RESUMO
The substituted amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy), is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic, however, promising outcomes for the therapeutic utility of MDMA for post-traumatic stress disorder and the long-time use of the dopaminergic and noradrenergic-directed amphetamines in treatment of attention-deficit hyperactivity disorder and narcolepsy increases the importance of understanding this phenomenon. Previously, we identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. Here, we performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. Our findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Drosophila/metabolismo , Alucinógenos/metabolismo , N-Metil-3,4-Metilenodioxianfetamina/metabolismo , Serotoninérgicos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Substituição de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HEK293 , Alucinógenos/farmacologia , Humanos , Mutagênese Sítio-Dirigida , Mutação , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Serotonina/metabolismo , Serotoninérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Especificidade da Espécie , Especificidade por Substrato , Xenopus laevisRESUMO
The dopaminergic system is essential for cognitive processes, including reward, attention and motor control. In addition to DA release and availability of synaptic DA receptors, timing and magnitude of DA neurotransmission depend on extracellular DA-level regulation by the dopamine transporter (DAT), the membrane expression and trafficking of which are highly dynamic. Data presented here from real-time TIRF (TIRFM) and confocal microscopy coupled with surface biotinylation and electrophysiology suggest that changes in the membrane potential alone, a universal yet dynamic cellular property, rapidly alter trafficking of DAT to and from the surface membrane. Broadly, these findings suggest that cell-surface DAT levels are sensitive to membrane potential changes, which can rapidly drive DAT internalization from and insertion into the cell membrane, thus having an impact on the capacity for DAT to regulate extracellular DA levels.
Assuntos
Membrana Celular/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Linhagem Celular , Membrana Celular/química , Membrana Celular/genética , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Humanos , Potenciais da Membrana , Transporte ProteicoRESUMO
Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (S)-citalopram (1). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands 15, 22, and 26. These ligands retained high to moderate affinity binding (K(i) = 24-227 nM) for hSERT, as assessed by [(3)H]5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with Ki values ranging from 3.8 to 9.9 µM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (S)-citalopram inhibition of labeling by [(125)I]15 compared to that by [(125)I]22 and [(125)I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug-protein binding interactions for (S)-citalopram at hSERT.
Assuntos
Citalopram/metabolismo , Desenho de Fármacos , Processos Fotoquímicos , Inibidores Seletivos de Recaptação de Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Azidas/química , Citalopram/síntese química , Citalopram/química , Células HEK293 , Humanos , Ligantes , Inibidores Seletivos de Recaptação de Serotonina/síntese química , Inibidores Seletivos de Recaptação de Serotonina/químicaRESUMO
Neurotransmitter transporters of the SLC6 family of proteins, including the human serotonin transporter (hSERT), utilize Na(+), Cl(-), and K(+) gradients to induce conformational changes necessary for substrate translocation. Dysregulation of ion movement through monoamine transporters has been shown to impact neuronal firing potentials and could play a role in pathophysiologies, such as depression and anxiety. Despite multiple crystal structures of prokaryotic and eukaryotic SLC transporters indicating the location of both (or one) conserved Na(+)-binding sites (termed Na1 and Na2), much remains uncertain in regard to the movements and contributions of these cation-binding sites in the transport process. In this study, we utilize the unique properties of a mutation of hSERT at a single, highly conserved asparagine on TM1 (Asn-101) to provide several lines of evidence demonstrating mechanistically distinct roles for Na1 and Na2. Mutations at Asn-101 alter the cation dependence of the transporter, allowing Ca(2+) (but not other cations) to functionally replace Na(+) for driving transport and promoting 5-hydroxytryptamine (5-HT)-dependent conformational changes. Furthermore, in two-electrode voltage clamp studies in Xenopus oocytes, both Ca(2+) and Na(+) illicit 5-HT-induced currents in the Asn-101 mutants and reveal that, although Ca(2+) promotes substrate-induced current, it does not appear to be the charge carrier during 5-HT transport. These findings, in addition to functional evaluation of Na1 and Na2 site mutants, reveal separate roles for Na1 and Na2 and provide insight into initiation of the translocation process as well as a mechanism whereby the reported SERT stoichiometry can be obtained despite the presence of two putative Na(+)-binding sites.
Assuntos
Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Sódio/metabolismo , Substituição de Aminoácidos , Asparagina/genética , Asparagina/metabolismo , Sítios de Ligação , Transporte Biológico Ativo/fisiologia , Dopamina/genética , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Proteínas da Membrana Plasmática de Transporte de Serotonina/genéticaRESUMO
Ligand-induced changes in the conformation of extracellular loop (EL) 2 in the rat (r) dopamine transporter (DAT) were examined using limited proteolysis with endoproteinase Asp-N and detection of cleavage products by epitope-specific immunoblotting. The principle N-terminal fragment produced by Asp-N was a 19kDa peptide likely derived by proteolysis of EL2 residue D174, which is present just past the extracellular end of TM3. Production of this fragment was significantly decreased by binding of cocaine and other uptake blockers, but was not affected by substrates or Zn(2+), indicating the presence of a conformational change at D174 that may be related to the mechanism of transport inhibition. DA transport activity and cocaine analog binding were decreased by Asp-N treatment, suggesting a requirement for EL2 integrity in these DAT functions. In a previous study we demonstrated that ligand-induced protease resistance also occurred at R218 on the C-terminal side of rDAT EL2. Here using substituted cysteine accessibility analysis of human (h) DAT we confirm cocaine-induced alterations in reactivity of the homologous R219 and identify conformational sensitivity of V221. Focused molecular modeling of D174 and R218 based on currently available Aquifex aeolicus leucine transporter crystal structures places these residues within 2.9Å of one another, suggesting their proximity as a structural basis for their similar conformational sensitivities and indicating their potential to form a salt bridge. These findings extend our understanding of DAT EL2 and its role in transport and binding functions.
Assuntos
Antagonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Matriz Extracelular/metabolismo , Animais , Dopamina/metabolismo , Antagonistas de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Inibidores da Captação de Dopamina/farmacologia , Matriz Extracelular/efeitos dos fármacos , Masculino , Metaloendopeptidases/metabolismo , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Na(+)- and Cl(-)-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl(-) coordination of human serotonin transport have been identified, the role of Cl(-) in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl(-) binding to concentrative neurotransmitter uptake.