RESUMO
Antipsychotics represent the mainstay of schizophrenia pharmacological therapy, and their role has been expanded in the last years to mood disorders treatment. Although introduced in 1952, many years of research were required before an accurate picture of how antipsychotics work began to emerge. Despite the well-recognized characterization of antipsychotics in typical and atypical based on their liability to induce motor adverse events, their main action at dopamine D2R to elicit the "anti-psychotic" effect, as well as the multimodal action at other classes of receptors, their effects on intracellular mechanisms starting with receptor occupancy is still not completely understood. Significant lines of evidence converge on the impact of these compounds on multiple molecular signaling pathways implicated in the regulation of early genes and growth factors, dendritic spine shape, brain inflammation, and immune response, tuning overall the function and architecture of the synapse. Here we present, based on PRISMA approach, a comprehensive and systematic review of the above mechanisms under a translational perspective to disentangle those intracellular actions and signaling that may underline clinically relevant effects and represent potential targets for further innovative strategies in antipsychotic therapy.
Assuntos
Antipsicóticos/farmacologia , Animais , Antipsicóticos/química , Antipsicóticos/uso terapêutico , Encéfalo/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Epigênese Genética , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Precoces , Humanos , Plasticidade Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Proteínas de Transporte de Neurotransmissores/antagonistas & inibidores , Proteínas de Transporte de Neurotransmissores/metabolismoRESUMO
Sodium-coupled neurotransmitter transporters play a fundamental role in the termination of synaptic neurotransmission, which makes them a major drug target. The reconstitution of these secondary active transporters into liposomes has shed light on their molecular transport mechanisms. From the earliest days of the reconstitution technique up to today's single-molecule studies, insights from live functioning transporters have been indispensable for our understanding of their physiological impact. The two classes of sodium-coupled neurotransmitter transporters, the neurotransmitter: sodium symporters and the excitatory amino acid transporters, have vastly different molecular structures, but complementary proteoliposome studies have sought to unravel their ion-dependence and transport kinetics. Furthermore, reconstitution experiments have been used on both protein classes to investigate the role of e.g. the lipid environment, of posttranslational modifications, and of specific amino acid residues in transport. Techniques that allow the detection of transport at a single-vesicle resolution have been developed, and single-molecule studies have started to reveal single transporter kinetics, which will expand our understanding of how transport across the membrane is facilitated at protein level. Here, we review a selection of the results and applications where the reconstitution of the two classes of neurotransmitter transporters has been instrumental.
Assuntos
Proteínas de Transporte de Neurotransmissores , Sódio , Transporte Biológico , Proteínas de Membrana Transportadoras/metabolismo , Neurotransmissores/metabolismo , Proteínas de Transporte de Neurotransmissores/química , Proteínas de Transporte de Neurotransmissores/metabolismoRESUMO
Excitatory amino acid transporters (EAAT) play a key role in glutamatergic synaptic communication. Driven by transmembrane cation gradients, these transporters catalyze the reuptake of glutamate from the synaptic cleft once this neurotransmitter has been utilized for signaling. Two decades ago, pioneering studies in the Kanner lab identified a conserved methionine within the transmembrane domain as key for substrate turnover rate and specificity; later structural work, particularly for the prokaryotic homologs GltPh and GltTk, revealed that this methionine is involved in the coordination of one of the three Na+ ions that are co-transported with the substrate. Albeit extremely atypical, the existence of this interaction is consistent with biophysical analyses of GltPh showing that mutations of this methionine diminish the binding cooperativity between substrates and Na+. It has been unclear, however, whether this intriguing methionine influences the thermodynamics of the transport reaction, i.e., its substrate:ion stoichiometry, or whether it simply fosters a specific kinetics in the binding reaction, which, while influential for the turnover rate, do not fundamentally explain the ion-coupling mechanism of this class of transporters. Here, studies of GltTk using experimental and computational methods independently arrive at the conclusion that the latter hypothesis is the most plausible, and lay the groundwork for future efforts to uncover the underlying mechanism.
Assuntos
Metionina , Sódio , Transporte Biológico , Íons/metabolismo , Metionina/metabolismo , Proteínas de Transporte de Neurotransmissores/metabolismoRESUMO
Psychostimulant, cardiovascular, and temperature actions of stimulants involve adrenergic (norepinephrine), dopaminergic (dopamine), and serotonergic (serotonin) pathways. Stimulants such as amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), or mephedrone can act on the neuronal membrane monoamine transporters NET, DAT, and SERT and/or the vesicular monoamine transporter 2 to inhibit reuptake of neurotransmitter or cause release by reverse transport. Stimulants may have additional effects involving pre- and postsynaptic/junctional receptors for norepinephrine, dopamine, and serotonin and other receptors. As a result, stimulants may have a wide range of possible actions. Agents with cocaine or MDMA-like actions can induce serious and potentially fatal adverse events via thermodysregulatory, cardiovascular, or other mechanisms. MDMA-like stimulants may cause hyperthermia that can be life threathening. Recreational users of stimulants should be aware of the dangers of hyperthermia in a rave/club environment.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Dopamina/metabolismo , Humanos , Proteínas de Transporte de Neurotransmissores/metabolismo , Norepinefrina/metabolismo , Serotonina/metabolismo , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Transtornos Relacionados ao Uso de Substâncias/patologiaRESUMO
Regulation of the ability of a neurotransmitter [our focus: serotonin, norepinephrine, dopamine, acetylcholine, glycine, and gamma-aminobutyric acid (GABA)] to reach its receptor targets is regulated in part by controlling the access the neurotransmitter has to receptors. Transporters, located at both the cellular plasma membrane and in subcellular vesicles, carry a myriad of responsibilities that include enabling neurotransmitter release and controlling uptake of neurotransmitter back into a cell or vesicle. Driven largely by electrochemical gradients, these transporters move neurotransmitters. The regulation of the transporters themselves through changes in expression and/or posttranslational modification allows for fine-tuning of this system. Transporters have been best recognized as targets for psychoactive stimulants and remain a mainstay target of primarily central nervous system (CNS) acting drugs for treatment of debilitating diseases such as depression and anxiety. Studies reveal, however, that transporters are found and functional in tissues outside the CNS (gastrointestinal and cardiovascular tissues, for example). The importance of neurotransmitter transporters is underscored with discoveries that dysfunction of transporters can cause life-changing disease. This article provides a high-level review of major classes of both plasma membrane transporters and vesicular transporters. © 2021 American Physiological Society. Compr Physiol 11:2279-2295, 2021.
Assuntos
Proteínas de Membrana Transportadoras , Proteínas de Transporte de Neurotransmissores , Transporte Biológico , Proteínas de Transporte/metabolismo , Neurotransmissores , Proteínas de Transporte de Neurotransmissores/genética , Proteínas de Transporte de Neurotransmissores/metabolismoRESUMO
Norepinephrine is a biogenic amine neurotransmitter that has widespread effects on alertness, arousal and pain sensation. Consequently, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ the Drosophila melanogaster dopamine transporter as a surrogate for the norepinephrine transporter and determine X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. We also report structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, we observe that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. Our experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Transporte de Neurotransmissores/química , Proteínas de Transporte de Neurotransmissores/metabolismo , Norepinefrina/química , Norepinefrina/metabolismo , Analgésicos Opioides/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Dopamina , Proteínas da Membrana Plasmática de Transporte de Dopamina , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HEK293 , Humanos , Camundongos Knockout , Modelos Moleculares , Proteínas de Transporte de Neurotransmissores/genética , Células Sf9 , TranscriptomaRESUMO
The 3,4-methylenedioxypyrovalerone (MDPV), and other structurally related synthetic cathinones, are popular alternatives to prototypical illicit psychostimulants, such as cocaine and methamphetamine. These drugs are often referred to as 'bath salts' and function either as cocaine-like inhibitors of monoamine uptake, or amphetamine-like substrates for dopamine, norepinephrine and serotonin transporters. These studies used male Sprague-Dawley rats trained to discriminate MDPV from saline to evaluate the substitution profiles of structurally related synthetic cathinones, cocaine, and other direct-acting dopamine and noradrenergic receptor agonists in order to characterize the relative contributions of dopamine, norepinephrine and serotonin to the discriminative stimulus effects of MDPV. As expected, each of the cathinones and cocaine dose-dependently increased MDPV-appropriate responding, with a rank-order potency that was positively correlated with their potency to inhibit dopamine and norepinephrine, but not serotonin, a relationship that is consistent with the rank order to maintain self-administration. The dopamine D2/3 receptor-preferring agonist quinpirole produced a modest increase in MDPV-appropriate responding, whereas the dopamine D1/5 receptor agonist, SKF 82958, nonselective dopamine receptor agonist, apomorphine, as well as the α-1, and α-2 adrenergic receptor agonists, phenylephrine and clonidine, respectively, failed to increase MDPV-appropriate responding at doses smaller than those that suppressed responding altogether. Although these studies do not support a role for serotonergic or adrenergic systems in mediating/modulating the discriminative stimulus effects of MDPV, convergent evidence is provided to suggest that the discriminative stimulus effects of MDPV are primarily mediated by its capacity to inhibit dopamine uptake, and the subsequent activation of dopamine D2 or D3 receptors.
Assuntos
Benzodioxóis , Monoaminas Biogênicas/metabolismo , Inibidores da Captação de Dopamina , Proteínas de Transporte de Neurotransmissores/metabolismo , Pirrolidinas , Alcaloides/química , Anfetaminas/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Benzodioxóis/química , Benzodioxóis/farmacologia , Estimulantes do Sistema Nervoso Central/química , Estimulantes do Sistema Nervoso Central/farmacologia , Cocaína/análogos & derivados , Cocaína/farmacologia , Aprendizagem por Discriminação , Inibidores da Captação de Dopamina/química , Inibidores da Captação de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Drogas Ilícitas , Masculino , Norepinefrina/antagonistas & inibidores , Pirrolidinas/química , Pirrolidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Medicamentos Sintéticos/química , Medicamentos Sintéticos/farmacologia , Catinona SintéticaRESUMO
Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix-spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre-fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release-type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions.
Assuntos
Proteínas de Transporte de Neurotransmissores/química , Proteínas de Transporte de Neurotransmissores/metabolismo , Animais , HumanosRESUMO
Neurotransmitters, such as γ-aminobutyric acid, glutamate, acetyl choline, glycine and the monoamines, facilitate the crosstalk within the central nervous system. The designated neurotransmitter transporters (NTTs) both release and take up neurotransmitters to and from the synaptic cleft. NTT dysfunction can lead to severe pathophysiological consequences, e.g. epilepsy, intellectual disability, or Parkinson's disease. Genetic point mutations in NTTs have recently been associated with the onset of various neurological disorders. Some of these mutations trigger folding defects in the NTT proteins. Correct folding is a prerequisite for the export of NTTs from the endoplasmic reticulum (ER) and the subsequent trafficking to their pertinent site of action, typically at the plasma membrane. Recent studies have uncovered some of the key features in the molecular machinery responsible for transporter protein folding, e.g., the role of heat shock proteins in fine-tuning the ER quality control mechanisms in cells. The therapeutic significance of understanding these events is apparent from the rising number of reports, which directly link different pathological conditions to NTT misfolding. For instance, folding-deficient variants of the human transporters for dopamine or GABA lead to infantile parkinsonism/dystonia and epilepsy, respectively. From a therapeutic point of view, some folding-deficient NTTs are amenable to functional rescue by small molecules, known as chemical and pharmacological chaperones.
Assuntos
Proteínas de Transporte de Neurotransmissores , Humanos , Neurotransmissores/metabolismo , Proteínas de Transporte de Neurotransmissores/genética , Proteínas de Transporte de Neurotransmissores/metabolismo , Dobramento de Proteína , Transporte Proteico/genéticaRESUMO
This protocol provides two independent methods to functionally detect the neuronal expression of CO2-sensitive hemichannels. These hemichannels (consisting of connexins 26 or 30) are directly gated by CO2, independent of pH changes and until recently were thought to be only expressed by glia. This protocol outlines a method to change the concentration of CO2 without changing pH, using isohydric solutions and then utilizing this to detect opening and closing of functional hemichannels using whole-cell patch clamp recording and dye loading. For complete details on the use and execution of this protocol, please refer to Hill et al. (2020).
Assuntos
Neurônios/metabolismo , Proteínas de Transporte de Neurotransmissores/análise , Técnicas de Patch-Clamp/métodos , Encéfalo/citologia , Encéfalo/metabolismo , Cálcio/metabolismo , Dióxido de Carbono/metabolismo , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Microtomia/métodos , Neuroglia/metabolismo , Neurônios/química , Neurônios/citologia , Proteínas de Transporte de Neurotransmissores/metabolismoRESUMO
The antidepressant medications that are currently prescribed to patients suffering from major depressive disorder (MDD) have limitations and as a result, there is an urgent need to increase the options that are available. A number of studies have found that natural polyphenols have neuroprotective properties and there is evidence to suggest that they modulate neurotransmitter systems. There are more than 200 phenolic compounds that have been identified in Olea europaea, many of which have not yet been investigated for their potential biological effects. In this study, in silico methods were used to screen the phenolic library from the OliveNet™ database and identify novel lead compounds for proteins implicated in the pathophysiology of MDD. The molecular docking results revealed that the monoamine oxidase enzyme isoforms (MAO-A/MAO-B) had binding specificities for certain phenolic subclasses. The lead ligands that were identified from these subclasses were positioned near the flavin adenine dinucleotide (FAD) cofactor, interacting in a similar manner as known inhibitors. In addition to the MAO enzymes, several phenolic compounds were docked to neurotransmitter transporters and postsynaptic receptors, as well as proteins involved in neuroinflammation, oxidative stress and the endocannabinoid system. Based on the binding affinity, position, orientation and interactions of the lead phenolic compounds identified in this study, it is predicted that they may have antidepressant properties. The results should be validated further using molecular dynamics (MD) simulations, as well as in vivo and in vitro techniques.
Assuntos
Antidepressivos/metabolismo , Olea , Fenóis/metabolismo , Compostos Fitoquímicos/metabolismo , Amidoidrolases/metabolismo , Transtorno Depressivo Maior/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Isoenzimas/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Simulação de Acoplamento Molecular , Monoaminoxidase/metabolismo , Proteínas de Transporte de Neurotransmissores/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
Inborn errors of monoamine neurotransmitter biosynthesis and degradation belong to the rare inborn errors of metabolism. They are caused by monogenic variants in the genes encoding the proteins involved in (1) neurotransmitter biosynthesis (like tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC)), (2) in tetrahydrobiopterin (BH4) cofactor biosynthesis (GTP cyclohydrolase 1 (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SPR)) and recycling (pterin-4a-carbinolamine dehydratase (PCD), dihydropteridine reductase (DHPR)), or (3) in co-chaperones (DNAJC12). Clinically, they present early during childhood with a lack of monoamine neurotransmitters, especially dopamine and its products norepinephrine and epinephrine. Classical symptoms include autonomous dysregulations, hypotonia, movement disorders, and developmental delay. Therapy is predominantly based on supplementation of missing cofactors or neurotransmitter precursors. However, diagnosis is difficult and is predominantly based on quantitative detection of neurotransmitters, cofactors, and precursors in cerebrospinal fluid (CSF), urine, and blood. This review aims at summarizing the diverse analytical tools routinely used for diagnosis to determine quantitatively the amounts of neurotransmitters and cofactors in the different types of samples used to identify patients suffering from these rare diseases.
Assuntos
Catecolaminas/análise , Erros Inatos do Metabolismo/metabolismo , Proteínas de Transporte de Neurotransmissores/metabolismo , Pterinas/análise , Animais , Humanos , Erros Inatos do Metabolismo/diagnósticoRESUMO
Voltage-gated potassium (Kv) channels open in response to changes in membrane potential to permit passage of K+ ions across the cell membrane, down their electrochemical gradient. Sodium-coupled solute transporters utilize the downhill sodium gradient to co-transport solutes, ranging from ions to sugars to neurotransmitters, into the cell. A variety of recent studies have uncovered cooperation between these two structurally and functionally unrelated classes of protein, revealing previously unnoticed functional crosstalk and in many cases physical interaction to form channel-transporter (chansporter) complexes. Adding to this field, Bartolomé-Martín and colleagues now report that the heteromeric KCNQ2/KCNQ3 (Kv7.2/7.3) potassium channel - the primary molecular correlate of the neuronal M-current - can physically interact with two sodium-coupled neurotransmitter transporters expressed in the brain, DAT and GLT1 (dopamine and glutamate transporters, respectively). The authors provide evidence that the interactions may enhance transporter activity while dampening the depolarizing effects of sodium influx. Cumulative evidence discussed here suggests that chansporter complexes represent a widespread form of cellular signaling hub, in the CNS and other tissues. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
Assuntos
Química Encefálica/fisiologia , Canais Iônicos/metabolismo , Proteínas de Transporte de Neurotransmissores/metabolismo , Animais , HumanosRESUMO
Using electrocorticogram (ECoG) analysis, we compared age-related dynamics of general neuronal activity and convulsive epileptiform responsiveness induced by intracortical microinjections of 4-aminopyridine (4-AP) in control Wistar rats and those subjected to prenatal hypoxia (Hx; E14; 7% O2, 3 h). The studies were carried out in three age periods roughly corresponding to childhood (P20-27), adolescence (P30-45), and adulthood (P90-120). It was found that in the process of postnatal development of the control rats, the peak of the ECoG power spectrum density (PSD) of the theta rhythm during wakefulness shifted from the low to the higher frequency, while in the Hx rats this shift had the opposite direction. Moreover, the Hx rats had different frequency characteristics of the ECoG PSD and longer episodes of spike-and-wave discharges caused by 4-AP injections compared to the controls. The total ECoG PSD of slow-wave sleep (1-5 Hz) was also dramatically decreased in the process of development of the Hx rats. Such alterations in PSD could be explained by the changes in balance of the excitation and inhibition processes in the cortical networks. Analyzing protein levels of neurotransmitter transporters in the brain structures of the Hx rats, we found that the content of the glutamate transporter EAAT1 was higher in the parietal cortex in all age groups of Hx rats while in the hippocampus it decreased during postnatal development compared to controls. Furthermore, the content of the vesicular acetylcholine transporter in the parietal cortex, and of the inhibitory GABA transporter 1 in the hippocampus, was also affected by prenatal Hx. These data suggest that prenatal Hx results in a shift in the excitatory and inhibitory balance in the rat cortex towards excitation, making the rat's brain more vulnerable to the effects of proconvulsant drugs and predisposing animals to epileptogenesis during postnatal life.
Assuntos
Hipóxia Fetal/metabolismo , Hipóxia Fetal/fisiopatologia , Proteínas de Transporte de Neurotransmissores/metabolismo , 4-Aminopiridina/toxicidade , Animais , Convulsivantes/toxicidade , Eletrocorticografia , Feminino , Bloqueadores dos Canais de Potássio/toxicidade , Gravidez , Ratos , Ratos Wistar , Convulsões/induzido quimicamente , Convulsões/fisiopatologiaRESUMO
Alzheimer's disease (AD) is the most common cause of dementia and is characterized by the progressive loss of memory and cognition in the aging population. However, the etiology of and therapies for AD remain far from understood. Astrocytes, the most abundant neuroglia in the brain, have recently aroused substantial concern due to their involvement in synaptotoxicity, amyloidosis, neuroinflammation, and oxidative stress. In this review, we summarize the candidate molecules of astrocytes, especially receptors and transporters, that may be involved in AD pathogenesis. These molecules include excitatory amino acid transporters (EAATs), metabotropic glutamate receptor 5 (mGluR5), the adenosine 2A receptor (A2AR), the α7-nicotinic acetylcholine receptor (α7-nAChR), the calcium-sensing receptor (CaSR), S100ß, and cannabinoid receptors. We describe the characteristics of these molecules and the neurological and pharmacological underpinnings of these molecules in AD. Among these molecules, EAATs, A2AR, and mGluR5 are strongly related to glutamate-mediated synaptotoxicity and are involved in glutamate transmission or the clearance of extrasynaptic glutamate in the AD brain. The α7-nAChR, CaSR, and mGluR5 are receptors of Aß and can induce a plethora of toxic effects, such as the production of excess Aß, synaptotoxicity, and NO production triggered by changes in intracellular calcium signaling. Antagonists or positive allosteric modulators of these receptors can repair cognitive ability and modify neurobiological changes. Moreover, blocking S100ß or activating cannabinoid receptors reduces neuroinflammation, oxidative stress, and reactive astrogliosis. Thus, targeting these molecules might provide alternative approaches for treating AD.
Assuntos
Doença de Alzheimer , Astrócitos , Neurotransmissores/farmacologia , Proteínas de Transporte de Neurotransmissores/metabolismo , Receptores de Superfície Celular/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Humanos , Terapia de Alvo Molecular/métodosRESUMO
Trafficking of glutamate, glutamine and GABA between astrocytes and neurons is essential to maintain proper neurotransmission. Chronic hyperammonemia alters neurotransmission and cognitive function. The aims of this work were to analyze in cerebellum of rats the effects of chronic hyperammonemia on: a) extracellular glutamate, glutamine and GABA concentrations; b) membrane expression of glutamate, glutamine and GABA transporters; c) how they are modulated by extracellular cGMP. Hyperammonemic rats show increased levels of extracellular glutamate, glutamine, GABA and citrulline in cerebellum in vivo. Hyperammonemic rats show: a) increased membrane expression of the astrocytic glutamine transporter SNAT3 and reduced membrane expression of the neuronal transporter SNAT1; b) reduced membrane expression of the neuronal GABA transporter GAT1 and increased membrane expression of the astrocytic GAT3 transporter; c) reduced membrane expression of the astrocytic glutamate transporters GLAST and GLT-1 and of the neuronal transporter EAAC1. Increasing extracellular cGMP normalizes membrane expression of SNAT3, GAT3, GAT1 and GLAST and extracellular glutamate, glutamine, GABA and citrulline hyperammonemic rats. Extracellular cGMP also modulates membrane expression of most transporters in control rats, reducing membrane expression of SNAT1, GLT-1 and EAAC1 and increasing that of GAT1 and GAT3. Modulation of SNAT3, SNAT1, GLT-1 and EAAC1 by extracellular cGMP would be mediated by inhibition of glycine receptors. These data suggest that, in pathological situations such as hyperammonemia, hepatic encephalopathy or Alzheimer's disease, reduced levels of extracellular cGMP contribute to alterations in membrane expression of glutamine, glutamate and GABA transporters, in the extracellular levels of glutamine, glutamate and GABA and in neurotransmission. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
Assuntos
Membrana Celular/metabolismo , Cerebelo/metabolismo , GMP Cíclico/farmacologia , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Hiperamonemia/metabolismo , Proteínas de Transporte de Neurotransmissores/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Citrulina/metabolismo , Espaço Extracelular , Masculino , Proteínas de Transporte de Neurotransmissores/fisiologia , Ratos , Ratos Wistar , Transmissão Sináptica/genética , Transmissão Sináptica/fisiologiaRESUMO
γ-Hydroxybutyric acid (GHB) is an endogenous compound proposed to act as a neurotransmitter. Na+ -dependent, high-affinity GHB transport has long been considered important evidence supporting this hypothesis. However, the molecular identity of such a high-affinity transporter remains unknown. In this study, we sought to identify and characterize GHB synaptic transport through a series of studies using both native and recombinant systems with the ultimate aim of providing evidence to clarify the proposed role of GHB as a neurotransmitter in the mammalian brain. Native [3 H]GHB transport was studied in isolated rat brain synaptosomes and compared to synaptic membranes. As a targeted approach, GHB was also screened against a panel of Na+ -dependent SLC6 neurotransmitter transporters recombinantly expressed in Xenopus laevis oocytes or tsA201 cells. Finally, the low-affinity GHB transporters, MCT1/2 and SMCT1, were probed as GHB transporters in L-[14 C]lactate uptake assays in synaptosomes. We found no evidence of high-affinity [3 H]GHB transport in purified rat brain cortical or striatal synaptosomes or at any of the 11 SLC6 transporters tested. Instead, our results indicate the binding of [3 H]GHB to an unidentified membrane component, distinct from any of the known GHB targets. In accordance with others, we found that GHB and the analog 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) can, in millimolar concentrations, inhibit L-[14 C]lactate uptake at MCT1 and/or MCT2 and that this also can occur in synaptosomes. In conclusion, through a variety of in vitro pharmacological studies, we were unsuccessful in identifying a specific synaptic high-affinity transporter for GHB. Our findings emphasize the need to reevaluate GHB's role as a potential neurotransmitter. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
Assuntos
Encéfalo/metabolismo , Hidroxibutiratos/metabolismo , Proteínas de Transporte de Neurotransmissores/metabolismo , Sinapses/metabolismo , Sinaptossomos/metabolismo , Animais , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Much of the structure-based mechanistic understandings of the function of SLC6A neurotransmitter transporters emerged from the study of their bacterial LeuT-fold homologs. It has become evident, however, that structural differences such as the long N- and C-termini of the eukaryotic neurotransmitter transporters are involved in an expanded set of functional properties to the eukaryotic transporters. These functional properties are not shared by the bacterial homologs, which lack the structural elements that appeared later in evolution. However, mechanistic insights into some of the measured functional properties of the eukaryotic transporters that have been suggested to involve these structural elements are sparse or merely descriptive. RESULTS: To learn how the structural elements added in evolution enable mechanisms of the eukaryotic transporters in ways not shared with their bacterial LeuT-like homologs, we focused on the human dopamine transporter (hDAT) as a prototype. We present the results of a study employing large-scale molecular dynamics simulations and comparative Markov state model analysis of experimentally determined properties of the wild-type and mutant hDAT constructs. These offer a quantitative outline of mechanisms in which a rich spectrum of interactions of the hDAT N-terminus and C-terminus contribute to the regulation of transporter function (e.g., by phosphorylation) and/or to entirely new phenotypes (e.g., reverse uptake (efflux)) that were added in evolution. CONCLUSIONS: The findings are consistent with the proposal that the size of eukaryotic neurotransmitter transporter termini increased during evolution to enable more functions (e.g., efflux) not shared with the bacterial homologs. The mechanistic explanations for the experimental findings about the modulation of function in DAT, the serotonin transporter, and other eukaryotic transporters reveal separate roles for the distal and proximal segments of the much larger N-terminus in eukaryotic transporters compared to the bacterial ones. The involvement of the proximal and distal segments - such as the role of the proximal segment in sustaining transport in phosphatidylinositol 4,5-bisphosphate-depleted membranes and of the distal segment in modulating efflux - may represent an evolutionary adaptation required for the function of eukaryotic transporters expressed in various cell types of the same organism that differ in the lipid composition and protein complement of their membrane environment.
Assuntos
Proteínas de Transporte de Neurotransmissores/química , Proteínas de Transporte de Neurotransmissores/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Evolução Molecular , Humanos , Simulação de Dinâmica Molecular , Fosforilação , Conformação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Methylphenidate-based designer drugs are new psychoactive substances (NPS) that are used outside medical settings and their pharmacology is largely unexplored. The aim of the present study was to characterize the pharmacology of methylphenidate-based substances in vitro. METHODS: We determined the potencies of the methylphenidate-based NPS N-benzylethylphenidate, 3,4-dichloroethylphenidate, 3,4-dichloromethylphenidate, ethylnaphthidate, ethylphenidate, 4-fluoromethylphenidate, isopropylphenidate, 4-methylmethylphenidate, methylmorphenate, and propylphenidate and the potencies of the related compounds cocaine and modafinil with respect to norepinephrine, dopamine, and serotonin transporter inhibition in transporter-transfected human embryonic kidney 293 cells. We also investigated monoamine efflux and monoamine receptor and transporter binding affinities. Furthermore, we assessed the cell integrity under assay conditions. RESULTS: All methylphenidate-based substances inhibited the norepinephrine and dopamine transporters 4 to >1000-fold more potently than the serotonin transporter. Similar to methylphenidate and cocaine, methylphenidate-based NPS did not elicit transporter-mediated efflux of monoamines. Besides binding to monoamine transporters, several test drugs had affinity for adrenergic, serotonergic, and rat trace amine-associated receptors but not for dopaminergic or mouse trace amine-associated receptors. No cytotoxicity was observed after drug treatment at assay concentrations. CONCLUSION: Methylphenidate-based substances had pharmacological profiles similar to methylphenidate and cocaine. The predominant actions on dopamine transporters vs. serotonin transporters may be relevant when considering abuse liability. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Drogas Desenhadas/farmacologia , Metilfenidato/farmacologia , Animais , Aminas Biogênicas/metabolismo , Estimulantes do Sistema Nervoso Central/química , Drogas Desenhadas/química , Células HEK293 , Humanos , Metilfenidato/química , Proteínas de Transporte de Neurotransmissores/antagonistas & inibidores , Proteínas de Transporte de Neurotransmissores/metabolismo , Ligação Proteica/efeitos dos fármacos , Receptores de Amina Biogênica/metabolismo , TransfecçãoRESUMO
BACKGROUND: Mephedrone is a synthetic cathinone and one of the most popular recreationally used new psychoactive substances. The aim of the present study was to characterize the in vitro pharmacology of novel analogs of mephedrone and related newly emerged designer stimulants. METHODS: We determined norepinephrine, dopamine, and serotonin transporter inhibition potencies and monoamine release in transporter-transfected human embryonic kidney 293 cells. We also assessed monoamine receptor and transporter binding affinities. RESULTS: Mephedrone analogs potently inhibited the norepinephrine transporter and, with the exception of 3-methylmethcathinone (3-MMC), inhibited the serotonin transporter more potently than the dopamine transporter. Similar to classic amphetamines, mephedrone analogs were substrate-type monoamine releasers. 5-(2-Aminopropyl)indole (5-IT) was a highly potent monoamine transporter inhibitor and a releaser of dopamine and serotonin. 4-Methylamphetamine (4-MA) mediated efflux of all three monoamines and inhibited the serotonin transporter more potently than the dopamine transporter, unlike amphetamine. N-methyl-2-aminoindane (N-methyl-2-AI) was a selective norepinephrine transporter inhibitor and norepinephrine releaser, whereas 5-methoxy-6-methyl-2-aminoindane (MMAI) was a selective serotonin transporter inhibitor and serotonin releaser. All of the drugs interacted with monoamine receptors. CONCLUSION: The predominant actions on serotonin vs. dopamine transporters suggest that dimethylmethcathinones, 4-MA, and MMAI cause entactogenic effects similar to 3,4-methylenedioxymethamphetamine, whereas 3-MMC, 5-IT, and N-methyl-2-AI have more stimulant-type properties like amphetamine. Because of pharmacological and structural similarity to mephedrone, similar health risks can be expected for these analogs. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'