggests that large conformational changes in the transporter protein accompany cotransport. This model rests on decades of research and has received substantial support because many transporter characteristics are explained using its premises. New experiments, however, have revealed the existence of channels in transporters, an idea that is in conflict with traditional models. The alternating access model is the subject of previous detailed reviews. Here we concentrate on the relatively recent data that document primarily the channel properties of transporters. In some cases, namely, the observation of single-transporter currents, the evidence is direct. In other cases the evidence--for example, from fluctuation analysis or transporter currents too large to be described as anything other than channel-like--is indirect. Although the existence of channels in transporters is not in doubt, we are far from understanding the significance of this property. In the online Supplemental Material , we review some pertinent aspects of ion channel theory and cotransport physiology to provide background for the channels and transporters presented here. We discuss the existence of channels in transporters, and we speculate on the biological significance of this newly unveiled property of transport proteins.


Assuntos
Proteínas de Transporte/metabolismo , Canais Iônicos/metabolismo , Animais , Humanos , Metais/metabolismo , Proteínas de Transporte de Neurotransmissores/metabolismo , Canais de Potássio/metabolismo , Transmissão Sináptica/fisiologia
8.
Neuron ; 49(6): 791-6, 2006 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-16543127

RESUMO

Efforts to define the mechanisms governing neurotransmitter uptake and drug action have moved into high gear with the publication of a high-resolution structure of a leucine transporter from Aquifex aeolicus, a bacterial member of the SLC6 transporter family. Solved with the substrate leucine bound, the new structure corroborates extensive biochemical and mutagenesis studies performed with related mammalian neurotransmitter transporters and provides exciting suggestions as to how coupling arises between ions and substrates to permit efficient neurotransmitter clearance.


Assuntos
Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Animais , Leucina/química , Leucina/metabolismo , Proteínas de Membrana Transportadoras/classificação , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Modelos Moleculares , Conformação Proteica
9.
Physiology (Bethesda) ; 20: 225-31, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16024510

RESUMO

Plasma membrane transporters have long been known to support the reuptake of biogenic amine neurotransmitters following release in the central and peripheral nervous systems. Using high-resolution imaging, patch-clamp and amperometric approaches, as well as molecular manipulations of transporter-regulatory pathways, surprising new details have been uncovered as to how transporters work and are influenced by signaling pathways and psychostimulants.


Assuntos
Monoaminas Biogênicas/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , Animais , Humanos
10.
J Biol Chem ; 280(19): 19177-84, 2005 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-15757904

RESUMO

The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768-9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 micros. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates.


Assuntos
1-Metil-4-fenilpiridínio/análogos & derivados , Simportadores/química , 1-Metil-4-fenilpiridínio/farmacologia , Anfetamina/farmacologia , Transporte Biológico , Calibragem , Linhagem Celular , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Corantes Fluorescentes/farmacologia , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinética , Espectrometria de Massas , Microscopia Confocal , Modelos Estatísticos , Norepinefrina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Ligação Proteica , Compostos de Piridínio/farmacologia , Especificidade por Substrato , Fatores de Tempo
11.
Proc Natl Acad Sci U S A ; 101(45): 16046-51, 2004 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-15520385

RESUMO

Neurotransmitter transporters generate larger currents than expected if one assumes fixed stoichiometry models. It remains controversial, however, whether these depolarizing currents arise from high density and rapid turnover rates of a classical transporter, or whether transporters exhibit bona fide channel behavior. Although heterologously expressed transporters show single-channel behavior and noise analysis in native cells strongly suggests channel behavior, no directly observed single-channel events associated with transporters have been reported thus far in native cells. We describe single-channel events arising directly from the Caenorhabditis elegans dopamine transporter (DAT-1) as evidenced by DA-induced channel activity blocked by a high-affinity DAT-1 inhibitor, increased channel activity in neurons that overexpress DAT-1, and loss of channels in dat-1 knockout neurons. Our data indicate that authentic transporter channels underlie depolarizing whole-cell currents. Thus, DA transporters not only transport DA but also exhibit a channel mode of conduction that directly modulates membrane potential and neuronal function.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Sequência de Bases , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , DNA de Helmintos/genética , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Glicoproteínas de Membrana/genética , Potenciais da Membrana , Proteínas de Membrana Transportadoras/genética , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
13.
Trends Neurosci ; 27(6): 352-9, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15165740

RESUMO

During the past several decades, lac permease has assumed almost tutelary proportions as a model for cotransporters. This archetypical membrane protein now exerts its influence by the most dramatic means possible: its structure is solved. This article describes the configuration and implied transport mechanism of the bacterial lactose transporter and compares its structure and function with those of other transporters and those of ion channels. This juxtaposition of transporters and channels is likely to be helpful because LacY is the first cotransporter with known structure and exclusive carrier properties, and it shares topology with neurotransmitter transporters with unknown structure and channel properties.


Assuntos
Proteínas de Escherichia coli/química , Canais Iônicos/química , Canais Iônicos/fisiologia , Proteínas de Membrana Transportadoras/química , Modelos Moleculares , Animais , Sítios de Ligação , Transporte Biológico , Membrana Celular/metabolismo , Proteínas de Escherichia coli/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
14.
PLoS Biol ; 2(3): E50, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15024413

RESUMO

Divalent metal transporter-1 (DMT1/DCT1/Nramp2) is the major Fe(2+) transporter mediating cellular iron uptake in mammals. Phenotypic analyses of animals with spontaneous mutations in DMT1 indicate that it functions at two distinct sites, transporting dietary iron across the apical membrane of intestinal absorptive cells, and transporting endosomal iron released from transferrin into the cytoplasm of erythroid precursors. DMT1 also acts as a proton-dependent transporter for other heavy metal ions including Mn(2+), Co(2+), and Cu(2), but not for Mg(2+) or Ca(2+). A unique mutation in DMT1, G185R, has occurred spontaneously on two occasions in microcytic (mk) mice and once in Belgrade (b) rats. This mutation severely impairs the iron transport capability of DMT1, leading to systemic iron deficiency and anemia. The repeated occurrence of the G185R mutation cannot readily be explained by hypermutability of the gene. Here we show that G185R mutant DMT1 exhibits a new, constitutive Ca(2+) permeability, suggesting a gain of function that contributes to remutation and the mk and b phenotypes.


Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Ligação ao Ferro/genética , Mutação , Processamento Alternativo , Animais , Transporte Biológico , Células CHO , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cátions , Linhagem Celular , Cricetinae , DNA Complementar/metabolismo , Eletrofisiologia , Enterócitos/metabolismo , Humanos , Proteínas de Ligação ao Ferro/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Fenótipo , Prótons , Ratos , Fatores de Tempo
15.
Biophys J ; 85(3): 1548-59, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12944272

RESUMO

We have investigated the conduction states of human serotonin transporter (hSERT) using the voltage clamp, cut-open frog oocyte method under different internal and external ionic conditions. Our data indicate discrepancies in the alternating access model of cotransport, which cannot consistently explain substrate transport and electrophysiological data. We are able simultaneously to isolate distinct external and internal binding sites for substrate, which exert different effects upon currents conducted by hSERT, in contradiction to the alternating access model. External binding sites of coupled Na ions are likewise simultaneously accessible from the internal and external face. Although Na and Cl are putatively cotransported, they have opposite effects on the internal face of the transporter. Finally, the internal K ion does not compete with internal 5-hydroxytryptamine for empty transporters. These data can be explained more readily in the language of ion channels, rather than carrier models distinguished by alternating access mechanisms: in a channel model of coupled transport, the currents represent different states of the same permeation path through hSERT and coupling occurs in a common pore.


Assuntos
Proteínas de Transporte/química , Íons , Glicoproteínas de Membrana/química , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Animais , Sítios de Ligação , Cloro/química , Eletrofisiologia , Humanos , Modelos Biológicos , Oócitos/metabolismo , Potássio/química , Conformação Proteica , RNA Complementar/metabolismo , Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina , Sódio/química , Xenopus laevis
16.
J Biol Chem ; 278(11): 9768-77, 2003 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-12499385

RESUMO

Monoamine transporters, the molecular targets for drugs of abuse and antidepressants, clear norepinephrine, dopamine, or serotonin from the synaptic cleft. Neurotransmitters, amphetamines, and neurotoxins bind before being transported, whereas cocaine and antidepressants bind to block transport. Although binding is crucial to transport, few assays separate binding from transport, nor do they provide adequate temporal or spatial resolution to describe real-time kinetics or localize sites of active uptake. Here, we report a new method that distinguishes substrate binding from substrate transport using single-cell, space-resolved, real-time fluorescence microscopy. For these studies we use a fluorescent analogue of 1-methyl-4-phenylpyridinium, a neurotoxic metabolite and known substrate of monoamine transporters, to assess binding and transport with 50-ms, sub-micron resolution. We show that ASP(+) (4-(4-(dimethylamino)styrl)-N-methylpyridinium) has micromolar potency for the human norepinephrine transporter, that ASP(+) accumulation is Na(+)-, Cl(-)-, cocaine-, and desipramine-sensitive and temperature-dependent, and that ASP(+) competes with norepinephrine uptake. Using this method we demonstrate that norepinephrine transporters are efficient buffers for substrate, with binding rates exceeding transport rates by 100-fold. Furthermore, substrates bind deep within the transporter, isolated from both the bath and the lipid bilayer. Although transport per se depends on Na(+) and Cl(-), binding is independent of Na(+) and actually increases in low Cl(-). We further demonstrate that ASP(+) interacts with transporters not only in transfected cells but in cultured neurons. ASP(+) is also a substrate for dopamine and serotonin transporters and therefore represents a powerful new technique for studying the biophysical properties of monoamine transporters, an approach also amenable to high throughput assays for drug discovery.


Assuntos
Norepinefrina/metabolismo , Inibidores da Captação Adrenérgica/farmacologia , Agonistas alfa-Adrenérgicos/farmacologia , Anisotropia , Transporte Biológico , Linhagem Celular , Células Cultivadas , Cloro/farmacologia , Cocaína/farmacologia , Desipramina/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Corantes Fluorescentes/farmacologia , Humanos , Processamento de Imagem Assistida por Computador , Cinética , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Sódio/farmacologia , Temperatura , Fatores de Tempo , Transfecção , Azul Tripano/farmacologia
17.
Biophys J ; 83(6): 3268-82, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12496095

RESUMO

The serotonin (5-hydroxytryptamine; 5HT) transporter (SERT) catalyzes the movement of 5HT across cellular membranes. In the brain, SERT clears 5HT from extracellular spaces, modulating the strength and duration of serotonergic signaling. SERT is also an important pharmacological target for antidepressants and drugs of abuse. We have studied the flux of radio-labeled 5HT through the transporter stably expressed in HEK-293 cells. Analysis of the time course of net transport, the equilibrium 5HT gradient sustained, and the ratio of the unidirectional influx to efflux of 5HT indicate that mechanistically, human SERT functions as a 5HT channel rather than a classical carrier. This is especially apparent at relatively high [5HT](out) (> or =10 microM), but is not restricted to this regime of external 5HT.


Assuntos
Proteínas de Transporte/fisiologia , Membrana Celular/fisiologia , Rim/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Serotonina/farmacocinética , Simportadores/fisiologia , Linhagem Celular , Permeabilidade da Membrana Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Canais Iônicos/fisiologia , Rim/embriologia , Cinética , Modelos Biológicos , Porosidade , Receptores de Serotonina/metabolismo , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Serotonina/administração & dosagem , Proteínas da Membrana Plasmática de Transporte de Serotonina , Transdução de Sinais/fisiologia
18.
J Am Chem Soc ; 124(17): 4586-94, 2002 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-11971705

RESUMO

To explore the potential for use of ligand-conjugated nanocrystals to target cell surface receptors, ion channels, and transporters, we explored the ability of serotonin-labeled CdSe nanocrystals (SNACs) to interact with antidepressant-sensitive, human and Drosophila serotonin transporters (hSERT, dSERT) expressed in HeLa and HEK-293 cells. Unlike unconjugated nanocrystals, SNACs were found to dose-dependently inhibit transport of radiolabeled serotonin by hSERT and dSERT, with an estimated half-maximal activity (EC(50)) of 33 (dSERT) and 99 microM (hSERT). When serotonin was conjugated to the nanocrystal through a linker arm (LSNACs), the EC(50) for hSERT was determined to be 115 microM. Electrophysiology measurements indicated that LSNACs did not elicit currents from the serotonin-3 (5HT(3)) receptor but did produce currents when exposed to the transporter, which are similar to those elicited by antagonists. Moreover, fluorescent LSNACs were found to label SERT-transfected cells but did not label either nontransfected cells or transfected cells coincubated with the high-affinity SERT antagonist paroxetine. These findings support further consideration of ligand-conjugated nanocrystals as versatile probes of membrane proteins in living cells.


Assuntos
Proteínas de Transporte/química , Glicoproteínas de Membrana/química , Proteínas de Membrana Transportadoras , Nanotecnologia/métodos , Proteínas do Tecido Nervoso , Serotonina/química , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Linhagem Celular , Eletrofisiologia , Células HeLa , Humanos , Rim/citologia , Rim/metabolismo , Rim/fisiologia , Ligantes , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Oócitos/metabolismo , Oócitos/fisiologia , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina , Especificidade por Substrato , Transfecção , Xenopus
19.
Br J Pharmacol ; 135(8): 1927-34, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11959795

RESUMO

1. This paper reports on a primary cell culture system that predominantly expresses native norepinephrine (NE) transporters (NETs), and is amenable to biophysical as well as biochemical analyses. 2. Previous research has identified human and rat placentas as rich sources of NET. We have exploited this to develop primary cultures of rat placental trophoblasts. NE uptake in these cultures is about 10 times higher when compared to 5HT uptake. The presence of NET protein is revealed by immunoblot analysis, while there is no detectable SERT protein. 3. NE transport in rat trophoblasts is sensitive to NET-specific antagonists, desipramine (DS) and nisoxetine (NX), but not to the dopamine-transporter (DAT) specific antagonist, GBR12909 or to the serotonin (5HT) transporter (SERT) specific antagonist paroxetine (PX). Drugs of abuse such as cocaine and amphetamine also inhibit NE transport in these cells. Together these results suggest that rat placental trophoblasts predominantly express NET over other monoamine transporters. 4. Patch-clamp analysis reveals that NETs in intact rat trophoblasts are electrogenic. Comparison of NE uptake with NE-induced currents suggests that these two modes of transporter activity are differentially regulated.


Assuntos
Antidepressivos/metabolismo , Cocaína/metabolismo , Fluoxetina/análogos & derivados , Norepinefrina/metabolismo , Simportadores/metabolismo , Trofoblastos/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Inibidores da Captação de Dopamina/metabolismo , Feminino , Fluoxetina/metabolismo , Potenciais da Membrana/fisiologia , Norepinefrina/antagonistas & inibidores , Norepinefrina/fisiologia , Proteínas da Membrana Plasmática de Transporte de Norepinefrina , Técnicas de Patch-Clamp , Gravidez , Ratos , Ratos Sprague-Dawley , Simportadores/biossíntese , Trofoblastos/citologia
20.
J Biol Chem ; 277(17): 14475-82, 2002 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-11844791

RESUMO

We express mammalian serotonin transporters (SERTs) in Xenopus oocytes by cRNA injection and measure 5-hydroxytryptamine (5-HT) transport and 5-HT-induced current at varying expression levels. Transport and current both increase sigmoidally with the amount of cRNA injected, but current requires approximately 5-fold more cRNA to elicit a half-maximal response. Western blots of SERT protein demonstrate that current, but not transport, correlates linearly with the amount of SERT on the plasma membrane. In oocytes co-injected with wild-type SERT and an inactive SERT mutant, transport is similar to SERT alone, but current is attenuated. The charge/transport ratio reports the differential sensitivity of transport and current to increasing SERT cRNA injection and mutant co-expression. Manipulations that alter the charge/transport ratio also perturb substrate and inhibitor recognition. 5-HT, d-amphetamine, cocaine, and paroxetine inhibit transport more potently at lower expression levels; however, 5-HT potency for induction of current is similar at high and low expression. Moreover, the apparent potency of cRNA for transport depends on 5-HT concentration. We postulate that SERT interacts allosterically with an endogenous factor of limited abundance to alter substrate and inhibitor potency and the balance of 5-HT transport and channel-like activity.


Assuntos
Proteínas de Transporte/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Anfetamina/farmacologia , Animais , Western Blotting , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Cocaína/farmacologia , Glicoproteínas de Membrana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Paroxetina/farmacologia , RNA Complementar , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina , Xenopus laevis
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