RESUMO
The mesolimbic dopamine system is a crucial component of reward and reinforcement processing, including the psychotropic effects of drugs of abuse such as cocaine. Drugs of abuse can activate intracellular signaling cascades that engender long-term molecular changes to brain reward circuitry, which can promote further drug use. However, gaps remain about how the activity of these signaling pathways, such as ERK1/2 signaling, can affect cocaine-induced neurochemical plasticity and cocaine-associated behaviors specifically within dopaminergic cells. To enable specific modulation of ERK1/2 signaling in dopaminergic neurons of the ventral tegmental area, we utilize a viral construct that Cre dependently expresses Map kinase phosphatase 3 (MKP3) to reduce the activity of ERK1/2, in combination with transgenic rats that express Cre in tyrosine hydroxylase (TH)-positive cells. Following viral transfection, we found an increase in the surface expression of the dopamine transporter (DAT), a protein associated with the regulation of dopamine signaling, dopamine transmission, and cocaine-associated behavior. We found that inactivation of ERK1/2 reduced post-translational phosphorylation of the DAT, attenuated the ability of cocaine to inhibit the DAT, and decreased motivation for cocaine without affecting associative learning as tested by conditioned place preference. Together, these results indicate that ERK1/2 signaling plays a critical role in shaping the dopamine response to cocaine and may provide additional insights into the function of dopaminergic neurons. Further, these findings lay important groundwork toward the assessment of how signaling pathways and their downstream effectors influence dopamine transmission and could ultimately provide therapeutic targets for treating cocaine use disorders.
Assuntos
Cocaína , Dopamina , Ratos , Animais , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Motivação , Sistema de Sinalização das MAP Quinases , Fosfatase 6 de Especificidade Dupla/metabolismo , Cocaína/farmacologia , Área Tegmentar Ventral/fisiologia , Recompensa , Ratos TransgênicosRESUMO
Aberrant dopamine (DA) signaling is associated with several psychiatric disorders, such as autism, bipolar disorder, addiction, and Parkinson's disease, and several medications that target the DA transporter (DAT) can induce or treat these disorders. In addition, psychostimulants, such as cocaine and D-amphetamine (AMPH), rely on the competitive interactions with the transporter's substrate binding site to produce their rewarding effects. Agents that exhibit noncompetitive, allosteric modulation of DAT remain an important topic of investigation due to their potential therapeutic applications. We previously identified a novel allosteric modulator of human DAT, KM822, that can decrease the affinity of cocaine for DAT and attenuate cocaine-elicited behaviors; however, whether DAT is the sole mediator of KM822 actions in vivo is unproven given the large number of potential off-target sites. Here, we provide in silico and in vitro evidence that the allosteric site engaged by KM822 is conserved between human DAT and Caenorhabditis elegans DAT-1. KM822 binds to a similar pocket in DAT-1 as previously identified in human DAT. In functional dopamine uptake assays, KM822 affects the interaction between AMPH and DAT-1 by reducing the affinity of AMPH for DAT-1. Finally, through a combination of genetic and pharmacological in vivo approaches we provide evidence that KM822 diminishes the behavioral actions of AMPH on swimming-induced paralysis through a direct allosteric modulation of DAT-1. More broadly, our findings demonstrate allosteric modulation of DAT as a behavior modifying strategy and suggests that Caenorhabditis elegans can be operationalized to identify and investigate the interactions of DAT allosteric modulators. SIGNIFICANCE STATEMENT: We previously demonstrated that the dopamine transporter (DAT) allosteric modulator KM822 decreases cocaine affinity for human DAT. Here, using in silico and in vivo genetic approaches, we extend this finding to interactions with amphetamine, demonstrating evolutionary conservation of the DAT allosteric site. In Caenorhabditis elegans, we report that KM822 suppresses amphetamine behavioral effects via specific interactions with DAT-1. Our findings reveal Caenorhabditis elegans as a new tool to study allosteric modulation of DAT and its behavioral consequences.
Assuntos
Anfetamina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Dopaminérgicos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Anfetamina/farmacologia , Animais , Células COS , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Chlorocebus aethiops , Dopaminérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Relação Dose-Resposta a Droga , Drosophila melanogaster , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Secundária de ProteínaRESUMO
Echinococcus granulosus, the etiological agent of human cystic echinococcosis (formerly known as hydatid disease), represents a serious worldwide public health problem with limited treatment options. The essential role played by the neuromuscular system in parasite survival and the relevance of serotonin (5-HT) in parasite movement and development make the serotonergic system an attractive source of drug targets. In this study, we cloned and sequenced a cDNA coding for the serotonin transporter from E. granulosus (EgSERT). Bioinformatic analyses suggest that EgSERT has twelve transmembrane domains with highly conserved ligand and ionic binding sites but a less conserved allosteric site compared with the human orthologue (HsSERT). Modeling studies also suggest a good degree of conservation of the overall structure compared with HsSERT. Functional and pharmacological studies performed on the cloned EgSERT confirm that this protein is indeed a serotonin transporter. EgSERT is specific for 5-HT and does not transport other neurotransmitters. Typical monoamine transport inhibitors also displayed inhibitory activities towards EgSERT, but with lower affinity than for the human SERT (HsSERT), suggesting a high divergence of the cestode transporter compared with HsSERT. In situ hybridization studies performed in the larval protoscolex stage suggest that EgSERT is located in discrete regions that are compatible with the major ganglia of the serotonergic nervous system. The pharmacological properties, the amino acidic substitutions at important functional regions compared with the HsSERT, and the putative role of EgSERT in the nervous system suggest that it could be an important target for pharmacological intervention.
Assuntos
Cestoides , Equinococose , Echinococcus granulosus , Animais , Equinococose/parasitologia , Echinococcus granulosus/fisiologia , Humanos , Sistema Nervoso/metabolismo , Serotonina/metabolismo , Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismoRESUMO
The reinforcing efficacy of cocaine is largely determined by its capacity to inhibit the dopamine transporter (DAT), and emerging evidence suggests that differences in cocaine potency are linked to several symptoms of cocaine use disorder. Despite this evidence, the neural processes that govern cocaine potency in vivo remain unclear. In male rats, we used chemogenetics with intra-VTA microinfusions of the agonist clozapine-n-oxide to bidirectionally modulate dopamine neurons. Using ex vivo fast scan cyclic voltammetry, pharmacological probes of the DAT, biochemical assessments of DAT membrane availability and phosphorylation, and cocaine self-administration, we tested the effects of chemogenetic manipulations on cocaine potency at distal DATs in the nucleus accumbens as well as the behavioral economics of cocaine self-administration. We discovered that chemogenetic manipulation of dopamine neurons produced rapid, bidirectional modulation of cocaine potency at DATs in the nucleus accumbens. We then provided evidence that changes in cocaine potency are associated with alterations in DAT affinity for cocaine and demonstrated that this change in affinity coincides with DAT conformation biases and changes in DAT phosphorylation state. Finally, we showed that chemogenetic manipulation of dopamine neurons alters cocaine consumption in a manner consistent with changes in cocaine potency at distal DATs. Based on the spatial and temporal constraints inherent to our experimental design, we posit that changes in cocaine potency are driven by alterations in dopamine neuron activity. When considered together, these observations provide a novel mechanism through which GPCRs regulate cocaine's pharmacological and behavioral effects.SIGNIFICANCE STATEMENT Differences in the pharmacological effects of cocaine are believed to influence the development and progression of cocaine use disorder. However, the biological and physiological processes that determine sensitivity to cocaine remain unclear. In this work, we use a combination of chemogenetics, fast scan cyclic voltammetry, pharmacology, biochemistry, and cocaine self-administration with economic demand analysis to demonstrate a novel mechanism by which cocaine potency is determined in vivo These studies identify a novel process by which the pharmacodynamics of cocaine are derived in vivo, and thus this work has widespread implications for understanding the mechanisms that regulate cocaine consumption across stages of addiction.
Assuntos
Cocaína/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Inibidores da Captação de Dopamina/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Clozapina/farmacologia , Transtornos Relacionados ao Uso de Cocaína/genética , Agonistas de Dopamina/farmacologia , Masculino , Microinjeções , Fosforilação , Ratos , Ratos Long-Evans , Autoadministração , Área Tegmentar VentralRESUMO
Augmented vasoconstriction is a hallmark of hypertension and is mediated partly by hyper-stimulation of G protein couple receptors (GPCRs) and downstream signaling components. Although GPCR blockade is a key component of current anti-hypertensive strategies, whether hypertension is better managed by directly targeting G proteins has not been thoroughly investigated. Here, we tested whether inhibiting Gq/11 proteins in vivo and ex vivo using natural cyclic depsipeptide, FR900359 (FR) from the ornamental plant, Ardisia crenata, and YM-254890 (YM) from Chromobacterium sp. QS3666, or it's synthetic analog, WU-07047 (WU), was sufficient to reverse hypertension in mice. All three inhibitors blocked G protein-dependent vasoconstriction, but to our surprise YM and WU and not FR inhibited K+-induced Ca2+ transients and vasoconstriction of intact vessels. However, each inhibitor blocked whole-cell L-type Ca2+ channel current in vascular smooth muscle cells. Subcutaneous injection of FR or YM (0.3 mg/kg, s.c.) in normotensive and hypertensive mice elicited bradycardia and marked blood pressure decrease, which was more severe and long lasting after the injection of FR relative to YM (FRt1/2 â 12 h vs. YMt1/2 â 4 h). In deoxycorticosterone acetate (DOCA)-salt hypertension mice, chronic injection of FR (0.3 mg/kg, s.c., daily for seven days) reversed hypertension (vehicle SBP: 149 ± 5 vs. FR SBP: 117 ± 7 mmHg), without any effect on heart rate. Our results together support the hypothesis that increased LTCC and Gq/11 activity is involved in the pathogenesis of hypertension, and that dual targeting of both proteins can reverse hypertension and associated cardiovascular disorders.
Assuntos
Anti-Hipertensivos/uso terapêutico , Depsipeptídeos/uso terapêutico , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hipertensão/tratamento farmacológico , Peptídeos Cíclicos/uso terapêutico , Animais , Anti-Hipertensivos/química , Ardisia/química , Chromobacterium/química , Depsipeptídeos/química , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos Cíclicos/química , Vasoconstrição/efeitos dos fármacosRESUMO
Excitatory amino acid transporters (EAATs) regulate glutamatergic signal transmission by clearing extracellular glutamate. Dysfunction of these transporters has been implicated in the pathogenesis of various neurological disorders. Previous studies have shown that venom from the spider Parawixia bistriata and a purified compound (Parawixin1) stimulate EAAT2 activity and protect retinal tissue from ischemic damage. In the present study, the EAAT2 subtype specificity of this compound was explored, employing chimeric proteins between EAAT2 and EAAT3 transporter subtypes and mutants to characterize the structural region targeted by the compound. This identified a critical residue (Histidine-71 in EAAT2 and Serine-45 in EAAT3) in transmembrane domain 2 (TM2) to be important for the selectivity between EAAT2 and EAAT3 and for the activity of the venom. Using the identified residue in TM2 as a structural anchor, several neighboring amino acids within TM5 and TM8 were identified to also be important for the activity of the venom. This structural domain of the transporter lies at the interface of the rigid trimerization domain and the central substrate-binding transport domain. Our studies suggest that the mechanism of glutamate transport enhancement involves an interaction with the transporter that facilitates the movement of the transport domain. We identified a domain (purple star) in the glutamate transporter EAAT2 that is important for transport stimulation through a spider venom, and suggest a mechanism for enhanced transporter function through facilitated substrate translocation (arrow). Because the dysfunction of glutamate transporters is implicated in the pathogenesis of neurological disorders, understanding the mechanisms of enhanced transport could have therapeutic implications.
Assuntos
Transportador 2 de Aminoácido Excitatório/química , Transportador 2 de Aminoácido Excitatório/metabolismo , Modelos Moleculares , Multimerização Proteica , Animais , Transporte Biológico/efeitos dos fármacos , Células COS , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Transportador 2 de Aminoácido Excitatório/efeitos dos fármacos , Transportador 2 de Aminoácido Excitatório/genética , Ácido Glutâmico/metabolismo , Humanos , Mutação/genética , Multimerização Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Venenos de Aranha/química , Toxinas Biológicas/análise , Toxinas Biológicas/farmacologia , TransfecçãoRESUMO
A major theme of addiction research has focused on the neural substrates of individual differences in the risk for addiction; however, little is known about how vulnerable populations differ from those that are relatively protected. Here, we prospectively measured dopamine (DA) neurotransmission prior to cocaine exposure to predict the onset and course of cocaine use. Using in vivo voltammetry, we first generated baseline profiles of DA release and uptake in the dorsomedial striatum (DMS) and nucleus accumbens of drug-naïve male rats prior to exposing them to cocaine using conditioned place preference (CPP) or operant self-administration. We found that the innate rate of DA uptake in the DMS strongly predicted motivation for cocaine and drug-primed reinstatement, but not CPP, responding when "price" was low, or extinction. We then assessed the impact of baseline variations in DA uptake on cocaine potency in the DMS using ex vivo voltammetry in naïve rats and in rats with DA transporter (DAT) knockdown. DA uptake in the DMS of naïve rats predicted the neurochemical response to cocaine, such that rats with innately faster rates of DA uptake demonstrated higher cocaine potency at the DAT and rats with DAT knockdown displayed reduced potency compared to controls. Together, these data demonstrate that inherent variability in DA uptake in the DMS predicts the behavioral response to cocaine, potentially by altering the apparent potency of cocaine.
Assuntos
Cocaína , Animais , Cocaína/farmacologia , Dopamina , Inibidores da Captação de Dopamina/farmacologia , Individualidade , Masculino , Motivação , Ratos , Ratos Sprague-DawleyRESUMO
Excitatory Amino Acid Transporters (EAATs) are plasma membrane proteins responsible for maintenance of low extracellular concentrations of glutamate in the CNS. Dysfunction in their activity is implicated in various neurological disorders. Glutamate transport by EAATs occurs through the movement of the central transport domain relative to the scaffold domain in the EAAT membrane protein. Previous studies suggested that residues located within the interface of these two domains in EAAT2, the main subtype of glutamate transporter in the brain, are involved in regulating transport rates. We used mutagenesis, structure-function relationship, surface protein expression and electrophysiology studies, in transfected COS-7 cells and oocytes, to examine residue glycine at position 298, which is located within this interface. Mutation G298A results in increased transport rate without changes in surface expression, suggesting a more hydrophobic and larger alanine results in facilitated transport movement. The increased transport rate does not involve changes in sodium affinity. Electrophysiological currents show that G298A increase both transport and anion currents, suggesting faster transitions through the transport cycle. This work identifies a region critically involved in setting the glutamate transport rate.
Assuntos
Transportador 2 de Aminoácido Excitatório/genética , Transportador 2 de Aminoácido Excitatório/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Transportador 2 de Aminoácido Excitatório/química , Feminino , Proteínas Associadas à Matriz Nuclear/química , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Especificidade por Substrato/fisiologia , XenopusRESUMO
Catecholamine transmitters dopamine (DA) and norepinephrine (NE) regulate prefrontal cortical (PFC) circuit activity and PFC-mediated executive functions. Accordingly, pharmacological agents that influence catecholamine neurotransmission exert prominent effects on cognition. Many such agents are used clinically to treat attention disorders. For example, methylphenidate blocks DA and NE reuptake and is the leading choice for attention deficit hyperactivity disorder (ADHD) treatment. Recently, we have designed SK609 - a selective small molecule agonist of the DA D3 receptor (D3R). In this study, we further characterized SK609's ability to selectively inhibit the reuptake of NE by NE transporters (NET). Our results indicate SK609 selectively inhibits NET with a Ki value of â¼500â¯nM and behaves as a NET substrate. Systemic dosing of SK609 (4â¯mg/kg; i.p.) in naïve rats produced a 300% and 160% increase in NE and DA, respectively, in the PFC as measured by microdialysis. Based on these neurochemical results, SK609 was tested in a PFC-dependent, visually-guided sustained attention task in rats. SK609 improved performance in a dose-dependent manner with a classical inverted-U dose response function with a peak effect at 4â¯mg/kg. SK609's peak effect was blocked by a pre-treatment with either the D2/D3R antagonist raclopride (0.05â¯mg/kg; i.p) or the alpha-1 adrenergic receptor antagonist prazosin (0.25â¯mg/kg; i.p), confirming a role for both DA and NE in promoting sustained attention. Additionally, SK609 improved sustained attention more prominently among low-performing animals. Doses of SK609 (2, 4, and 8â¯mg/kg) associated with cognitive enhancement did not produce an increase in spontaneous locomotor activity, suggesting a lack of side effects mediated by DA transporter (DAT) activity. These results demonstrate that the novel catecholaminergic modulator SK609 has the potential to treat sustained attention deficits without affecting DAT activity, distinguishing it from amphetamines and methylphenidate.
Assuntos
Atenção/fisiologia , Butilaminas/farmacologia , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/antagonistas & inibidores , Receptores de Dopamina D3/fisiologia , Animais , Butilaminas/antagonistas & inibidores , Células Cultivadas , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Masculino , Atividade Motora/efeitos dos fármacos , Norepinefrina/metabolismo , Prazosina/farmacologia , Córtex Pré-Frontal/metabolismo , Racloprida/farmacologia , Ratos , Receptores de Dopamina D3/agonistasRESUMO
The dopamine transporter (DAT) serves a pivotal role in controlling dopamine (DA)-mediated neurotransmission by clearing DA from synaptic and perisynaptic spaces and controlling its action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DAT to mediate their effects by enhancing extracellular DA concentrations. We previously identified a novel allosteric site in the related human serotonin transporter that lies outside the central substrate and inhibitor binding pocket. We used the hybrid structure based (HSB) method to screen for allosteric modulator molecules that target a similar site in DAT. We identified a compound, KM822, that was found to be a selective, noncompetitive inhibitor of DAT. We confirmed the structural determinants of KM822 allosteric binding within the allosteric site by structure/function and substituted cysteine scanning accessibility biotinylation experiments. In the in vitro cell-based assay and ex vivo in both rat striatal synaptosomal and slice preparations, KM822 was found to decrease the affinity of cocaine for DAT. The in vivo effects of KM822 on cocaine were tested on psychostimulant-associated behaviors in a planarian model where KM822 specifically inhibited the locomotion elicited by DAT-interacting stimulants amphetamine and cocaine. Overall, KM822 provides a unique opportunity as a molecular probe to examine allosteric modulation of DAT function.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Sinaptossomos/efeitos dos fármacos , Animais , Cocaína/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Humanos , Masculino , Atividade Motora/efeitos dos fármacos , Planárias , Ratos , Ratos Sprague-Dawley , Sinaptossomos/metabolismoRESUMO
Dysfunction of excitatory amino acid transporters (EAATs) has been implicated in the pathogenesis of various neurological disorders, such as stroke, brain trauma, epilepsy, and neurodegenerative diseases, among others. EAAT2 is the main subtype responsible for glutamate clearance in the brain, having a key role in regulating transmission and preventing excitotoxicity. Therefore, compounds that increase the expression or activity of EAAT2 have therapeutic potential for neuroprotection. Previous studies identified molecular determinants for EAAT2 transport stimulation in a structural domain that lies at the interface of the rigid trimerization domain and the central substrate binding transport domain. In this work, a hybrid structure based approach was applied, based on this molecular domain, to create a high-resolution pharmacophore. Subsequently, virtual screening of a library of small molecules was performed, identifying 10 hit molecules that interact at the proposed domain. Among these, three compounds were determined to be activators, four were inhibitors, and three had no effect on EAAT2-mediated transport in vitro. Further characterization of the two best ranking EAAT2 activators for efficacy, potency, and selectivity for glutamate over monoamine transporters subtypes and NMDA receptors and for efficacy in cultured astrocytes is demonstrated. Mutagenesis studies suggest that the EAAT2 activators interact with residues forming the interface between the trimerization and transport domains. These compounds enhance the glutamate translocation rate, with no effect on substrate interaction, suggesting an allosteric mechanism. The identification of these novel positive allosteric modulators of EAAT2 offers an innovative approach for the development of therapies based on glutamate transport enhancement.
Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Animais , Linhagem Celular , Haplorrinos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologiaRESUMO
Detailed in this unit are protocols for studying the in vitro uptake of dopamine (DA) as a means for defining the functional characteristics of dopamine transporters. All assays are performed using commercially available cell lines that transiently express the transporter under investigation. The three main assays provided are: a kinetic assay to calculate the affinity (KM ) and maximal velocity (Vmax ) of radiolabeled DA uptake into cells; concentration-response assays to measure the potencies (IC50 /Ki values) of test compounds as transport inhibitors; and an efflux assay to assess the ability and potency (EC50 ) of a ligand to elicit reverse transport of DA accumulated in the cell. Although the methods are described using DAT and its ligands, the same procedure can be employed for studying serotonin and norepinephrine transporters as well. © 2017 by John Wiley & Sons, Inc.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Animais , Bioensaio , Linhagem Celular , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Humanos , Cinética , LigantesRESUMO
The dopamine (DAT), serotonin (SERT), and norepinephrine (NET) transporters, which are collectively referred to as monoamine transporters (MATs), play significant roles in regulating the neuronal response to these neurotransmitters. MATs terminate the action of these neurotransmitters by translocating them from the synaptic space into the presynaptic neurons. These three transmitters are responsible for controlling a number of physiological, emotional, and behavioral functions, with their transporters being the site of action of drugs employed for the treatment of a variety of conditions, including depression, anxiety, ADHD, schizophrenia, and psychostimulant abuse. Provided in this unit is information on the localization and regulation of MATs and the structural components of these proteins most responsible for the translocation process. Also included is a brief description of the evolution of ligands that interact with these transporters, as well as current theories concerning the pharmacological effects of substances that interact with these sites, including the molecular mechanisms of action of uptake inhibitors and allosteric modulators. Data relating to the presence, structure, and functions of allosteric modulators are included as well. The aim of this review is to provide background information on MATs to those who are new to this field, with a focus on the therapeutic potential of compounds that interact with these substrate transport sites. © 2017 by John Wiley & Sons, Inc.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismo , Animais , Transporte Biológico , HumanosRESUMO
The norepinephrine transporter (NET) mediates the clearance of norepinephrine (NE) from the extracellular space and is a target of therapeutic antidepressants and psychostimulants. Previously we identified a MAP kinase phosphatase 3 (MKP3), as an important modulator of protein kinase C (PKC) mediated internalization of the related dopamine transporter (DAT). Here we show that MKP3 decreases PKC-mediated down regulation of NET expressed in PC12 cells. We demonstrate that this process involves a PKC-stimulated decrease of NET surface expression that is dependent on dynamin. Surprisingly, MAP kinase inhibitors have no effect on the PKC-mediated regulation of NET activity, suggesting that, like PKC-mediated regulation of the DAT, the acute activation of MAP kinases is not likely to be involved. To elucidate potential mechanisms we used a substrate trap-based assay to identify extracellular-signal-regulated kinase (ERK)1/2 as the predominant substrate of MKP3. Furthermore we also established that brief chemical stabilization of a modified destabilized MKP3 does not alter PKC-mediated down regulation of NET. Finally, the expression of a dominant negative version of H-Ras, an upstream activator of ERK1/2, abolishes phorbol 12-myristate 13-acetate (PMA)-mediated down regulation of NET in a manner similar to MKP3. Taken together we propose that chronic MKP3 expression regulates surface NET through the sustained inhibition of ERK1/2 MAP kinase signaling that alters gene expression in PC12 cells. This is supported by gene expression data from naïve and MKP3-expressing PC12 cells that reveal robust decreases in gene expression of several genes in the MKP3-tranfected cells. Interestingly, caveolin-1, a protein with a critical role in membrane protein trafficking is down regulated by MKP3 expression. We further show that selective silencing of the caveolin-1 gene in naïve PC12 cells attenuates PKC-mediated downregulation of NET activity, consistent with a potential role for caveolins in regulating NET surface expression. In summary, these results suggest that chronic MKP3 expression alters the expression of genes in PC12 cells that are involved in the regulation of NET surface expression.
RESUMO
The plasma-membrane monoamine transporters (MATs), including the serotonin (SERT), norepinephrine (NET) and dopamine (DAT) transporters, serve a pivotal role in limiting monoamine-mediated neurotransmission through the reuptake of their respective monoamine neurotransmitters. The transporters are the main target of clinically used psychostimulants and antidepressants. Despite the availability of several potent and selective MAT substrates and inhibitors the continuing need for therapeutic drugs to treat brain disorders involving aberrant monoamine signaling provides a compelling reason to identify novel ways of targeting and modulating the MATs. Designing novel modulators of MAT function have been limited by the lack of three dimensional structure information of the individual MATs. However, crystal structures of LeuT, a bacterial homolog of MATs, in a substrate-bound occluded, substrate-free outward-open, and an apo inward-open state and also with competitive and non-competitive inhibitors have been determined. In addition, several structures of the Drosophila DAT have also been resolved. Together with computational modeling and experimental data gathered over the past decade, these structures have dramatically advanced our understanding of several aspects of SERT, NET, and DAT transporter function, including some of the molecular determinants of ligand interaction at orthosteric substrate and inhibitor binding pockets. In addition progress has been made in the understanding of how allosteric modulation of MAT function can be achieved. Here we will review all the efforts up to date that has been made through computational approaches employing structural models of MATs to design small molecule modulators to the orthosteric and allosteric sites using virtual screening techniques.
RESUMO
In the mammalian central nervous system the dopamine transporter (DAT) is the primary mechanism for clearance of dopamine from the extracellular space. Presynaptic receptors for dopamine and other neurotransmitters (auto-receptors and hetero-receptors) present on dopaminergic neurons are poised to regulate the activity of the dopamine transporter acutely through their actions on intracellular signaling systems. The mechanisms proposed for acute presynaptic regulation of dopamine transport include direct effects of phosphorylation on enzymatic rate, indirect effects through the alteration of the electrical and chemical gradients that drive transport and/or the modulation of transporter number through the trafficking of carriers to and from the cell surface. This review focuses on recent evidence for several distinct mechanisms which dynamically regulate dopamine transporter activity and thus have an important role in shaping the duration and amplitude of dopamine signals in the brain.
Assuntos
Glicoproteínas de Membrana , Moduladores de Transporte de Membrana , Proteínas de Membrana Transportadoras/antagonistas & inibidores , Proteínas de Membrana Transportadoras/fisiologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/fisiologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina , Inibidores da Captação de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
Release of neurotransmitters is a fundamental and regulated process that is essential for normal brain functioning. Regulation of this process is potentially important for any neuronal process, and disruption of the release process may contribute to the pathophysiology associated with psychiatric diseases. In this work it is shown that expression of the negative regulator of mitogen-activated protein kinase (MAPK) signaling the MAPK phosphatase MKP3/DUSP6 eliminates depolarization-dependent release of dopamine in rat PC12 cells. Pharmacologic interventions with latrotroxin (LTX) or A23187, which make the cells permeable to calcium, reestablish the dopamine release. Calcium imaging also reveals that calcium influx is impaired in MKP3-expressing cells. Because acute pharmacologic inhibition of MAPKs has no effect on dopamine release in naïve PC12 cells, the MKP3-mediated elimination of neurotransmitter release must be caused by a long-term process, such as changes in gene expression. In support of this the expression of the L-type calcium channel cav1.2 alpha subunit (Cacna1c) is decreased in MKP3-expressing PC12 cells. With the reintroduction of cav1.2 expression, neurotransmitter release is restored in the MKP3-expressing PC12 cells. Thus, MKP3 expression reduces neurotransmitter release by decreasing the expression of cav1.2. Because MKP3 is increased when neuronal activity is elevated, this process could play a role in regulating neurotransmitter homeostasis.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Regulação para Baixo , Fosfatase 6 de Especificidade Dupla/metabolismo , Neurotransmissores/metabolismo , Animais , Células PC12 , RatosRESUMO
The trematode Schistosoma mansoni is the primary cause of schistosomiasis, a devastating neglected tropical disease that affects 200 million individuals. Identifying novel therapeutic targets for the treatment of schistosomiasis is therefore of great public interest. The catecholamines norepinephrine (NE) and dopamine (DA) are essential for the survival of the parasite as they cause muscular relaxation and a lengthening in the parasite and thereby control movement. Here we characterize a novel dopamine/norepinephrine transporter (SmDAT) gene transcript, from S. mansoni. The SmDAT is expressed in the adult form and in the sporocyst form (infected snails) of the parasite, and also in the egg and miracidium stage. It is absent in the cercariae stage but curiously a transcript missing the exon encoding transmembrane domain 8 was identified in this stage. Heterologous expression of the cDNA in mammalian cells resulted in saturable, dopamine transport activity with an apparent affinity for dopamine comparable to that of the human dopamine transporter. Efflux experiments reveal notably higher substrate selectivity compared with its mammalian counterparts as amphetamine is a much less potent efflux elicitor against SmDAT compared to the human DAT. Pharmacological characterization of the SmDAT revealed that most human DAT inhibitors including psychostimulants such as cocaine were significantly less potent in inhibiting SmDAT. Like DATs from other simpler organisms the pharmacology for SmDAT was more similar to the human norepinephrine transporter. We were not able to identify other dopamine transporting carriers within the completed parasite genome and we hypothesize that the SmDAT is the only catecholamine transporter in the parasite and could be responsible for not only clearing DA but also NE.
Assuntos
Fármacos do Sistema Nervoso Autônomo/metabolismo , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/química , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/metabolismo , Catecolaminas/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Schistosoma mansoni/metabolismo , Sequência de Aminoácidos , Animais , Fármacos do Sistema Nervoso Autônomo/química , Proteínas da Membrana Plasmática de Transporte de Catecolaminas/genética , Catecolaminas/química , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Helminto/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Schistosoma mansoni/genética , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose mansoni , Caramujos/parasitologia , Especificidade por SubstratoRESUMO
The human blood fluke Schistosoma mansoni is the primary cause of schistosomiasis, a debilitating disease that affects 200 million individuals in over 70 countries. The biogenic amine serotonin is essential for the survival of the parasite and serotonergic proteins are potential novel drug targets for treating schistosomiasis. Here we characterize two novel serotonin transporter gene transcripts, SmSERT-A and SmSERT-B, from S.mansoni. Southern blot analysis shows that the two mRNAs are the products of different alleles of a single SmSERT gene locus. The two SmSERT forms differ in three amino acid positions near the N-terminus of the protein. Both SmSERTs are expressed in the adult form and in the sporocyst form (infected snails) of the parasite, but are absent from all other stages of the parasite's complex life cycle. Heterologous expression of the two cDNAs in mammalian cells resulted in saturable, sodium-dependent serotonin transport activity with an apparent affinity for serotonin comparable to that of the human serotonin transporter. Although the two SmSERTs are pharmacologically indistinguishable from each other, efflux experiments reveal notably higher substrate selectivity for serotonin compared with their mammalian counterparts. Several well-established substrates for human SERT including (+/-)MDMA, S-(+)amphetamine, RU 24969, and m-CPP are not transported by SmSERTs, underscoring the higher selectivity of the schistosomal isoforms. Voltage-clamp recordings of SmSERT substrate-elicited currents confirm the substrate selectivity observed in efflux experiments and suggest that it may be possible to exploit the electrogenic nature of SmSERT to screen for compounds that target the parasite in vivo.
Assuntos
Alelos , Schistosoma mansoni/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Serotonina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/genética , Condutividade Elétrica , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Estágios do Ciclo de Vida/genética , Masculino , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/crescimento & desenvolvimento , Esquistossomose/tratamento farmacológico , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Especificidade por Substrato , Xenopus laevis/genética , Xenopus laevis/metabolismo , Xenopus laevis/fisiologiaRESUMO
Two atypical inhibitors of the dopamine transporter, benztropine, used in the treatment of Parkinson's disease, and bupropion, used as an antidepressant, show very different psychostimulant effects when compared with another inhibitor, cocaine. Taking advantage of the differential sensitivity of the dopamine and the norepinephrine transporters (DAT and NET) to benztropine and bupropion, we have used site-directed mutagenesis to produce gain-of-function mutants in NET which demonstrate that Ala279 in the trans-membrane domain 5 (TM5) and Ser359 in the TM7 of DAT are responsible for the higher sensitivity of DAT to both bupropion and benztropine. Substitution of these two DAT residues into the NET background does not alter the potency of NET-selective inhibitors, such as desipramine. The results from experiments examining the ability of DAT-selective inhibitors to displace [3H]nisoxetine binding in NET gain-of-function mutants suggest that Ser359 contributes to the initial binding of the inhibitor, and that Ala279 may influence subsequent steps involved in the blockade of translocation. Thus, these studies begin to identify residues that are important for the unique molecular interactions of benztropine and bupropion with the DAT, and that ultimately may contribute to the distinct behavioral actions of these drugs.