RESUMO
Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation (OC) transporter that transports a variety of endogenous biogenic amines and xenobiotic cations. Previous radiotracer uptake studies showed that PMAT-mediated OC transport is sensitive to changes in membrane potential and extracellular pH, but the precise role of membrane potential and protons on PMAT-mediated OC transport is unknown. Here, we characterized the electrophysiological properties of PMAT in Xenopus laevis oocytes using a two-microelectrode voltage-clamp approach. PMAT-mediated histamine uptake is associated with inward currents under voltage-clamp conditions, and the currents increased in magnitude as the holding membrane potential became more negative. A similar effect was also observed for another cation, nicotine. Substrate-induced currents were largely independent of Na+ but showed strong dependence on membrane potential and pH of the perfusate. Detailed kinetic analysis of histamine uptake revealed that the energizing effect of membrane potentials on PMAT transport is mainly due to an augmentation of Imax with little effect on K0.5. At most holding membrane potentials, Imax at pH 6.0 is approximately 3- to 4-fold higher than that at pH 7.5, whereas K0.5 is not dependent on pH. Together, these data unequivocally demonstrate PMAT as an electrogenic transporter and establish the physiological inside-negative membrane potential as a driving force for PMAT-mediated OC transport. The important role of membrane potential and pH in modulating the transport activity of PMAT toward OCs suggests that the in vivo activity of PMAT could be regulated by pathophysiological processes that alter physiological pH or membrane potential.
Assuntos
Membrana Celular/fisiologia , Proteínas de Transporte de Nucleosídeo Equilibrativas/fisiologia , Proteínas de Transporte de Cátions Orgânicos/fisiologia , Proteínas de Xenopus/fisiologia , Animais , Fenômenos Eletrofisiológicos/fisiologia , Feminino , Humanos , Potenciais da Membrana/fisiologia , Xenopus laevisRESUMO
Plasma membrane monoamine transporter (PMAT) is a new polyspecific transporter that interacts with a wide range of structurally diverse organic cations. To map the physicochemical descriptors of cationic compounds that allow interaction with PMAT, we systematically analyzed the interactions between PMAT and three series of structural analogs of known organic cation substrates including phenylalkylamines, n-tetraalkylammonium (n-TAA) compounds, and ß-carbolines. Our results showed that phenylalkylamines with a distance between the aromatic ring and the positively charged amine nitrogen atom of â¼6.4 Å confer optimal interactions with PMAT, whereas studies with n-TAA compounds revealed an excellent correlation between IC(50) values and hydrophobicity. The five ß-carbolines that we tested, which possess a pyridinium-like structure and are structurally related to the neurotoxin 1-methyl-4-phenylpyridinium, inhibited PMAT with high affinity (IC(50) values of 39.1-65.5 µM). Cytotoxicity analysis further showed that cells expressing PMAT are 14- to 15-fold more sensitive to harmalan and norharmanium, suggesting that these two ß-carbolines are also transportable substrates of PMAT. We then used computer-aided modeling to generate qualitative and quantitative three-dimensional pharmacophore models on the basis of 23 previously reported and currently identified PMAT inhibitors and noninhibitors. These models are characterized by a hydrogen bond donor and two to three hydrophobic features with distances between the hydrogen bond donor and hydrophobic features ranging between 5.20 and 7.02 Å. The consistency between the mapping results and observed PMAT affinity of a set of test compounds indicates that the models performed well in inhibitor prediction and could be useful for future virtual screening of new PMAT inhibitors.
Assuntos
Proteínas de Transporte de Nucleosídeo Equilibrativas/antagonistas & inibidores , Proteínas de Transporte de Nucleosídeo Equilibrativas/química , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Compostos de Anilina/farmacologia , Animais , Derivados de Benzeno/química , Derivados de Benzeno/metabolismo , Derivados de Benzeno/farmacologia , Carbolinas/química , Carbolinas/metabolismo , Carbolinas/farmacologia , Linhagem Celular , Clonagem Molecular , Cães , Avaliação Pré-Clínica de Medicamentos , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Conformação Molecular , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/metabolismo , Compostos de Amônio Quaternário/farmacologia , Relação Estrutura-AtividadeRESUMO
Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter in the solute carrier 29 (SLC29) family. Previous studies suggested that the major substrate recognition sites are located within transmembrane domains (TM) 1-6, and interaction of PMAT with organic cations may involve aromatic residues. In this study, we analyzed the roles of tyrosine and tryptophan residues located within TM1-6 with a goal of identifying potential residues involved in substrate recognition and translocation. The six tyrosines and one tryptophan in this region were each mutated to alanine followed by analysis of the mutant's membrane localization and transport activity toward 1-methyl-4-phenylpyridinium (MPP(+)), serotonin (5-HT), and dopamine. Two mutants, Y85A and Y112A, exhibited normal cell surface expressions but lost their transport activities toward organic cations. At position Y85, aromatic substitution with phenylalanine or tryptophan fully restored organic cation transport activity. Interestingly, at position Y112, phenylalanine substitution was not allowed. Tryptophan substitution at Y112 partially restored transport activity toward 5-HT and dopamine but severely impaired MPP(+) transport. Detailed kinetic analyses revealed that tryptophan substitution at Y85 and Y112 affected the apparent binding affinity (K(m)) and maximal transport velocity (V(max)) in a substrate-dependent manner. Together, our data suggest that Y85 and Y112 are important molecular determinants for PMAT function, and Y112 is indispensable for optimal interaction with organic cation substrates. Our analyses also suggest the involvement of transmembrane domains 1 and 2 in forming the substrate permeation pathway of PMAT.
Assuntos
Proteínas de Transporte de Cátions Orgânicos/química , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Tirosina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico , Células Cultivadas , Cães , Proteínas de Transporte de Nucleosídeo Equilibrativas/química , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Humanos , Cinética , Microscopia Confocal , Dados de Sequência Molecular , Proteínas de Transporte de Cátions Orgânicos/genética , Relação Estrutura-Atividade , Tirosina/genéticaRESUMO
Depression and use of antidepressant medications are both associated with increased risk of obesity, potentially attributed to a reduced serotonin transporter (SERT) function. However, how SERT deficiency promotes obesity is unknown. Here, we demonstrated that SERT -/- mice display abnormal fat accumulation in both white and brown adipose tissues, glucose intolerance and insulin resistance while exhibiting suppressed aromatase (Cyp19a1) expression and reduced circulating 17ß-estradiol levels. 17ß-estradiol replacement in SERT -/- mice reversed the obesity and glucose intolerance, supporting a role for estrogen in SERT deficiency-associated obesity and glucose intolerance. Treatment of wild type mice with paroxetine, a chemical inhibitor of SERT, also resulted in Cyp19a1 suppression, decreased circulating 17ß-estradiol levels, abnormal fat accumulation, and glucose intolerance. Such effects were not observed in paroxetine-treated SERT -/- mice. Conversely, pregnant SERT -/- mice displayed normalized estrogen levels, markedly reduced fat accumulation, and improved glucose tolerance, which can be eliminated by an antagonist of estrogen receptor α (ERα). Together, these findings support that estrogen suppression is involved in SERT deficiency-induced obesity and glucose intolerance, and suggest approaches to restore 17ß-estradiol levels as a novel treatment option for SERT deficiency associated obesity and metabolic abnormalities.
Assuntos
Estradiol/metabolismo , Intolerância à Glucose/fisiopatologia , Obesidade/fisiopatologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/deficiência , Animais , Aromatase/metabolismo , Resistência à Insulina , Camundongos , Camundongos KnockoutRESUMO
ING1b is a candidate tumor suppressor that can stimulate the transcriptional activity of p53 and inhibit cell proliferation. The molecular basis of how ING1b activates p53 function remains unclear. Here we show that ING1b could stimulate the activity of p53 by increasing the level and stability of the p53 protein. The stabilization and activation of p53 by ING1b could be reversed by MDM2 in a dose-dependent manner. Conversely, ING1b could reverse the inhibition and degradation of p53 caused by MDM2 in a dose-dependent manner. Furthermore, ING1b and MDM2 bound to p53 in a mutually exclusive manner. In agreement with these observations, we found that similarly to MDM2, ING1b binds to the NH(2)-terminal region of p53. These data suggest a model in which ING1b disrupts the interaction between p53 and MDM2, leading to the stabilization of p53 and growth inhibition.
Assuntos
Proteínas/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Ligação Competitiva , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Proteínas de Ciclo Celular , Divisão Celular/fisiologia , Proteínas de Ligação a DNA , Genes Supressores de Tumor , Humanos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Nucleares , Isoformas de Proteínas , Proteínas/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de TumorRESUMO
Camptothecin and Adriamycin are clinically important inhibitors for topoisomerase (Topo) I and Topo II, respectively. The ataxia-telangiectasia mutated (ATM) product is essential for ionizing radiation-induced DNA damage responses, but the role of ATM in Topo poisons-induced checkpoints remains unresolved. We found that distinct mechanisms are involved in the activation of different cell cycle checkpoints at different concentrations of Adriamycin and camptothecin. Adriamycin promotes the G(1) checkpoint through activation of the p53-p21(CIP1/WAF1) pathway and decrease of pRb phosphorylation. Phosphorylation of p53(Ser20) after Adriamycin treatment is ATM dependent, but is not required for the full activation of p53. The G(1) checkpoint is dependent on ATM at low doses but not at high doses of Adriamycin. In contrast, the Adriamycin-induced G(2) checkpoint is independent on ATM but sensitive to caffeine. Adriamycin inhibits histone H3(Ser10) phosphorylation through inhibitory phosphorylation of CDC2 at low doses and down-regulation of cyclin B1 at high doses. The camptothecin-induced intra-S checkpoint is partially dependent on ATM, and is associated with inhibitory phosphorylation of cyclin-dependent kinase 2 and reduction of BrdUrd incorporation after mid-S phase. Finally, apoptosis associated with high doses of Adriamycin or camptothecin is not influenced by the absence of ATM. These data indicate that the involvement of ATM following treatment with Topo poisons differs extensively with dosage and for different cell cycle checkpoints.
Assuntos
Dano ao DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genes cdc/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Inibidores da Topoisomerase I , Inibidores da Topoisomerase II , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/farmacologia , Camptotecina/antagonistas & inibidores , Camptotecina/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Relação Dose-Resposta a Droga , Doxorrubicina/antagonistas & inibidores , Doxorrubicina/farmacologia , Fase G1/efeitos dos fármacos , Fase G2/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fase S/efeitos dos fármacos , Proteínas Supressoras de TumorRESUMO
Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter belonging to the equilibrative nucleoside transporter (ENT) family. Despite its distinct substrate specificity from the classic nucleoside transporters ENT1 and 2, PMAT appears to share similar protein architecture with ENT1/2 and retains low affinity binding to classic ENT inhibitors such as nitrobenzylmercaptopurine riboside (NBMPR) and the coronary vasodilators dilazep and dipyridamole. Here we investigated the role of residue Ile89, a position known to be important for ENT interaction with dilazep, dipyridamole, and nucleoside substrates, in PMAT transport function and its interaction with classic ENT inhibitors using Madin-Darby canine kidney (MDCK) cells stably expressing human PMAT. Substitution of Ile89 in PMAT with Met, the counterpart residue in ENT1, resulted in normal plasma membrane localization and protein expression. Transport kinetic analysis revealed that I89M mutant had a 2.7-fold reduction in maximal transport velocity (V(max)) with no significant change in apparent binding affinity (K(m)) towards the prototype PMAT substrate 1-methyl-4-phenylpyridinium (MPP+), suggesting that I89 is an important determinant for the catalytic activity of PMAT. Dose-dependent inhibition studies further showed that the I89M mutation significantly increased PMAT's sensitivity to dilazep by 2.5-fold without affecting its sensitivity to dipyridamole and NBMPR. Located at the extracellular end of transmembrane domain 1 of PMAT, I89 may occupy an important position close to the substrate permeation pathway and may be involved in direct interaction with the vasodilator dilazep.
Assuntos
Dilazep/farmacologia , Proteínas de Transporte de Nucleosídeo Equilibrativas/antagonistas & inibidores , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Isoleucina/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Cães , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Humanos , Transporte de Íons/genética , Isoleucina/genética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , MutaçãoRESUMO
The choroid plexus (CP) is a highly vascularized tissue in the brain ventricles and acts as the blood-cerebrospinal fluid (CSF) barrier (BCSFB). A main function of the CP is to secrete CSF, which is accomplished by active transport of small ions and water from the blood side to the CSF side. The CP also supplies the brain with certain nutrients, hormones, and metal ions, while removing metabolites and xenobiotics from the CSF. Numerous membrane transporters are expressed in the CP in order to facilitate the solute exchange between the blood and the CSF. The solute carrier (SLC) superfamily represents a major class of transporters in the CP that constitutes the molecular mechanisms for CP function. Recently, we systematically and quantitatively examined Slc gene expression in 20 anatomically comprehensive brain areas in the adult mouse brain using high-quality in situ hybridization data generated by the Allen Brain Atlas. Here we focus our analysis on Slc gene expression at the BCSFB using previously obtained data. Of the 252 Slc genes present in the mouse brain, 202 Slc genes were found at detectable levels in the CP. Unsupervised hierarchical cluster analysis showed that the CP Slc gene expression pattern is substantially different from the other 19 analyzed brain regions. The majority of the Slc genes in the CP are expressed at low to moderate levels, whereas 28 Slc genes are present in the CP at the highest levels. These highly expressed Slc genes encode transporters involved in CSF secretion, energy production, and transport of nutrients, hormones, neurotransmitters, sulfate, and metal ions. In this review, the functional characteristics and potential importance of these Slc transporters in the CP are discussed, with particular emphasis on their localization and physiological functions at the BCSFB.
RESUMO
Plasma membrane monoamine transporter (PMAT) is a novel polyspecific organic cation transporter that transports organic cations and the purine nucleoside, adenosine. PMAT is expressed in the kidney, but the specific localization and function of this transporter in renal cells are unclear. In this study, we developed a polyclonal antibody toward a 14-amino acid sequence in the last intracellular loop of PMAT and determined the precise cellular localization of PMAT in human and rat kidneys. Surprisingly, we found that the PMAT protein was predominantly expressed in the glomerulus with minimal expression in tubular cells. Within the glomerulus, dual-color immunofluorescence labeling showed that the PMAT protein was specifically localized to the visceral glomerular epithelial cells, i.e., podocytes. There was no significant PMAT immunoreactivity in mesangial or glomerular endothelial cells. We further showed that puromycin aminonucleoside (PAN), a classic podocyte toxin that induces massive proteinuria and severe glomerulopathy, is transported by PMAT. Expression of PMAT in Madin-Darby canine kidney cells significantly increased cell sensitivity to PAN. Decynium 22, a potent PMAT inhibitor, abolished PAN toxicity in PMAT-expressing cells. Together, our data suggest that PMAT is specifically expressed in podocytes and may play an important role in PAN-induced kidney injury.
Assuntos
Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Regulação da Expressão Gênica/fisiologia , Nefropatias/induzido quimicamente , Podócitos/metabolismo , Puromicina Aminonucleosídeo/toxicidade , Animais , Anticorpos/imunologia , Linhagem Celular , Cães , Células Epiteliais/metabolismo , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Humanos , Concentração de Íons de Hidrogênio , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Puromicina Aminonucleosídeo/metabolismo , RatosRESUMO
Inhibition of cyclin-dependent kinases (CDKs) by Thr14/Tyr15 phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G2 DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominant-negative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr14/Tyr15 phosphorylation in normal cell cycle and during the DNA damage checkpoint.