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1.
J Chem Inf Model ; 60(7): 3577-3586, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32525311

RESUMO

Dopamine clearance in the brain is controlled by the dopamine transporter (DAT), a protein residing in the plasma membrane, which drives reuptake of extracellular dopamine into presynaptic neurons. Studies have revealed that the ßγ subunits of heterotrimeric G proteins modulate DAT function through a physical association with the C-terminal region of the transporter. Regulation of neurotransmitter transporters by Gßγ subunits is unprecedented in the literature; therefore, it is interesting to investigate the structural details of this particular protein-protein interaction. Here, we refined the crystal structure of the Drosophila melanogaster DAT (dDAT), modeling de novo the N- and C-terminal domains; subsequently, we used the full-length dDAT structure to generate a comparative model of human DAT (hDAT). Both proteins were assembled with Gß1γ2 subunits employing protein-protein docking, and subsequent molecular dynamics simulations were run to identify the specific interactions governing the formation of the hDAT:Gßγ and dDAT:Gßγ complexes. A [L/F]R[Q/E]R sequence motif containing the residues R588 in hDAT and R587 in dDAT was found as key to bind the Gßγ subunits through electrostatic interactions with a cluster of negatively charged residues located at the top face of the Gß subunit. Alterations of DAT function have been associated with multiple devastating neuropathological conditions; therefore, this work represents a step toward better understanding DAT regulation by signaling proteins, allowing us to predict therapeutic target regions.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas de Drosophila/química , Drosophila melanogaster , Proteínas de Ligação ao GTP/química , Animais , Dopamina , Drosophila melanogaster/metabolismo , Simulação de Dinâmica Molecular
2.
J Biol Chem ; 292(30): 12471-12482, 2017 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-28584050

RESUMO

The human dopamine (DA) transporter (hDAT) is a key regulator of neurotransmission and a target for antidepressants and addictive drugs. Despite the recent resolution of dDAT structures from Drosophila melanogaster, complete understanding of its mechanism of function and even information on its biological assembly is lacking. The resolved dDAT structures are monomeric, but growing evidence suggests that hDAT might function as a multimer, and its oligomerization may be relevant to addictive drug effects. Here, using structure-based computations, we examined the possible mechanisms of hDAT dimerization and its dynamics in a lipid bilayer. Using a combination of site-directed mutagenesis, DA-uptake, and cross-linking experiments that exploited the capacity of Cys-306 to form intermonomeric disulfide bridges in the presence of an oxidizing agent, we tested the effects of mutations at transmembrane segment (TM) 6 and 12 helices in HEK293 cells. The most probable structural model for hDAT dimer suggested by computations and experiments differed from the dimeric structure resolved for the bacterial homolog, LeuT, presumably because of a kink at TM12 preventing favorable monomer packing. Instead, TM2, TM6, and TM11 line the dimer interface. Molecular dynamics simulations of the dimeric hDAT indicated that the two subunits tend to undergo cooperative structural changes, both on local (extracellular gate opening/closure) and global (transition between outward-facing and inward-facing states) scales. These observations suggest that hDAT transport properties may be allosterically modulated under conditions promoting dimerization. Our study provides critical insights into approaches for examining the oligomerization of neurotransmitter transporters and sheds light on their drug modulation.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Regulação Alostérica , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Multimerização Proteica
3.
J Biol Chem ; 290(38): 22977-90, 2015 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-26203187

RESUMO

In the mammalian central nervous system, excitatory amino acid transporters (EAATs) are responsible for the clearance of glutamate after synaptic release. This energetically demanding activity is crucial for precise neuronal communication and for maintaining extracellular glutamate concentrations below neurotoxic levels. In addition to their ability to recapture glutamate from the extracellular space, EAATs exhibit a sodium- and glutamate-gated anion conductance. Here we show that substitution of a conserved positively charged residue (Arg-388, hEAAT1) in transmembrane domain 7 with a negatively charged amino acid eliminates the ability of glutamate to further activate the anion conductance. When expressed in oocytes, R388D or R388E mutants show large anion currents that display no further increase in amplitude after application of saturating concentrations of Na(+) and glutamate. They also show a substantially reduced transport activity. The mutant transporters appear to exist preferentially in a sodium- and glutamate-independent constitutive open channel state that rarely transitions to complete the transport cycle. In addition, the accessibility of cytoplasmic residues to membrane-permeant modifying reagents supports the idea that this substrate-independent open state correlates with an intermediate outward facing conformation of the transporter. Our data provide additional insights into the mechanism by which substrates gate the anion conductance in EAATs and suggest that in EAAT1, Arg-388 is a critical element for the structural coupling between the substrate translocation and the gating mechanisms of the EAAT-associated anion channel.


Assuntos
Transportador 1 de Aminoácido Excitatório/metabolismo , Ativação do Canal Iônico/fisiologia , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Animais , Transportador 1 de Aminoácido Excitatório/genética , Expressão Gênica , Humanos , Transporte de Íons/fisiologia , Oócitos , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis
4.
J Exp Pharmacol ; 16: 13-24, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38249320

RESUMO

Background: Viloxazine ER (viloxazine extended-release capsules; Qelbree®), a nonstimulant attention-deficit/hyperactivity disorder (ADHD) treatment, has known activity as a norepinephrine (NE) transporter (NET) inhibitor. In vitro studies have also shown direct pharmacological effects on specific serotonin (5-HT) receptors, but not on the serotonin transporter (SERT). An in vivo microdialysis study in rats showed viloxazine (50 mg/kg i.p.) increased extracellular 5-HT, NE, and dopamine (DA) in the prefrontal cortex (PFC), a key brain region in ADHD pathology. This study evaluated whether these effects occur at clinically relevant concentrations. Methods: Microdialysis experiments were conducted in freely-moving, Sprague-Dawley rats (males, 8 weeks). Viloxazine (1, 3, 10, 30 mg/kg) was administered intraperitoneally to establish the dose range in rats at which viloxazine plasma concentrations aligned with those of individuals with ADHD administered therapeutic doses of viloxazine ER. Concentrations of unbound viloxazine, NE, 5-HT, DA, and NE and 5-HT metabolites (3,5-dihydroxyphenylglycol [DHPG] and 5-hydroxyindoleacetic acid [5-HIAA]) were measured in PFC interstitial fluid. After identifying a therapeutically relevant dose (30 mg/kg), the experiment was repeated using 30 and 50 mg/kg viloxazine (as 50 mg/kg increased NE, 5-HT, and DA in prior studies). Results: Viloxazine unbound (free drug) plasma concentrations in rats at 30 mg/kg were comparable to free drug concentrations in individuals with ADHD taking clinically effective doses (based on validated population PK models). Viloxazine 30 mg/kg significantly increased extracellular NE, 5-HT, and DA PFC levels compared to vehicle. Concomitant decreases in DHPG, but not 5-HIAA, support the inhibitory effect of viloxazine on NET but not SERT. Conclusion: At clinically relevant concentrations, viloxazine increases PFC NE, DA, and 5-HT. Prefrontal augmentation of 5-HT does not appear to result from 5-HT reuptake inhibition but may be related to activation of 5-HT neurons. The potential therapeutic role of serotonergic effects in ADHD treatment merits further exploration.

5.
Epilepsia Open ; 9(5): 1826-1836, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39096485

RESUMO

OBJECTIVE: We evaluated huperzine A treatment in the Genetic Absence Epilepsy Rat from Strasbourg (GAERS) model of genetic generalized epilepsy (GGE) with absence seizures. METHODS: Adult male GAERS (N = 15) were implanted with EEG recording electrodes 10 days before receiving study drug. Each animal received the following six treatments as a single, intraperitoneal dose, 7 days apart (in random order): huperzine A (0.3, 1.0, or 3.0 mg/kg), two periods of vehicle (0.9% NaCl), or ethosuximide (100 mg/kg) as a positive control. Electroencephalograms (EEGs) were acquired for 24 h before and after each treatment and analyzed for seizure activity during the 90-min period immediately post-treatment, including 30-min intervals at 30, 60, and 90 min. Additional analyses evaluated seizure activity over the 24-h post-treatment period using 60-min intervals at 6, 12, and 24 h. The cumulative 24-h periods before and after each administered treatment were also compared. RESULTS: Two-way ANOVA showed a treatment difference [F(91,182) = 3.592, p < 0.0001] on the number of seizures over the first 90-min post-treatment (primary outcome); Tukey's post hoc analyses showed that, compared to vehicle, huperzine A (3.0 mg/kg) significantly reduced seizures in the 30-min (p = 0.02) and 60-min (p = 0.001) intervals, and ethosuximide significantly reduced seizures at all measured time intervals except the 1-h blocks at 12 and 24 h. Huperzine A 3.0 mg/kg and ethosuximide significantly reduced seizures during the cumulative 24-h post-treatment period relative to pretreatment baseline. While huperzine A 3.0 mg/kg did not differ significantly from ethosuximide at any time point, the study was not designed to evaluate non-inferiority. The only adverse event after huperzine A or ethosuximide was mild, dose-dependent sedation. SIGNIFICANCE: Huperzine A potently suppressed absence-like seizures in GAERS, albeit with a shorter duration of action relative to ethosuximide, showing promise for clinical efficacy in GGE. PLAIN LANGUAGE SUMMARY: This study looked at how huperzine A affects seizures in rats with similar abnormal brain activity as seen in humans with absence epilepsy. Rats received different treatments, placebo (i.e., saline solution), huperzine A, and ethosuximide. Ethosuximide is considered a gold standard treatment for absence epilepsy. We recorded brain activity to measure seizures before and after each treatment. We found that huperzine A (3.0 mg/kg) reduced seizures soon after treatment, like ethosuximide. Both treatments appeared safe, causing only mild sleepiness. The study shows that huperzine A could be a good new treatment for a type of absence epilepsy.


Assuntos
Alcaloides , Anticonvulsivantes , Modelos Animais de Doenças , Eletroencefalografia , Epilepsia Tipo Ausência , Etossuximida , Sesquiterpenos , Animais , Alcaloides/farmacologia , Alcaloides/uso terapêutico , Epilepsia Tipo Ausência/tratamento farmacológico , Ratos , Masculino , Anticonvulsivantes/uso terapêutico , Anticonvulsivantes/farmacologia , Sesquiterpenos/uso terapêutico , Sesquiterpenos/farmacologia , Etossuximida/uso terapêutico , Etossuximida/farmacologia , Epilepsia Generalizada/tratamento farmacológico , Relação Dose-Resposta a Droga , Convulsões/tratamento farmacológico
7.
CNS Drugs ; 35(6): 643-653, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34003459

RESUMO

Viloxazine has a long history of clinical use in Europe as an antidepressant, and has recently been repurposed into an extended-release form for the treatment of attention-deficit/hyperactivity disorder in the USA. An immediate-release formulation was approved for the treatment of depression in the UK in 1974, and was subsequently marketed there and in several European countries for 30 years with no major safety concerns. In contrast to first-generation antidepressants (e.g., tricyclic antidepressants, monoamine oxidase inhibitors), viloxazine was associated with a relatively low risk for cardiotoxicity. Gastrointestinal symptoms were the most commonly reported side effects. The therapeutic effects of viloxazine are thought to be primarily the result of its action as a norepinephrine reuptake inhibitor, although in vitro and preclinical in vivo animal data suggest that viloxazine may also impact the serotoninergic system. This review summarizes the evolving knowledge of viloxazine based on information from previously published preclinical and clinical investigations, and acquired unpublished historical study reports from both open-label and blinded controlled clinical trials. We review the chemical properties, mechanism of action, safety, and tolerability across these studies, and discuss the contemporary rationale for the development of this agent as an extended-release oral formulation for the treatment of attention-deficit/hyperactivity disorder.


Assuntos
Inibidores da Captação Adrenérgica/administração & dosagem , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Viloxazina/administração & dosagem , Administração Oral , Inibidores da Captação Adrenérgica/efeitos adversos , Inibidores da Captação Adrenérgica/farmacologia , Animais , Doenças do Sistema Nervoso Central/tratamento farmacológico , Preparações de Ação Retardada , Humanos , Viloxazina/efeitos adversos , Viloxazina/farmacologia
8.
J Exp Pharmacol ; 12: 285-300, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32943948

RESUMO

BACKGROUND: Viloxazine was historically described as a norepinephrine reuptake inhibitor (NRI). Since NRIs have previously demonstrated efficacy in attention deficit/hyperactivity disorder (ADHD), viloxazine underwent contemporary investigation in the treatment of ADHD. Its clinical and safety profile, however, was found to be distinct from other ADHD medications targeting norepinephrine reuptake. Considering the complexity of neuropsychiatric disorders, understanding the mechanism of action (MoA) is an important differentiating point between viloxazine and other ADHD medications and provides pharmacology-based rationale for physicians prescribing appropriate therapy. METHODS: Viloxazine was evaluated in a series of in vitro binding and functional assays. Its effect on neurotransmitter levels in the brain was evaluated using microdialysis in freely moving rats. RESULTS: We report the effects of viloxazine on serotoninergic (5-HT) system. In vitro, viloxazine demonstrated antagonistic activity at 5-HT2B and agonistic activity at 5-HT2C receptors, along with predicted high receptor occupancy at clinical doses. In vivo, viloxazine increased extracellular 5-HT levels in the prefrontal cortex (PFC), a brain area implicated in ADHD. Viloxazine also exhibited moderate inhibitory effects on the norepinephrine transporter (NET) in vitro and in vivo, and elicited moderate activity at noradrenergic and dopaminergic systems. CONCLUSION: Viloxazine's ability to increase 5-HT levels in the PFC and its agonistic and antagonistic effects on certain 5-HT receptor subtypes, which were previously shown to suppress hyperlocomotion in animals, indicate that 5-HT modulating activity of viloxazine is an important (if not the predominant) component of its MoA, complemented by moderate NET inhibition. Supported by clinical data, these findings suggest the updated psychopharmacological profile of viloxazine can be best explained by its action as a serotonin norepinephrine modulating agent (SNMA).

9.
Hum Mutat ; 30(3): 397-405, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19191339

RESUMO

Heterozygous mutations in the CLCN2 gene encoding the voltage-gated chloride channel CLC2 have been identified in patients with idiopathic generalized epilepsy (IGE). Yet the involvement of CLCN2 in epilepsy remains controversial. To investigate the involvement of CLCN2 in another independent sample, we screened 52 unrelated patients from IGE families and 23 patients with Doose syndrome for mutations in CLCN2. No mutations were found in patients with Doose syndrome. In three unrelated IGE families, we identified two novel missense mutations, p.Arg235Gln and p.Arg577Gln, which were absent in large ethnically-matched control populations, and one novel p.Arg644Cys variant, which was also found in five Indian controls. Functional characterization of mutant channels using heterologous expression in mammalian cells and whole-cell patch-clamp recordings revealed faster deactivation kinetics as the major phenotype of both missense mutations. This finding predicts a loss of function that may contribute to intracellular chloride accumulation or neuronal hyperexcitability. However, the incomplete segregation of the mutations among affected members and the transmission by unaffected parents suggests that these CLCN2 mutations alone are not sufficient to induce epilepsy. They may instead represent susceptibility factors among other so far undetected genetic alterations in the respective families.


Assuntos
Canais de Cloreto/genética , Epilepsia Generalizada/genética , Mutação de Sentido Incorreto , Adolescente , Adulto , Sequência de Aminoácidos , Canais de Cloro CLC-2 , Linhagem Celular , Canais de Cloreto/fisiologia , Análise Mutacional de DNA , Epilepsia Generalizada/patologia , Epilepsia Generalizada/fisiopatologia , Saúde da Família , Feminino , Humanos , Masculino , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Linhagem , Homologia de Sequência de Aminoácidos , Transfecção , Adulto Jovem
10.
J Neurochem ; 110(2): 581-94, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19457116

RESUMO

Synaptic transmission depends on the efficient loading of transmitters into synaptic vesicles by vesicular neurotransmitter transporters. The vesicular monoamine transporter-2 (VMAT2) is essential for loading monoamines into vesicles and maintaining normal neurotransmission. In an effort to understand the regulatory mechanisms associated with VMAT2, we have embarked upon a systematic search for interacting proteins. Glutathione-S-transferase pull-down assays combined with mass spectrometry led to the identification of the 70-kDa heat shock cognate protein (Hsc70) as a VMAT2 interacting protein. Co-immunoprecipitation experiments in brain tissue and heterologous cells confirmed this interaction. A direct binding was observed between the amino terminus and the third cytoplasmic loop of VMAT2, as well as, a region containing the substrate binding and the carboxy-terminal domains of Hsc70. Furthermore, VMAT2 and Hsc70 co-fractionated with purified synaptic vesicles obtained from a sucrose gradient, suggesting that this interaction occurs at the synaptic vesicle membrane. The functional significance of this novel VMAT2/Hsc70 interaction was examined by performing vesicular uptake assays in heterologous cells and purified synaptic vesicles from brain tissue. Recombinant Hsc70 produced a dose-dependent inhibition of VMAT2 activity. This effect was mimicked by the closely related Hsp70 protein. In contrast, VMAT2 activity was not altered in the presence of previously denatured Hsc70 or Hsp70, as well as the unrelated Hsp60 protein; confirming the specificity of the Hsc70 effect. Finally, a purified Hsc70 fragment that binds VMAT2 was sufficient to inhibit VMAT2 activity in synaptic vesicles. Our results suggest an important role for Hsc70 in VMAT2 function and regulation.


Assuntos
Proteínas de Choque Térmico HSC70/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Vesiculares de Transporte de Monoamina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Proteínas de Choque Térmico HSC70/fisiologia , Humanos , Masculino , Chaperonas Moleculares/fisiologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas Vesiculares de Transporte de Monoamina/antagonistas & inibidores
11.
Front Cell Neurosci ; 13: 35, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30828290

RESUMO

The dopamine transporter (DAT) is a plasma membrane protein responsible for the uptake of released dopamine back to the presynaptic terminal and ending dopamine neurotransmission. The DAT is the molecular target for cocaine and amphetamine as well as a number of pathological conditions including autism spectrum disorders, attention-deficit hyperactivity disorder (ADHD), dopamine transporter deficiency syndrome (DTDS), and Parkinson's disease. The DAT uptake capacity is dependent on its level in the plasma membrane. In vitro studies show that DAT functional expression is regulated by a balance of endocytosis, recycling, and lysosomal degradation. However, recent reports suggest that DAT regulation by endocytosis in neurons is less significant than previously reported. Therefore, additional mechanisms appear to determine DAT steady-state level and functional expression in the neuronal plasma membrane. Here, we hypothesize that the ubiquitin-like protein small ubiquitin-like modifier 1 (SUMO1) increases the DAT steady-state level in the plasma membrane. In confocal microscopy, fluorescent resonance energy transfer (FRET), and Western blot analyses, we demonstrate that DAT is associated with SUMO1 in the rat dopaminergic N27 and DAT overexpressing Human Embryonic Kidney cells (HEK)-293 cells. The overexpression of SUMO1 and the Ubc9 SUMO-conjugase induces DAT SUMOylation, reduces DAT ubiquitination and degradation, enhancing DAT steady-state level. In addition, the Ubc9 knock-down by interference RNA (RNAi) increases DAT degradation and reduces DAT steady-state level. Remarkably, the Ubc9-mediated SUMOylation increases the expression of DAT in the plasma membrane and dopamine uptake capacity. Our results strongly suggest that SUMOylation is a novel mechanism that plays a central role in regulating DAT proteostasis, dopamine uptake, and dopamine signaling in neurons. For that reason, the SUMO pathway including SUMO1, SUMO2, Ubc9, and DAT SUMOylation, can be critical therapeutic targets in regulating DAT stability and dopamine clearance in health and pathological states.

12.
J Physiol ; 586(22): 5325-36, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18801843

RESUMO

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability.


Assuntos
Canais de Cloreto/química , Canais de Cloreto/metabolismo , Sítios de Ligação , Canais de Cloro CLC-2 , Linhagem Celular , Canais de Cloreto/genética , Cistationina beta-Sintase/química , Dimerização , Humanos , Ativação do Canal Iônico , Cinética , Modelos Biológicos , Mutagênese , Neurônios/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência
13.
PLoS One ; 8(3): e59788, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23555781

RESUMO

Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson's disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the ßγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gßγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gßγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gßγ subunit overexpression, or the Gßγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gßγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gßγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gßγ signaling in the control of dopamine homeostasis.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Animais , Biotinilação , Encéfalo/metabolismo , Corpo Estriado/metabolismo , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Glutationa Transferase/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Homeostase , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/citologia , Estrutura Terciária de Proteína , Transdução de Sinais , Sinaptossomos/metabolismo , Xenopus laevis
14.
J Biol Chem ; 281(34): 24104-10, 2006 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-16793763

RESUMO

High voltage-gated calcium channels consist of a pore-forming subunit (alpha(1)) and three nonhomologous subunits (alpha(2)/delta, beta, and gamma). Although it is well established that the beta-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the alpha(1)-subunit remains controversial. Here, we address this issue by examining the effect of purified beta(2a) protein on Ca(V)1.2 and Ca(V)2.3 channels expressed in Xenopus oocytes. The beta(2a)-subunit binds to the alpha(1)-interaction domain (AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of Ca(V)1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the alpha(1)-beta interaction through the AID site can take place at the plasma membrane. Furthermore, injection of beta(2a) protein inhibited inactivation of Ca(V)2.3 channels and converted fast inactivating Ca(V)2.3/beta(1b) channels to slow inactivating channels. Inhibition of inactivation required larger concentration of beta(2a) in oocytes expressing Ca(V)2.3/beta(1b) channels than expressing Ca(V)2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the alpha(1)-beta interaction is reversible in intact cells and defines calcium channels beta-subunits as regulatory proteins rather than stoichiometric subunits.


Assuntos
Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo R/química , Ativação do Canal Iônico , Animais , Sítios de Ligação , Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo R/fisiologia , Eletrofisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Macrolídeos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , Ratos , Triptofano , Xenopus
15.
J Biol Chem ; 279(21): 21689-94, 2004 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-15016803

RESUMO

Voltage-gated calcium channels mediate the influx of Ca(2+) ions into eukaryotic cells in response to membrane depolarization. They are hetero-multimer membrane proteins formed by at least three subunits, the poreforming alpha(1)-subunit and the auxiliary beta- and alpha(2)delta-subunits. The beta-subunit is essential for channel performance because it regulates two distinct features of voltage-gated calcium channels, the surface expression and the channel activity. Four beta-subunit genes have been cloned, beta(1-4), with molecular masses ranging from 52 to 78 kDa, and several splice variants have been identified. The beta(1b)-subunit, expressed at high levels in mammalian brain, has been used extensively to study the interaction between the pore forming alpha(1)- and the regulatory beta-subunit. However, structural characterization has been impaired for its tendency to form aggregates when expressed in bacteria. We applied an on-column refolding procedure based on size exclusion chromatography to fold the beta(1b)-subunit of the voltage gated-calcium channels from Escherichia coli inclusion bodies. The beta(1b)-subunit refolds into monomers, as shown by sucrose gradient analysis, and binds to a glutathione S-transferase protein fused to the known target in the alpha(1)-subunit (the alpha-interaction domain). Using the cut-open oocyte voltage clamp technique, we measured gating and ionic currents in Xenopus oocytes expressing cardiac alpha(1)-subunit (alpha(1C)) co-injected with folded-beta(1b)-protein or beta(1b)-cRNA. We demonstrate that the co-expression of the alpha(1C)-subunit with either folded-beta(1b)-protein or beta(1b)-cRNA increases ionic currents to a similar extent and with no changes in charge movement, indicating that the beta(1b)-subunit primarily modulates channel activity, rather than expression.


Assuntos
Canais de Cálcio/química , Canais de Cálcio/fisiologia , Processamento Alternativo , Animais , Western Blotting , Cálcio/química , Calibragem , Centrifugação com Gradiente de Concentração , Cromatografia , Clonagem Molecular , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Eletrofisiologia , Escherichia coli/metabolismo , Feminino , Íons , Cinética , Oócitos/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/química , RNA Complementar/metabolismo , Ratos , Proteínas Recombinantes de Fusão/química , Sacarose/química , Sacarose/farmacologia , Fatores de Tempo , Xenopus laevis
16.
J Neurosci Res ; 71(3): 353-64, 2003 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-12526024

RESUMO

The contribution of distinct Ca(2+)-sensitive protein kinases to the regulation of the expression of the synaptosomal-associated protein SNAP-25 was examined in bovine chromaffin cells. Prolonged incubation with high K(+) (38 mM) or 1,1-dimethyl-4-phenyl-piperazinium (DMPP), a nicotinic receptor agonist, significantly increased SNAP-25 protein and mRNA expression, as assessed by immunoblotting and semi-quantitative RT-PCR analysis. Both stimuli preferentially enhanced mRNA coding for the SNAP-25a isoform. Increase of SNAP-25 expression induced by K(+) or DMPP was inhibited over 70% by KN-62 and KN-93, two Ca(2+)/calmodulin-dependent protein kinase (CaMK) inhibitors, whereas the inactive analogue KN-92 only reduced the expression by 34%. The three compounds also inhibited the high K(+)-elicited [Ca(2+)](i) signal by 40%, suggesting that the effect of KN-62 and KN-93 was a combination of CaMK/ Ca(2+) influx inhibitory actions. Incubation of the cells with mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126 reduced protein expression elicited by high K(+) by 50%, but did not modify the response to DMPP. Interestingly, although protein kinase A (PKA) inhibition by H-89 did not affect the high K(+) or DMPP-induced SNAP-25 expression, basal protein levels were significantly modified upon activation or inhibition of this pathway. Basal expression of SNAP-25 was also modified by the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, but not by Gö6976, a PKC-alpha inhibitor, suggesting that the Ca(2+)-insensitive PKC-epsilon isoform control basal expression of SNAP-25 in these cells. Taken together, these results provide the first evidence that diverse protein kinases might converge in the induction of SNAP-25 expression in chromaffin cells. The preferential contribution of one or another kinase would depend on the physiological or experimental conditions.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Células Cromafins/enzimologia , Proteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Proteínas Quinases/fisiologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Bovinos , Células Cultivadas , Células Cromafins/efeitos dos fármacos , Células Cromafins/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Inibidores de Proteínas Quinases , Proteína 25 Associada a Sinaptossoma
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