RESUMO
The availability of monoamine neurotransmitters in the brain is under the control of dopamine, norepinephrine, and serotonin transporters expressed on the plasma membrane of monoaminergic neurons. By regulating transmitter levels these proteins mediate crucial functions including cognition, attention, and reward, and dysregulation of their activity is linked to mood and psychiatric disorders of these systems. Amphetamine-based transporter substrates stimulate non-exocytotic transmitter efflux that induces psychomotor stimulation, addiction, altered mood, hallucinations, and psychosis, thus constituting a major component of drug neurochemical and behavioral outcomes. Efflux is under the control of transporter post-translational modifications that synergize with other regulatory events, and this review will summarize our knowledge of these processes and their role in drug mechanisms.
Assuntos
Anfetamina , Dopamina , Humanos , Anfetamina/farmacologia , Transporte Biológico , Dopamina/metabolismo , Neurotransmissores , Processamento de Proteína Pós-TraducionalRESUMO
In a consanguineous Pakistani family with two affected individuals, a homozygous variant Gly399Val in the eighth transmembrane domain of the taurine transporter SLC6A6 was identified resulting in a hypomorph transporting capacity of ~15% compared with normal. Three-dimensional modeling of this variant has indicated that it likely causes displacement of the Tyr138 (TM3) side chain, important for transport of taurine. The affected individuals presented with rapidly progressive childhood retinal degeneration, cardiomyopathy and almost undetectable plasma taurine levels. Oral taurine supplementation of 100 mg/kg/day resulted in maintenance of normal blood taurine levels. Following approval by the ethics committee, a long-term supplementation treatment was introduced. Remarkably, after 24-months, the cardiomyopathy was corrected in both affected siblings, and in the 6-years-old, the retinal degeneration was arrested, and the vision was clinically improved. Similar therapeutic approaches could be employed in Mendelian phenotypes caused by the dysfunction of the hundreds of other molecular transporters.
Assuntos
Cardiomiopatias/tratamento farmacológico , Glicoproteínas de Membrana/deficiência , Proteínas de Membrana Transportadoras/deficiência , Degeneração Retiniana/tratamento farmacológico , Taurina/uso terapêutico , Adolescente , Transporte Biológico , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Criança , Feminino , Humanos , Masculino , Linhagem , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologiaRESUMO
The antidepressant-sensitive serotonin (5-HT) transporter (SERT) dictates rapid, high-affinity clearance of the neurotransmitter in both the brain and periphery. In a study of families with multiple individuals diagnosed with autism spectrum disorder (ASD), we previously identified several, rare, missense coding variants that impart elevated 5-HT transport activity, relative to wild-type SERT, upon heterologous expression as well as in ASD subject lymphoblasts. The most common of these variants, SERT Ala56, located in the transporter's cytosolic N-terminus, has been found to confer in transgenic mice hyperserotonemia, an ASD-associated biochemical trait, an elevated brain 5-HT clearance rate, and ASD-aligned behavioral changes. Hyperfunction of SERT Ala56 has been ascribed to a change in 5-HT KM, though the physical basis of this change has yet to be elucidated. Through assessments of fluorescence resonance energy transfer (FRET) between cytosolic N- and C-termini, sensitivity to methanethiosulfonates, and capacity for N-terminal tryptic digestion, we obtain evidence for mutation-induced conformational changes that support an open-outward 5-HT binding conformation in vitro and in vivo. Aspects of these findings were also evident with another naturally occurring C-terminal SERT coding variant identified in our ASD study, Asn605. We conclude that biased conformations of surface resident transporters that can impact transporter function and regulation are an unappreciated consequence of heritable and disease-associated SERT coding variation.
Assuntos
Hipocampo/metabolismo , Neurônios/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Serotonina/metabolismo , Animais , Células CHO , Cricetulus , Fenfluramina/farmacologia , Hipocampo/efeitos dos fármacos , Humanos , Camundongos , Neurônios/efeitos dos fármacos , Conformação Proteica , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
The dopamine transporter (DAT) is a neuronal membrane protein that is responsible for reuptake of dopamine (DA) from the synapse and functions as a major determinant in control of DA neurotransmission. Cocaine and many psychostimulant drugs bind to DAT and block reuptake, inducing DA overflow that forms the neurochemical basis for euphoria and addiction. Paradoxically, however, some ligands such as benztropine (BZT) bind to DAT and inhibit reuptake but do not produce these effects, and it has been hypothesized that differential mechanisms of binding may stabilize specific transporter conformations that affect downstream neurochemical or behavioral outcomes. To investigate the binding mechanisms of BZT on DAT we used the photoaffinity BZT analog [125I]N-[n-butyl-4-(4â´-azido-3â´-iodophenyl)]-4',4â³-difluoro-3α-(diphenylmethoxy)tropane ([125I]GA II 34) to identify the site of cross-linking and predict the binding pose relative to that of previously-examined cocaine photoaffinity analogs. Biochemical findings show that adduction of [125I]GA II 34 occurs at residues Asp79 or Leu80 in TM1, with molecular modeling supporting adduction to Leu80 and a pharmacophore pose in the central S1 site similar to that of cocaine and cocaine analogs. Substituted cysteine accessibility method protection analyses verified these findings, but identified some differences in structural stabilization relative to cocaine that may relate to BZT neurochemical outcomes.
Assuntos
Benzotropina/farmacologia , Sítios de Ligação/efeitos dos fármacos , Cocaína/farmacologia , Dopamina/metabolismo , Relação Estrutura-Atividade , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Humanos , Radioisótopos do Iodo/farmacologiaRESUMO
Dopamine neurotransmission is highly dysregulated by the psychostimulant methamphetamine, a substrate for the dopamine transporter (DAT). Through interactions with DAT, methamphetamine increases extracellular dopamine levels in the brain, leading to its rewarding and addictive properties. Methamphetamine also interacts with the sigma-1 receptor (σ1R), an inter-organelle signaling modulator. Using complementary strategies, we identified a novel mechanism for σ1R regulation of dopamine neurotransmission in response to methamphetamine. We found that σ1R activation prevents methamphetamine-induced, DAT-mediated increases in firing activity of dopamine neurons. In vitro and in vivo amperometric measurements revealed that σ1R activation decreases methamphetamine-stimulated dopamine efflux without affecting basal dopamine neurotransmission. Consistent with these findings, σ1R activation decreases methamphetamine-induced locomotion, motivated behavior, and enhancement of brain reward function. Notably, we revealed that the σ1R interacts with DAT at or near the plasma membrane and decreases methamphetamine-induced Ca2+ signaling, providing potential mechanisms. Broadly, these data provide evidence for σ1R regulation of dopamine neurotransmission and support the σ1R as a putative target for the treatment of methamphetamine addiction.
Assuntos
Dopaminérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Metanfetamina/farmacologia , Receptores sigma/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Comportamento Animal , Células CHO , Células Cultivadas , Cricetulus , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Células HEK293 , Humanos , Locomoção , Camundongos , Camundongos Knockout , Motivação , Técnicas de Patch-Clamp , Receptores sigma/genética , Receptores sigma/metabolismo , Recompensa , Receptor Sigma-1RESUMO
Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility strategies to identify the specific adduction site and binding pose for the crosslinkable, photoactive cocaine analog, RTI 82, which contains a photoactive azide attached at the 2ß position of the tropane pharmacophore. Here, we utilize similar methodology with a different cocaine analog N-[4-(4-azido-3-I-iodophenyl)-butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane, MFZ 2-24, where the photoactive azide is attached to the tropane nitrogen. In contrast to RTI 82, which crosslinked into residue Phe319 of transmembrane domain (TM) 6, our findings show that MFZ 2-24 adducts to Leu80 in TM1 with modeling and biochemical data indicating that MFZ 2-24, like RTI 82, occupies the central S1 binding pocket with the (+)-charged tropane ring nitrogen coordinating with the (-)-charged carboxyl side chain of Asp79. The superimposition of the tropane ring in the three-dimensional binding poses of these two distinct ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site and the importance of the tropane moiety in competitive mechanisms of DA uptake inhibition. These findings set a structure-function baseline for comparison of typical and atypical DAT inhibitors and how their interactions with DAT could lead to the loss of cocaine-like behaviors.
Assuntos
Cocaína/análogos & derivados , Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Tropanos/metabolismo , Animais , Azidas/química , Azidas/metabolismo , Sítios de Ligação , Cocaína/química , Cocaína/metabolismo , Reagentes de Ligações Cruzadas/química , Reagentes de Ligações Cruzadas/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Radioisótopos do Iodo , Células LLC-PK1 , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mapeamento de Peptídeos , Marcadores de Fotoafinidade , Ligação Proteica , Relação Estrutura-Atividade , Transtornos Relacionados ao Uso de Substâncias/psicologia , Suínos , Tropanos/químicaRESUMO
BACKGROUND AND PURPOSE: The psychostimulant cocaine induces complex molecular, cellular and behavioural responses as a consequence of inhibiting presynaptic dopamine, noradrenaline and 5-HT transporters. To elucidate 5-HT transporter (SERT)-specific contributions to cocaine action, we evaluated cocaine effects in the SERT Met172 knock-in mouse, which expresses a SERT coding substitution that eliminates high-affinity cocaine recognition. EXPERIMENTAL APPROACH: We measured the effects of SERT Met172 on cocaine antagonism of 5-HT re-uptake using ex vivo synaptosome preparations and in vivo microdialysis. We assessed SERT dependence of cocaine actions behaviourally through acute and chronic locomotor activation, sensitization, conditioned place preference (CPP) and oral cocaine consumption. We used c-Fos, quantitative RT-PCR and RNA sequencing methods for insights into cellular and molecular networks supporting SERT-dependent cocaine actions. KEY RESULTS: SERT Met172 mice demonstrated functional insensitivity for cocaine at SERT. Although they displayed wild-type levels of acute cocaine-induced hyperactivity or chronic sensitization, the pattern of acute motor activation was different, with a bias toward thigmotaxis. CPP was increased, and a time-dependent elevation in oral cocaine consumption was observed. SERT Met172 mice displayed relatively higher levels of neuronal activation in the hippocampus, piriform cortex and prelimbic cortex (PrL), accompanied by region-dependent changes in immediate early gene expression. Distinct SERT-dependent gene expression networks triggered by acute and chronic cocaine administration were identified, including PrL Akt and nucleus accumbens ERK1/2 signalling. CONCLUSION AND IMPLICATIONS: Our studies reveal distinct SERT contributions to cocaine action, reinforcing the possibility of targeting specific aspects of cocaine addiction by modulation of 5-HT signalling.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Cocaína/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Serotonina/metabolismo , Animais , Estimulantes do Sistema Nervoso Central/administração & dosagem , Cocaína/administração & dosagem , Condicionamento Psicológico , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Atividade Motora , Neurônios , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genéticaRESUMO
Previous studies of transgenic mice carrying a single isoleucine to methionine substitution (I172M) in the serotonin transporter (SERT) demonstrated a loss of sensitivity to multiple antidepressants (ADs) at SERT. However, the ability of AD metabolites to antagonize SERT was not assessed. Here, we evaluated the selectivity and potency of these metabolites for inhibition of SERT in mouse brain-derived synaptosomes and blood platelets from wild-type (I172 mSERT) and the antidepressant-insensitive mouse M172 mSERT. The metabolites norfluoxetine and desmethylsertraline lost the selectivity demonstrated by the parent compounds for inhibition of wild-type mSERT over M172 mSERT, whereas desvenlafaxine and desmethylcitalopram retained selectivity. Furthermore, we show that the metabolite desmethylcitalopram accumulates in the brain and that the metabolites desmethylcitalopram, norfluoxetine, and desvenlafaxine inhibit serotonin uptake in wild-type mSERT at potencies similar to those of their parent compounds, suggesting that metabolites may play a role in effects observed following AD administration in wild-type and M172 mice.
Assuntos
Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Sinaptossomos/efeitos dos fármacos , Animais , Citalopram/análogos & derivados , Citalopram/farmacologia , Fluoxetina/análogos & derivados , Fluoxetina/farmacologia , Camundongos , Sertralina/análogos & derivados , Sertralina/farmacologia , Sinaptossomos/metabolismoRESUMO
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
The dopamine transporter (DAT) functions as a key regulator of dopaminergic neurotransmission via re-uptake of synaptic dopamine (DA). Cocaine binding to DAT blocks this activity and elevates extracellular DA, leading to psychomotor stimulation and addiction, but the mechanisms by which cocaine interacts with DAT and inhibits transport remain incompletely understood. Here, we addressed these questions using computational and biochemical methodologies to localize the binding and adduction sites of the photoactivatable irreversible cocaine analog 3ß-(p-chlorophenyl)tropane-2ß-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([(125)I]RTI 82). Comparative modeling and small molecule docking indicated that the tropane pharmacophore of RTI 82 was positioned in the central DA active site with an orientation that juxtaposed the aryliodoazide group for cross-linking to rat DAT Phe-319. This prediction was verified by focused methionine substitution of residues flanking this site followed by cyanogen bromide mapping of the [(125)I]RTI 82-labeled mutants and by the substituted cysteine accessibility method protection analyses. These findings provide positive functional evidence linking tropane pharmacophore interaction with the core substrate-binding site and support a competitive mechanism for transport inhibition. This synergistic application of computational and biochemical methodologies overcomes many uncertainties inherent in other approaches and furnishes a schematic framework for elucidating the ligand-protein interactions of other classes of DA transport inhibitors.
Assuntos
Azidas/metabolismo , Cocaína/análogos & derivados , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Simulação de Acoplamento Molecular , Animais , Azidas/química , Sítios de Ligação , Cocaína/química , Cocaína/metabolismo , Brometo de Cianogênio/metabolismo , Células HeLa , Humanos , Células LLC-PK1 , Ligantes , Mesilatos/metabolismo , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ratos , Especificidade por Substrato , SuínosRESUMO
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
The serotonin (5-HT) transporter (SERT) is an integral membrane protein that functions to reuptake 5-HT released into the synapse following neurotransmission. This role serves an important regulatory mechanism in neuronal homeostasis. Previous studies have demonstrated that several clinically important antimalarial compounds inhibit serotonin (5-hydroxytryptamine, 5-HT) reuptake. In this study, we examined the details of antimalarial inhibition of 5-HT transport in both Drosophila (dSERT) and human SERT (hSERT) using electrophysiologic, biochemical and computational approaches. We found that the cinchona alkaloids quinidine and cinchonine, which have identical stereochemistry about carbons 8 and 9, exhibited the greatest inhibition of dSERT and hSERT transporter function whereas quinine and cinchonidine, enantiomers of quinidine and cinchonine, respectively, were weaker inhibitors of dSERT and hSERT. Furthermore, SERT mutations known to decrease the binding affinity of many antidepressants affected the cinchona alkaloids in a stereo-specific manner where the similar inhibitory profiles for quinine and cinchonidine (8S,9R) were distinct from quinidine and cinchonine (8R,9S). Small molecule docking studies with hSERT homology models predict that quinine and cinchonidine bind to the central 5-HT binding site (S1) whereas quinidine and cinchonine bind to the S2 site. Taken together, the data presented here support binding of cinchona alkaloids to two different sites on SERT defined by their stereochemistry which implies separate modes of transporter inhibition. Notably, the most potent antimalarial inhibitors of SERT appear to preferentially bind to the S2 site. Our findings provide important insight related to how this class of drugs can modulate the serotonergic system as well as identify compounds that may discriminate between the S1 and S2 binding sites and serve as lead compounds for novel SERT inhibitors.
Assuntos
Antimaláricos/farmacologia , Antagonistas da Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Animais , Alcaloides de Cinchona/farmacologia , Células HeLa , Humanos , Oócitos/metabolismo , Técnicas de Patch-Clamp , RNA Complementar/biossíntese , RNA Complementar/genética , Estereoisomerismo , Relação Estrutura-Atividade , Xenopus laevisRESUMO
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
The SLC6 family of secondary active transporters are integral membrane solute carrier proteins characterized by the Na(+)-dependent translocation of small amino acid or amino acid-like substrates. SLC6 transporters, which include the serotonin, dopamine, norepinephrine, GABA, taurine, creatine, as well as amino acid transporters, are associated with a number of human diseases and disorders making this family a critical target for therapeutic development. In addition, several members of this family are directly involved in the action of drugs of abuse such as cocaine, amphetamines, and ecstasy. Recent advances providing structural insight into this family have vastly accelerated our ability to study these proteins and their involvement in complex biological processes.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/fisiologia , Modelos Moleculares , Família Multigênica/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/fisiologia , Conformação Proteica , Sódio/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Transporte Biológico/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Lipoilação , Filogenia , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismoRESUMO
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.
Assuntos
Asparagina/química , Cloretos/química , Neurotransmissores/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Animais , Cisteína/química , Eletrofisiologia/métodos , Células HeLa , Humanos , Íons , Mutagênese Sítio-Dirigida , Norepinefrina/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp , Plasmídeos/metabolismo , Ratos , Serotonina/metabolismo , Xenopus laevisRESUMO
The plasma membrane serotonin (5-HT) transporter (SERT, SLC6A4) clears 5-HT after release at nerve termini and is targeted by both antidepressant medications and psychostimulants (e.g. MDMA, cocaine). Homology modeling of human SERT (hSERT), based on high resolution structures of the microbial SLC6 family member LeuT(Aa), along with biochemical studies of wild type and mutant transporters, predicts transmembrane (TM) domains 1, 3, 6, and 8 comprise the 5-HT-binding pocket. We utilized the substituted cysteine accessibility method along with surface and site-specific biotinylation to probe TM6 for aqueous accessibility and differential interactions with 5-HT and psychostimulants. Our results are consistent with TM6 being composed of an aqueous-accessible, alpha-helical extracellular domain (TM6a) that is separated by a central, unwound section from a cytoplasmically localized domain (TM6b) with limited aqueous accessibility. The substitution G338C appears to lock hSERT in an outward-facing conformation that, although accessible to aminoethylmethanethiosulfonate-biotin, 5-HT, and citalopram, is incapable of inward 5-HT transport. Transport of 5-HT by G338C can be partially restored by the TM1 mutation Y95F. With regard to methanethiosulfonate (MTS) inactivation of uptake, TM6a Cys mutants demonstrate Na(+)-dependent [2-(trimethylammonium)ethyl]-MTS sensitivity. Studies with the centrally located substitution S336C reveal features of a common binding pocket for 5-HT and 3,4-methylenedioxymethamphetamine (MDMA). Interestingly, the substitution I333C reveals an MDMA-induced conformation not observed with 5-HT. In the context of prior studies on TM1, our findings document shared and unique features of TM6 contributing to hSERT aqueous accessibility, ligand recognition, and conformational dynamics.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Serotonina/química , Transporte Biológico , Citoplasma/metabolismo , Humanos , Cinética , Modelos Biológicos , Mutagênese Sítio-Dirigida , Mutação , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Serotoninérgicos/farmacologia , Água/químicaRESUMO
To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuT(Aa)) structure reported by Yamashita et al. (Nature 2005;437:215-223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuT(Aa) is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to critically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuT(Aa) structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 A of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected.