RESUMO
De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster, the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.
Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Dopamina/fisiologia , Animais , Transtornos Globais do Desenvolvimento Infantil/fisiopatologia , Pré-Escolar , Neurônios Dopaminérgicos/fisiologia , Drosophila melanogaster/genética , Homeostase/genética , Humanos , Masculino , Atividade Motora/genética , Mutação de Sentido Incorreto/genética , Fatores de RiscoAssuntos
Transtornos Relacionados ao Uso de Cocaína/psicologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Recompensa , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Transtornos Relacionados ao Uso de Cocaína/tratamento farmacológico , Condicionamento Operante/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Exenatida , Comportamento Exploratório/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/uso terapêutico , Peçonhas/uso terapêuticoRESUMO
Fast desensitization is an important regulatory mechanism of neuronal NMDA receptor function. Only recombinant NMDA receptors composed of NR1/NR2A exhibit a fast component of desensitization similar to neuronal NMDA receptors. Here we report that the fast desensitization of NR1/NR2A receptors is caused by ambient zinc, and that a positive allosteric interaction occurs between the extracellular zinc-binding site located in the amino terminal domain and the glutamate-binding domain of NR2A. The relaxation of macroscopic currents reflects a shift to a new equilibrium due to increased zinc affinity after binding of glutamate. We also show a similar interaction between the ifenprodil binding site and the glutamate binding site of NR1/NR2B receptors. These data raise the possibility that there is an allosteric interaction between the amino terminal domain and the ligand-binding domain of other glutamate receptors. Our findings may provide insight into how zinc and other extracellular modulators regulate NMDA receptor function.
Assuntos
Fragmentos de Peptídeos/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Regulação Alostérica/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Ácido Edético/farmacologia , Condutividade Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Espaço Extracelular/metabolismo , Ácido Glutâmico/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Piperidinas/farmacologia , Estrutura Terciária de Proteína/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Zinco/metabolismo , Zinco/farmacologiaRESUMO
Agonism of the glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) has been effective at treating aspects of addictive behavior for a number of abused substances, including cocaine. However, the molecular mechanisms and brain circuits underlying the therapeutic effects of GLP-1R signaling on cocaine actions remain elusive. Recent evidence has revealed that endogenous signaling at the GLP-1R within the forebrain lateral septum (LS) acts to reduce cocaine-induced locomotion and cocaine conditioned place preference, both considered dopamine (DA)-associated behaviors. DA terminals project from the ventral tegmental area to the LS and express the DA transporter (DAT). Cocaine acts by altering DA bioavailability by targeting the DAT. Therefore, GLP-1R signaling might exert effects on DAT to account for its regulation of cocaine-induced behaviors. We show that the GLP-1R is highly expressed within the LS. GLP-1, in LS slices, significantly enhances DAT surface expression and DAT function. Exenatide (Ex-4), a long-lasting synthetic analog of GLP-1 abolished cocaine-induced elevation of DA. Interestingly, acute administration of Ex-4 reduces septal expression of the retrograde messenger 2-arachidonylglycerol (2-AG), as well as a product of its presynaptic degradation, arachidonic acid (AA). Notably, AA reduces septal DAT function pointing to AA as a novel regulator of central DA homeostasis. We further show that AA oxidation product γ-ketoaldehyde (γ-KA) forms adducts with the DAT and reduces DAT plasma membrane expression and function. These results support a mechanism in which postsynaptic septal GLP-1R activation regulates 2-AG levels to alter presynaptic DA homeostasis and cocaine actions through AA.
Assuntos
Ácido Araquidônico/metabolismo , Dopamina/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Núcleos Septais/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Cocaína/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Endocanabinoides/metabolismo , Exenatida , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Glicerídeos/metabolismo , Homeostase , Incretinas/farmacologia , Camundongos , Microdiálise , Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Núcleos Septais/efeitos dos fármacos , Peçonhas/farmacologiaRESUMO
Emerging evidence associates dysfunction in the dopamine (DA) transporter (DAT) with the pathophysiology of autism spectrum disorder (ASD). The human DAT (hDAT; SLC6A3) rare variant with an Ala to Val substitution at amino acid 559 (hDAT A559V) was previously reported in individuals with bipolar disorder or attention-deficit hyperactivity disorder (ADHD). We have demonstrated that this variant is hyper-phosphorylated at the amino (N)-terminal serine (Ser) residues and promotes an anomalous DA efflux phenotype. Here, we report the novel identification of hDAT A559V in two unrelated ASD subjects and provide the first mechanistic description of its impaired trafficking phenotype. DAT surface expression is dynamically regulated by DAT substrates including the psychostimulant amphetamine (AMPH), which causes hDAT trafficking away from the plasma membrane. The integrity of DAT trafficking directly impacts DA transport capacity and therefore dopaminergic neurotransmission. Here, we show that hDAT A559V is resistant to AMPH-induced cell surface redistribution. This unique trafficking phenotype is conferred by altered protein kinase C ß (PKCß) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCß activity, inhibition of which restores the AMPH-induced hDAT A559V membrane redistribution. Mechanistically, we link the inability of hDAT A559V to traffic in response to AMPH to the phosphorylation of the five most distal DAT N-terminal Ser. Mutation of these N-terminal Ser to Ala restores AMPH-induced trafficking. Furthermore, hDAT A559V has a diminished ability to transport AMPH, and therefore lacks AMPH-induced DA efflux. Pharmacological inhibition of PKCß or Ser to Ala substitution in the hDAT A559V background restores AMPH-induced DA efflux while promoting intracellular AMPH accumulation. Although hDAT A559V is a rare variant, it has been found in multiple probands with neuropsychiatric disorders associated with imbalances in DA neurotransmission, including ADHD, bipolar disorder, and now ASD. These findings provide valuable insight into a new cellular phenotype (altered hDAT trafficking) supporting dysregulated DA function in these disorders. They also provide a novel potential target (PKCß) for therapeutic interventions in individuals with ASD.
Assuntos
Transtorno Autístico/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Mutação/genética , Transmissão Sináptica/genética , Movimento Celular/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/fisiologia , Humanos , Masculino , IrmãosRESUMO
1. The time course of macroscopic current responses of homomeric murine serotonin 5-HT3A receptors was studied in whole cells and excised membrane patches under voltage clamp in response to rapid application of serotonin. 2. Serotonin activated whole cell currents with an EC(50) value for the peak response of 2 microM and a Hill slope of 3.0 (n = 12), suggesting that the binding of at least three agonist molecules is required to open the channel. 3. Homomeric 5-HT3A receptors in excised membrane patches had a slow activation time course (mean +/- S.E.M. 10-90 % rise time 12.5 +/- 1.6 ms; n = 9 patches) for 100 microM serotonin. The apparent activation rate was estimated by fitting an exponential function to the rising phase of responses to supramaximal serotonin to be 136 s(-1). 4. The 5-HT3A receptor response to 100 microM serotonin in outside-out patches (n = 19) and whole cells (n = 41) desensitized with a variable rate that accelerated throughout the experiment. The time course for desensitization was described by two exponential components (for patches tau(slow) 1006 +/- 139 ms, amplitude 31 %; tau(fast) 176 +/- 25 ms, amplitude 69 %). 5. Deactivation of the response following serotonin removal from excised membrane patches (n = 8) and whole cells (n = 29) was described by a dual exponential time course with time constants similar to those for desensitization (for patches tau(slow) 838 +/- 217 ms, 55 % amplitude; tau(fast) 213 +/- 44 ms, 45 % amplitude). 6. In most patches (6 of 8), the deactivation time course in response to a brief 1-5 ms pulse of serotonin was similar to or slower than desensitization. This suggests that the continued presence of agonist can induce desensitization with a similar or more rapid time course than agonist unbinding. The difference between the time course for deactivation and desensitization was voltage independent over the range -100 to -40 mV in patches (n = 4) and -100 to +50 mV in whole cells (n = 4), suggesting desensitization of these receptors in the presence of serotonin does not reflect a voltage-dependent block of the channel by agonist. 7. Simultaneously fitting the macroscopic 5-HT3A receptor responses in patches to submaximal (2 microM) and maximal (100 microM) concentrations of serotonin to a variety of state models suggests that homomeric 5-HT3A receptors require the binding of three agonists to open and possess a peak open probability greater than 0.8. Our modelling also suggests that channel open probability varies with the number of serotonin molecules bound to the receptor, with a reduced open probability for fully liganded receptors. Increasing the desensitization rate constants in this model can generate desensitization that is more rapid than deactivation, as observed in a subpopulation of our patches.