RESUMO
The K+/Cl- cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl- levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.
Assuntos
Neurônios/metabolismo , Simportadores/metabolismo , Animais , Apoptose , Cloretos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurogênese/efeitos dos fármacos , Neurônios/fisiologia , Potássio/metabolismo , Cultura Primária de Células , Receptores de GABA/metabolismo , Convulsões , Simportadores/fisiologia , Ácido gama-Aminobutírico/metabolismo , Cotransportadores de K e Cl-RESUMO
The decreased expression of the KCC2 membrane transporter in subicular neurons has been proposed to be a key epileptogenic event in temporal lobe epilepsy (TLE). Here, we have addressed this question in a reduced model in vitro and have studied the properties and mechanistic involvement of a major class of interneurons, that is, parvalbumin-expressing cells (PVs). When exposed to the KCC2 blocker VU0463271, mouse subicular slices generated hypersynchronous discharges that could be recorded electrophysiologically and visualized as clusters of co-active neurons with calcium imaging. The pharmacological profile of these events resembled interictal-like discharges in human epileptic tissue because of their dependence on GABAA and AMPA receptors. On average, PVs fired before pyramidal cells (PCs) and the area of co-active clusters was comparable to the individual axonal spread of PVs, suggesting their mechanistic involvement. Optogenetic experiments confirmed this hypothesis, as the flash-stimulation of PVs in the presence of VU0463271 initiated interictal-like discharges, whereas their optogenetic silencing suppressed network hyper-excitability. We conclude that reduced KCC2 activity in subicular networks in vitro is sufficient to induce interictal-like activity via altered GABAergic signaling from PVs without other epilepsy-related changes. This conclusion supports an epileptogenic role for impaired subicular KCC2 function during the progression of TLE.
Assuntos
Hipocampo/fisiopatologia , Interneurônios/fisiologia , Parvalbuminas/metabolismo , Convulsões/fisiopatologia , Simportadores/fisiologia , Animais , Axônios/efeitos dos fármacos , Fenômenos Eletrofisiológicos , Hipocampo/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Masculino , Camundongos , Rede Nervosa/efeitos dos fármacos , Optogenética , Estimulação Luminosa , Células Piramidais/efeitos dos fármacos , Simportadores/antagonistas & inibidores , Cotransportadores de K e Cl-RESUMO
NaCT/SLC13A5 is a Na+-coupled transporter for citrate in hepatocytes, neurons, and testes. It is also called mINDY (mammalian ortholog of 'I'm Not Dead Yet' in Drosophila). Deletion of Slc13a5 in mice leads to an advantageous phenotype, protecting against diet-induced obesity, and diabetes. In contrast, loss-of-function mutations in SLC13A5 in humans cause a severe disease, EIEE25/DEE25 (early infantile epileptic encephalopathy-25/developmental epileptic encephalopathy-25). The difference between mice and humans in the consequences of the transporter deficiency is intriguing but probably explainable by the species-specific differences in the functional features of the transporter. Mouse Slc13a5 is a low-capacity transporter, whereas human SLC13A5 is a high-capacity transporter, thus leading to quantitative differences in citrate entry into cells via the transporter. These findings raise doubts as to the utility of mouse models to evaluate NaCT biology in humans. NaCT-mediated citrate entry in the liver impacts fatty acid and cholesterol synthesis, fatty acid oxidation, glycolysis, and gluconeogenesis; in neurons, this process is essential for the synthesis of the neurotransmitters glutamate, GABA, and acetylcholine. Thus, SLC13A5 deficiency protects against obesity and diabetes based on what the transporter does in hepatocytes, but leads to severe brain deficits based on what the transporter does in neurons. These beneficial versus detrimental effects of SLC13A5 deficiency are separable only by the blood-brain barrier. Can we harness the beneficial effects of SLC13A5 deficiency without the detrimental effects? In theory, this should be feasible with selective inhibitors of NaCT, which work only in the liver and do not get across the blood-brain barrier.
Assuntos
Simportadores/deficiência , Animais , Barreira Hematoencefálica , Osso e Ossos/metabolismo , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico/genética , Esmalte Dentário/metabolismo , Diabetes Mellitus/metabolismo , Transportadores de Ácidos Dicarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Dicarboxílicos/deficiência , Transportadores de Ácidos Dicarboxílicos/fisiologia , Modelos Animais de Doenças , Proteínas de Drosophila/fisiologia , Fígado Gorduroso/metabolismo , Feminino , Células Germinativas/metabolismo , Hepatócitos/metabolismo , Humanos , Recém-Nascido , Transporte de Íons , Longevidade/genética , Masculino , Camundongos , Camundongos Knockout , Mutação , Neoplasias/metabolismo , Neurônios/metabolismo , Conformação Proteica , Espasmos Infantis/genética , Especificidade da Espécie , Simportadores/antagonistas & inibidores , Simportadores/genética , Simportadores/fisiologiaRESUMO
BACKGROUND: Somatic mutations of the TP53 gene occur frequently in pancreatic ductal adenocarcinoma (PDA). Solute carrier family 45 member A4 (SLC45A4) is a H+ -dependent sugar cotransporter. The role of SLC45A4 in PDA, especially in TP53 mutant PDA, remains poorly understood. METHODS: We explored the TCGA datasets to identify oncogenes in TP53 mutant PDA. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium], colony formation and 5-ethynyl-2'-deoxyuridine (Edu) assays were performed to investigate the function of SLC45A4 in vitro. Glucose consumption, lactate production and ATP production were detected to evaluate glucose utilization. Extracellular acidification rate and oxygen consumption rate assays were used to evaluate glycolysis and oxidative phosphorylation. The subcutaneous xenotransplantation models were conducted to explore the function of SLC45A4 in vivo. RNA-sequencing and gene set enrichment analysis were employed to explore the biological alteration caused by SLC45A4 knockdown. Western blotting was performed to evaluate the activation of glycolysis, as well as the AMPK pathway and autophagy. RESULTS: SLC45A4 was overexpressed in PDA for which the expression was significantly higher in TP53 mutant PDA than that in wild-type PDA tissues. Moreover, high level of SLC45A4 expression was tightly associated with poor clinical outcomes in PDA patients. Silencing SLC45A4 inhibited proliferation in TP53 mutant PDA cells. Knockdown of SLC45A4 reduced glucose uptake and ATP production, which led to activation of autophagy via AMPK/ULK1 pathway. Deleting SLC45A4 in TP53 mutant HPAF-II cells inhibited the growth of xenografts in nude mice. CONCLUSIONS: The present study found that SLC45A4 prevents autophagy via AMPK/ULK1 axis in TP53 mutant PDA, which may be a promising biomarker and therapeutic target in TP53 mutant PDA.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia , Carcinoma Ductal Pancreático/fisiopatologia , Glucose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pancreáticas/fisiopatologia , Simportadores/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Glicólise , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Transdução de Sinais , Transplante Heterólogo , Proteína Supressora de Tumor p53/genéticaRESUMO
Stroke severely impairs quality of life and has a high mortality rate. On the other hand, dietary docosahexaenoic acid (DHA) prevents neuronal damage. In this review, we describe the effects of dietary DHA on ischemic stroke-associated neuronal damage and its role in stroke prevention. Recent epidemiological studies have been conducted to analyze stroke prevention through DHA intake. The effects of dietary intake and supply of DHA to neuronal cells, DHA-mediated inhibition of neuronal damage, and its mechanism, including the effects of the DHA metabolite, neuroprotectin D1 (NPD1), were investigated. These studies revealed that DHA intake was associated with a reduced risk of stroke. Moreover, studies have shown that DHA intake may reduce stroke mortality rates. DHA, which is abundant in fish oil, passes through the blood-brain barrier to accumulate as a constituent of phospholipids in the cell membranes of neuronal cells and astrocytes. Astrocytes supply DHA to neuronal cells, and neuronal DHA, in turn, activates Akt and Raf-1 to prevent neuronal death or damage. Therefore, DHA indirectly prevents neuronal damage. Furthermore, NDP1 blocks neuronal apoptosis. DHA, together with NPD1, may block neuronal damage and prevent stroke. The inhibitory effect on neuronal damage is achieved through the antioxidant (via inducing the Nrf2/HO-1 system) and anti-inflammatory effects (via promoting JNK/AP-1 signaling) of DHA.
Assuntos
Dano Encefálico Crônico/prevenção & controle , Ácidos Docosa-Hexaenoicos/uso terapêutico , AVC Isquêmico/dietoterapia , Degeneração Neural/prevenção & controle , Acidente Vascular Cerebral/prevenção & controle , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/uso terapêutico , Antioxidantes/administração & dosagem , Antioxidantes/farmacocinética , Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Transporte Biológico , Barreira Hematoencefálica , Dano Encefálico Crônico/etiologia , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/farmacocinética , Gorduras na Dieta/uso terapêutico , Ácidos Docosa-Hexaenoicos/administração & dosagem , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Docosa-Hexaenoicos/farmacocinética , Ácidos Docosa-Hexaenoicos/farmacologia , Proteínas de Ligação a Ácido Graxo/fisiologia , Óleos de Peixe/administração & dosagem , Óleos de Peixe/farmacocinética , Humanos , Incidência , AVC Isquêmico/complicações , AVC Isquêmico/epidemiologia , Lipídeos de Membrana/metabolismo , Camundongos , Proteínas de Neoplasias/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Óleos de Plantas/administração & dosagem , Óleos de Plantas/farmacocinética , Transdução de Sinais/efeitos dos fármacos , Simportadores/deficiência , Simportadores/fisiologia , Ácido alfa-Linolênico/farmacocinéticaRESUMO
Abnormal glycolytic metabolism contributes to angiogenic sprouting involved in atherogenesis. We investigated the potential anti-angiogenic properties of specific 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) inhibitors in endothelial cells (ECs). ECs were treated with PFKFB3 inhibitors (named PA-1 and PA-2) and their effects on metabolic and functional characteristics of ECs were investigated. The anti-glycolytic compound 3-(pyridinyl)- 1-(4-pyridinyl)- 2-propen-1-one (3PO) was used as reference compound. PFKFB3 expression and activity (IC50 about 3-21 nM) was inhibited upon treatment with both compounds. Glucose uptake and lactate export were measured using commercial assays and showed a partial reduction up to 40%. PFKFB3 inhibition increased intracellular lactate accumulation, and reduced expression of monocarboxylate transporters-1 (MCT1) and MCT4. Furthermore, endothelial cell migration and proliferation assays demonstrated significant reduction upon treatment with both compounds. Matrix- metalloproteinase (MMP) activity, measured by gelatin zymography, and expression was significantly reduced (up to 25%). In addition, PA compounds downregulated the expression of VCAM-1, VE-cadherin, VEGFa, VEGFR2, TGF-ß, and IL-1ß, in inflamed ECs. Finally, PA-1 and PA-2 treatment impaired the formation of angiogenic sprouts measured by both morphogenesis and spheroid-based angiogenesis assays. Our data demonstrate that the anti-glycolytic PA compounds may affect several steps involved in angiogenesis. Targeting the key glycolytic enzyme PFKFB3 might represent an attractive therapeutic strategy to improve the efficacy of cancer treatments, or to be applied in other pathologies where angiogenesis is a detrimental factor.
Assuntos
Inibidores da Angiogênese/farmacologia , Fosfofrutoquinase-2/antagonistas & inibidores , Células Cultivadas , Humanos , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/fisiologia , Proteínas Musculares/fisiologia , NAD/metabolismo , Neovascularização Patológica/tratamento farmacológico , Simportadores/fisiologiaRESUMO
One hallmark of renal cell carcinoma (RCC) is metabolic reprogramming, which involves elevation of glycolysis and upregulation of lipid metabolism. However, the mechanism of metabolic reprogramming is incompletely understood. Monocarboxylate transporter 1 (MCT1) promotes transport for lactate and pyruvate, which are crucial for cell metabolism. The aim of present study was to investigate the function of MCT1 on RCC development and its mechanism on metabolic reprogramming. The results showed that MCT1 messenger RNA and protein levels significantly increased in cancer tissues of ccRCC compared to normal tissue. MCT1 was further found to mainly located in the cell membrane of RCC. The knockdown of MCT1 by RNAi significantly inhibited proliferation and migration of 786-O and ACHN cells. MCT1 also induced the expressions of proliferation marker Ki-67 and invasion marker SNAI1. Moreover, we also showed that acetate treatment could upregulate the expression of MCT1, but not other MCT isoforms. On the other hand, MCT1 was involved in acetate transport and intracellular histone acetylation. In summary, this study revealed that MCT1 is abnormally high in ccRCC and promotes cancer development. The regulatory effect of MCT1 on cell proliferation and invasion maybe mediated by acetate transport.
Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Transportadores de Ácidos Monocarboxílicos/fisiologia , Simportadores/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , HumanosRESUMO
Stressors such as soil salinity and dehydration are major constraints on plant growth, causing worldwide crop losses. Compounding these insults, increasing climate volatility requires adaptation to fluctuating conditions. Salinity stress responses are relatively well understood in Arabidopsis thaliana, making this system suited for the rapid molecular dissection of evolutionary mechanisms. In a large-scale genomic analysis of Catalonian A. thaliana, we resequenced 77 individuals from multiple salinity gradients along the coast and integrated these data with 1,135 worldwide A. thaliana genomes for a detailed understanding of the demographic and evolutionary dynamics of naturally evolved salinity tolerance. This revealed that Catalonian varieties adapted to highly fluctuating soil salinity are not Iberian relicts but instead have immigrated to this region more recently. De novo genome assembly of three allelic variants of the high-affinity K+ transporter (HKT1;1) locus resolved structural variation between functionally distinct alleles undergoing fluctuating selection in response to seasonal changes in soil salinity. Plants harboring alleles responsible for low root expression of HKT1;1 and consequently high leaf sodium (HKT1;1HLS ) were migrants that have moved specifically into areas where soil sodium levels fluctuate widely due to geography and rainfall variation. We demonstrate that the proportion of plants harboring HKT1;1HLS alleles correlates with soil sodium level over time, HKT1;1HLS -harboring plants are better adapted to intermediate levels of salinity, and the HKT1;1HLS allele clusters with high-sodium accumulator accessions worldwide. Together, our evidence suggests that HKT1;1 is under fluctuating selection in response to climate volatility and is a worldwide determinant in adaptation to saline conditions.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Simportadores/genética , Simportadores/metabolismo , Adaptação Biológica/genética , Adaptação Fisiológica/genética , Alelos , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Frequência do Gene/genética , Transporte de Íons , Salinidade , Tolerância ao Sal , Sódio/metabolismo , Cloreto de Sódio , Solo , Simportadores/fisiologiaRESUMO
Major facilitator superfamily domain-containing protein-2a (Mfsd2a) which was considered as an orphan transporter has recently gained attention for its regulatory role in the maintenance of proper functioning of the blood-brain barrier. Besides the major role of Mfsd2a in maintaining the barrier function, increasing evidence has emerged with regard to the contributions of Mfsd2a to various biological processes such as transport, cell fusion, cell cycle, inflammation and regeneration, managing tumor growth, functioning of other organs with barrier functions or responses to injury. The purpose of this article is to review the different roles of Mfsd2a and its involvement in the physiological and pathophysiological processes primarily in the central nervous system and throughout the mammalian body under the lights of the current literature.
Assuntos
Barreira Hematoencefálica/fisiologia , Simportadores/fisiologia , Animais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Barreira Hematorretiniana/fisiologia , Encéfalo/fisiologia , Feminino , Humanos , Placenta/fisiologia , Gravidez , Transdução de Sinais/fisiologia , Simportadores/metabolismoRESUMO
Solute carrier (SLC) transporters play important roles in regulating the movement of small molecules and ions across cellular membranes. In mammals, they play an important role in regulating the uptake of nutrients and vitamins from the diet, and in controlling the distribution of their metabolic intermediates within the cell. Several SLC families also play an important role in drug transport and strategies are being developed to hijack SLC transporters to control and regulate drug transport within the body. Through the addition of amino acid and peptide moieties several novel antiviral and anticancer agents have been developed that hijack the proton-coupled oligopeptide transporters, PepT1 (SCL15A1) and PepT2 (SLC15A2), for improved intestinal absorption and renal retention in the body. A major goal is to understand the rationale behind these successes and expand the library of prodrug molecules that utilise SLC transporters. Recent co-crystal structures of prokaryotic homologues of the human PepT1 and PepT2 transporters have shed important new insights into the mechanism of prodrug recognition. Here, I will review recent developments in our understanding of ligand recognition and binding promiscuity within the SLC15 family, and discuss current models for prodrug recognition.
Assuntos
Transportador 1 de Peptídeos/fisiologia , Pró-Fármacos/farmacologia , Simportadores/fisiologia , Animais , Transporte Biológico , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Oligopeptídeos/química , Transportador 1 de Peptídeos/química , Simportadores/química , Valaciclovir/farmacologia , Valganciclovir/farmacologiaRESUMO
A single-nucleotide polymorphism in the promoter region of the Matrix Metalloproteinase-9 (MMP9) gene, rs3918242, has been shown to affect MMP9 expression in macrophages and was associated with schizophrenia by two independent groups. However, rs3918242's effects on MMP9 expression were not replicable in cell lines or brain tissue. Additionally, publically available data indicate that rs3918242 genotype is related not to MMP9 expression, but rather to expression of SLC12A5, a nearby gene coding for a K+/Cl- cotransporter, whose expression has also been related to schizophrenia. Here, we studied brain structure and function in healthy participants stratified by rs3918242 genotype using structural MRI (N = 298), functional MRI during an N-back working memory task (N = 554), and magnetoencephalography (MEG) during the same task (N = 190). We found rs3918242 was associated with gray matter volume (GMV) in the insula and dorsolateral prefrontal cortex bilaterally, closely replicated in discovery and replication samples; and with inferior parietal lobule (IPL) GMV when the samples were meta-analytically combined. Additionally, using both fMRI and MEG, rs3918242 was associated with right IPL working memory-related activation, replicated in two cohorts and across imaging modalities. These convergent results provide further impetus for examinations of the relationship of SLC12A5 with brain structure and function in neuropsychiatric disease.
Assuntos
Encéfalo/anatomia & histologia , Encéfalo/fisiologia , Expressão Gênica , Simportadores/fisiologia , Adulto , Mapeamento Encefálico , Feminino , Genótipo , Humanos , Imageamento por Ressonância Magnética , Magnetoencefalografia , Masculino , Memória de Curto Prazo/fisiologia , Polimorfismo de Nucleotídeo Único , Simportadores/genéticaRESUMO
Bovine peptide transporter 2 (bPepT2), which mediates the absorption of di- and tripeptides in the bovine mammary gland, was predicted to contain multiple putative N-glycosylation sites of asparagine residues. N-Linked glycosylation is proven to be essential for the folding, stability, localization, and substrate binding of nutrient transporters and could therefore potentially have an essential role in the function of bPepT2. This study investigated the effect of mutagenesis of N-glycosylation sites on the transport function of bPepT2 in Chinese hamster ovary (CHO) cells. The bPepT2 cDNA was cloned and sequenced. BioXM (http://202.195.246.60/BioXM/) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) software were used to predict the AA composition and transmembrane domain of bPepT2, respectively. The AA sequence of bPepT2 was predicted to have 12 transmembrane domains, with a large extracellular loop between the ninth and tenth transmembrane domains. All 5 putative N-glycosylation sites in this loop were altered by site-directed mutagenesis, and the mutant construct was transfected into CHO cells for transport activity assay. Compared with the wild type, the bPepT2 mutant had significantly lower uptake activity of ß-alanyl-l-lysyl-Nε-7-amino-4-methyl-coumarin-3-acetic acid (ß-Ala-Lys-AMCA), a model dipeptide. Treatment with tunicamycin, an inhibitor of N-linked glycosylation, reduced the uptake of ß-Ala-Lys-AMCA in CHO cells relative to the control group. Kinetic studies indicated that the Michaelis constant of bPepT2 was not affected by the mutation (98.03 ± 8.30 and 88.33 ± 4.23 µM for the wild type and the mutant, respectively), but the maximum transport activity was significantly reduced (40.29 ± 8.30 and 13.02 ± 2.95 pmol/min per milligram of protein for the wild type and the mutant, respectively). In summary, this study demonstrated that N-glycosylation is critical for the function of bPepT2.
Assuntos
Bovinos/metabolismo , Simportadores/fisiologia , Animais , Células CHO , Bovinos/genética , Cricetulus , Glicosilação , Mutagênese Sítio-Dirigida , Transporte Proteico/efeitos dos fármacos , Simportadores/genética , Transfecção , Tunicamicina/farmacologiaRESUMO
Maintenance of a low intracellular Cl- concentration ([Cl-]i) is critical for enabling inhibitory neuronal responses to GABAA receptor-mediated signaling. Cl- transporters, including KCC2, and extracellular impermeant anions ([A]o) of the extracellular matrix are both proposed to be important regulators of [Cl-]i Neurons of the reticular thalamic (RT) nucleus express reduced levels of KCC2, indicating that GABAergic signaling may produce excitation in RT neurons. However, by performing perforated patch recordings and calcium imaging experiments in rats (male and female), we find that [Cl-]i remains relatively low in RT neurons. Although we identify a small contribution of [A]o to a low [Cl-]i in RT neurons, our results also demonstrate that reduced levels of KCC2 remain sufficient to maintain low levels of Cl- Reduced KCC2 levels, however, restrict the capacity of RT neurons to rapidly extrude Cl- following periods of elevated GABAergic signaling. In a computational model of a local RT network featuring slow Cl- extrusion kinetics, similar to those we found experimentally, model RT neurons are predisposed to an activity-dependent switch from GABA-mediated inhibition to excitation. By decreasing the activity threshold required to produce excitatory GABAergic signaling, weaker stimuli are able to propagate activity within the model RT nucleus. Our results indicate the importance of even diminished levels of KCC2 in maintaining inhibitory signaling within the RT nucleus and suggest how this important activity choke point may be easily overcome in disorders such as epilepsy.SIGNIFICANCE STATEMENT Precise regulation of intracellular Cl- levels ([Cl-]i) preserves appropriate, often inhibitory, GABAergic signaling within the brain. However, there is disagreement over the relative contribution of various mechanisms that maintain low [Cl-]i We found that the Cl- transporter KCC2 is an important Cl- extruder in the reticular thalamic (RT) nucleus, despite this nucleus having remarkably low KCC2 immunoreactivity relative to other regions of the adult brain. We also identified a smaller contribution of fixed, impermeant anions ([A]o) to lowering [Cl-]i in RT neurons. Inhibitory signaling among RT neurons is important for preventing excessive activation of RT neurons, which can be responsible for generating seizures. Our work suggests that KCC2 critically restricts the spread of activity within the RT nucleus.
Assuntos
Neurônios GABAérgicos/fisiologia , Formação Reticular/fisiologia , Transdução de Sinais/fisiologia , Tálamo/fisiologia , Animais , Cloretos/metabolismo , Cloretos/farmacologia , Simulação por Computador , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/genética , Receptores de GABA-A/fisiologia , Simportadores/genética , Simportadores/fisiologia , Cotransportadores de K e Cl-RESUMO
Lactate is a key metabolite for the normal occurrence of spermatogenesis. In the testis, lactate is produced by the Sertoli cells and transported to germline cells. Monocarboxylate transporters (MCTs) are key players in that process. Among the family of MCTs, MCT1 is at least partly responsible for lactate uptake by the germ cells. We aimed to perform a first assessment of the role of MCT1 in male reproductive potential. Mct1 conditional knockout (cKO) mice were used for morphometric evaluation, testicular morphology, and sperm parameter assessment. Serum steroid hormones levels were also measured. cKO animals showed a decrease in gonadosomatic index, testis weight, and seminiferous tubular diameters. Deletion of MCT1 also causes morphological changes in the organization of the seminiferous tubules and on Sertoli cell morphology. These changes resulted in failure of spermatogenesis with depletion of germ cells and total absence of spermatozoa. MCT1 cKO animals presented also hormonal dysregulation, with a decrease in serum 17ß-estradiol levels. In conclusion, MCT1 is pivotal for male reproductive potential. Absence of MCT1 results in maintenance of undifferentiated spermatogonia pool and compromised sperm production.
Assuntos
Fertilidade/fisiologia , Transportadores de Ácidos Monocarboxílicos/fisiologia , Túbulos Seminíferos/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Simportadores/fisiologia , Animais , Estradiol/sangue , Ácido Láctico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transportadores de Ácidos Monocarboxílicos/genética , Células de Sertoli/citologia , Espermatozoides/citologia , Simportadores/genéticaRESUMO
KCC2 is the major chloride extruder in neurons. The spatiotemporal regulation of KCC2 expression orchestrates the developmental shift towards inhibitory GABAergic drive and the formation of glutamatergic synapses. Whether KCC2's role in synapse formation is similar in different brain regions is unknown. First, we found that KCC2 subcellular localization, but not overall KCC2 expression levels, differed between cortex and hippocampus during the first postnatal week. We performed site-specific in utero electroporation of KCC2 cDNA to target either hippocampal CA1 or somatosensory cortical pyramidal neurons. We found that a premature expression of KCC2 significantly decreased spine density in CA1 neurons, while it had the opposite effect in cortical neurons. These effects were cell autonomous, because single-cell biolistic overexpression of KCC2 in hippocampal and cortical organotypic cultures also induced a reduction and an increase of dendritic spine density, respectively. In addition, we found that the effects of its premature expression on spine density were dependent on BDNF levels. Finally, we showed that the effects of KCC2 on dendritic spine were dependent on its chloride transporter function in the hippocampus, contrary to what was observed in cortex. Altogether, these results demonstrate that KCC2 regulation of dendritic spine development, and its underlying mechanisms, are brain-region specific.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Região CA1 Hipocampal/crescimento & desenvolvimento , Espinhas Dendríticas/fisiologia , Córtex Somatossensorial/crescimento & desenvolvimento , Simportadores/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Região CA1 Hipocampal/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células Piramidais/fisiologia , Ratos Sprague-Dawley , Simportadores/metabolismo , Cotransportadores de K e Cl-RESUMO
Blasticidin S (BlaS) interferes in the cell growth of both eukaryotes and prokaryotes. Its mode of action as a protein synthesis inhibitor has been investigated extensively. However, the mechanism of BlaS transport into the target cells is not understood well. Here, we show that Ptr2, a member of the proton-dependent oligopeptide transporter (POT) family, is responsible for the uptake of BlaS in yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Notably, some mutants of Ptr2 that are dysfunctional in dipeptide uptake were still competent to transport BlaS. Mouse-derived oligopeptide transporter PepT1 conferred BlaS sensitivity in the S. cerevisiae ptr2∆ mutant. Furthermore, bacterial POT family proteins also potentiated the BlaS sensitivity of E. coli. The role of the POT family oligopeptide transporters in the uptake of BlaS is conserved across species from bacteria to mammals.
Assuntos
Antibacterianos/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Animais , Camundongos , Nucleosídeos/metabolismo , Transportador 1 de Peptídeos , Saccharomyces cerevisiae/metabolismo , Simportadores/fisiologiaRESUMO
K+/Cl- cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl- levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl--permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.
Assuntos
Etilmaleimida/metabolismo , Simportadores/metabolismo , Animais , Membrana Celular/metabolismo , Embrião de Mamíferos , Humanos , Moduladores de Transporte de Membrana/metabolismo , Neurônios/metabolismo , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA/metabolismo , Simportadores/fisiologia , Cotransportadores de K e Cl-RESUMO
A balance between excitation and inhibition is necessary to maintain stable brain network dynamics. Traditionally, seizure activity is believed to arise from the breakdown of this delicate balance in favor of excitation with loss of inhibition. Surprisingly, recent experimental evidence suggests that this conventional view may be limited, and that inhibition plays a prominent role in the development of epileptiform synchronization. Here, we explored the role of the KCC2 co-transporter in the onset of inhibitory network-induced seizures. Our experiments in acute mouse brain slices, of either sex, revealed that optogenetic stimulation of either parvalbumin- or somatostatin-expressing interneurons induced ictal discharges in rodent entorhinal cortex during 4-aminopyridine application. These data point to a proconvulsive role of GABAA receptor signaling that is independent of the inhibitory input location (i.e., dendritic vs. somatic). We developed a biophysically realistic network model implementing dynamics of ion concentrations to explore the mechanisms leading to inhibitory network-induced seizures. In agreement with experimental results, we found that stimulation of the inhibitory interneurons induced seizure-like activity in a network with reduced potassium A-current. Our model predicts that interneuron stimulation triggered an increase of interneuron firing, which was accompanied by an increase in the intracellular chloride concentration and a subsequent KCC2-dependent gradual accumulation of the extracellular potassium promoting epileptiform ictal activity. When the KCC2 activity was reduced, stimulation of the interneurons was no longer able to induce ictal events. Overall, our study provides evidence for a proconvulsive role of GABAA receptor signaling that depends on the involvement of the KCC2 co-transporter.
Assuntos
Sincronização Cortical , Epilepsia/fisiopatologia , Interneurônios/fisiologia , Potássio/metabolismo , Convulsões/fisiopatologia , Simportadores/fisiologia , 4-Aminopiridina/administração & dosagem , Animais , Córtex Entorrinal/metabolismo , Córtex Entorrinal/fisiopatologia , Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Feminino , Interneurônios/metabolismo , Masculino , Camundongos , Redes Neurais de Computação , Parvalbuminas/metabolismo , Bloqueadores dos Canais de Potássio/administração & dosagem , Receptores de GABA-A/fisiologia , Convulsões/induzido quimicamente , Convulsões/metabolismo , Somatostatina/metabolismo , Simportadores/metabolismo , Cotransportadores de K e Cl-RESUMO
Bacteria have evolved many routes for taking up nutrients, demonstrating great versatility in the types and mechanism of uptake used in different physiological conditions. The discovery of a single transporter in the bacterium Advenella mimigardefordensis for the uptake of five different sugars, including L-glucose and D-xylose, is described in this issue (Meinert et al., ), providing yet another example of the surprising adaptability of bacterial transport strategies. The transporter identified is a tripartite ATP-independent (TRAP) transporter, not previously associated with sugar transport, and in fact does not transport the sugars directly at all, rather requiring them to be converted in the periplasm to their respective sugar acid forms before transport through what appears to be a novel general sugar acid transporter. In this commentary, I describe how this process is consistent with the known mechanisms of TRAP transporters and consider how the role of sugar oxidation, or oxidative fermentation, operates with multiple hexose and pentose sugars. Finally I suggest that the periplasmic conversion of nutrients acquired across the outer membrane, before transport across the inner membrane, could have potentially useful biological functions in Gram negative bacteria.
Assuntos
Transporte Biológico/fisiologia , Açúcares Ácidos/metabolismo , Simportadores/metabolismo , Sequência de Aminoácidos , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Glucose , Proteínas de Membrana Transportadoras/metabolismo , Periplasma/metabolismo , Simportadores/fisiologia , XiloseRESUMO
Osmosensing transporter ProP protects bacteria from osmotically induced dehydration by mediating the uptake of zwitterionic osmolytes. ProP activity is a sigmoidal function of the osmolality. ProP orthologues share an extended, cytoplasmic C-terminal domain. Orthologues with and without a C-terminal, α-helical coiled-coil domain respond similarly to the osmolality. ProP concentrates at the poles and septa of Escherichia coli cells in a cardiolipin (CL)-dependent manner. The roles of phospholipids and the C-terminal domain in subcellular localization of ProP were explored. Liposome association of peptides representing the C-terminal domains of ProP orthologues and variants in vitro was compared with subcellular localization of the corresponding orthologues and variants in vivo. In the absence of coiled-coil formation, the C-terminal domain bound liposomes and ProP concentrated at the cell poles in a CL-independent manner. The presence of the coiled-coil replaced those phenomena with CL-dependent binding and localization. The effects of amino acid replacements on lipid association of the C-terminal peptide fully recapitulated their effects on the subcellular localization of ProP. These data suggest that polar localization of ProP results from association of its C-terminal domain with the anionic lipid-enriched membrane at the cell poles. The coiled-coil domain present on only some orthologues renders that phenomenon CL-dependent.