45 results on '"Moraga-Cid G"'
Search Results
2. G PROTEIN-DEPENDENT MECHANISMS FOR ETHANOL ACTIONS ON GLYCINE ACTIVATED ION CHANNELS: 264
- Author
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Aguayo, L. G., Yevenes, G., Moraga-Cid, G., and Schmalzing, Gunther
- Published
- 2007
Catalog
3. Allosteric Modulation of Retinal GABA Receptors by Ascorbic Acid
- Author
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Calero, C. I., primary, Vickers, E., additional, Moraga Cid, G., additional, Aguayo, L. G., additional, von Gersdorff, H., additional, and Calvo, D. J., additional
- Published
- 2011
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4. A Single phenylalanine residue in the main intracellular loop of α1 γ-aminobutyric acid type A and glycine receptors influences their sensitivity to propofol.
- Author
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Moraga-Cid G, Yevenes GE, Schmalzing G, Peoples RW, Aguayo LG, Moraga-Cid, Gustavo, Yevenes, Gonzalo E, Schmalzing, Günther, Peoples, Robert W, and Aguayo, Luis G
- Abstract
Background: The intravenous anesthetic propofol acts as a positive allosteric modulator of glycine (GlyRs) and γ-aminobutyric acid type A (GABAARs) receptors. Although the role of transmembrane residues is recognized, little is known about the involvement of other regions in the modulatory effects of propofol. Therefore, the influence of the large intracellular loop in propofol sensitivity of both receptors was explored.Methods: The large intracellular loop of α1 GlyRs and α1β2 GABAARs was screened using alanine replacement. Sensitivity to propofol was studied using patch-clamp recording in HEK293 cells transiently transfected with wild type or mutant receptors.Results: Alanine mutation of a conserved phenylalanine residue within the α1 large intracellular loop significantly reduced propofol enhancement in both GlyRs (360 ± 30 vs. 75 ± 10%, mean ± SEM) and GABAARs (361 ± 49% vs. 80 ± 23%). Remarkably, propofol-hyposensitive mutant receptors retained their sensitivity to other allosteric modulators such as alcohols, etomidate, trichloroethanol, and isoflurane. At the single-channel level, the ability of propofol to increase open probability was significantly reduced in both α1 GlyR (189 ± 36 vs. 22 ± 13%) and α1β2 GABAAR (279 ± 29 vs. 29 ± 11%) mutant receptors.Conclusion: In this study, it is demonstrated that the large intracellular loop of both GlyR and GABAAR has a conserved single phenylalanine residue (F380 and F385, respectively) that influences its sensitivity to propofol. Results suggest a new role of the large intracellular loop in the allosteric modulation of two members of the Cys-loop superfamily. Thus, these data provide new insights into the molecular framework behind the modulation of inhibitory ion channels by propofol. [ABSTRACT FROM AUTHOR] more...- Published
- 2011
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5. Molecular Pharmacology of Gelsemium Alkaloids on Inhibitory Receptors.
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Marileo AM, Lara CO, Sazo A, Contreras OV, González G, Castro PA, Aguayo LG, Moraga-Cid G, Fuentealba J, Burgos CF, and Yévenes GE
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- Animals, Plant Extracts chemistry, Indole Alkaloids chemistry, gamma-Aminobutyric Acid, Mammals metabolism, Gelsemium chemistry, Alkaloids chemistry
- Abstract
Indole alkaloids are the main bioactive molecules of the Gelsemium genus plants. Diverse reports have shown the beneficial actions of Gelsemium alkaloids on the pathological states of the central nervous system (CNS). Nevertheless, Gelsemium alkaloids are toxic for mammals. To date, the molecular targets underlying the biological actions of Gelsemium alkaloids at the CNS remain poorly defined. Functional studies have determined that gelsemine is a modulator of glycine receptors (GlyRs) and GABA
A receptors (GABAA Rs), which are ligand-gated ion channels of the CNS. The molecular and physicochemical determinants involved in the interactions between Gelsemium alkaloids and these channels are still undefined. We used electrophysiological recordings and bioinformatic approaches to determine the pharmacological profile and the molecular interactions between koumine, gelsemine, gelsevirine, and humantenmine and these ion channels. GlyRs composed of α1 subunits were inhibited by koumine and gelsevirine (IC50 of 31.5 ± 1.7 and 40.6 ± 8.2 μM, respectively), while humantenmine did not display any detectable activity. The examination of GlyRs composed of α2 and α3 subunits showed similar results. Likewise, GABAA Rs were inhibited by koumine and were insensitive to humantenmine. Further assays with chimeric and mutated GlyRs showed that the extracellular domain and residues within the orthosteric site were critical for the alkaloid effects, while the pharmacophore modeling revealed the physicochemical features of the alkaloids for the functional modulation. Our study provides novel information about the molecular determinants and functional actions of four major Gelsemium indole alkaloids on inhibitory receptors, expanding our knowledge regarding the interaction of these types of compounds with protein targets of the CNS. more...- Published
- 2024
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6. Elephant Black Garlic's Beneficial Properties for Hippocampal Neuronal Network, Chemical Characterization and Biological Evaluation.
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Gavilán J, Mardones C, Oyarce G, Triviño S, Espinoza-Rubilar N, Ramírez-Molina O, Pérez C, Becerra J, Varas P, Duran-Arcos R, Muñoz-Montesino C, Moraga-Cid G, Yévenes GE, and Fuentealba J
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Garlic has been used for decades as an important food and additionally for its beneficial properties in terms of nutrition and ancestral therapeutics. In this work, we compare the properties of fresh (WG) and aged (BG) extract obtained from elephant garlic, harvested on Chiloe Island, Chile. BG was prepared from WG with a 20-day aging process under controlled temperature and humidity conditions. We observed that in BG, compounds such as diallyl disulfide decrease, and compounds of interest such as 5-hydroxymethylfurfural (69%), diallyl sulfide (17%), 3H-1,2-Dithiole (22%) and 4-Methyl-1,2,3-trithiolane (16%) were shown to be increased. Using 2,2-diphenyl-1-picrylhydrazyl (DPPH, BG: 51 ± 5.7%, WG: 12 ± 2.6%) and 2,20-azino-bis-(3-ethylbenzothiazoline-6 sulfonate) diammonium salt (ABTS, BG: 69.4 ± 2.3%, WG: 21 ± 3.9%) assays, we observed that BG possesses significantly higher antioxidant activity than WG and increased cell viability in hippocampal slices (41 ± 9%). The effects of WG and BG were shown to improve the neuronal function through an increased in intracellular calcium transients (189 ± 4%). In parallel, BG induced an increase in synaptic vesicle protein 2 (SV-2; 75 ± 12%) and brain-derived neurotrophic factor (BDNF; 32 ± 12%) levels. Thus, our study provides the initial scientific bases to foster the use of BG from Chiloe Island as a functional food containing a mixture of bioactive compounds that may contribute to brain health and well-being. more...
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- 2023
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7. TG2 promotes amyloid beta aggregates: Impact on ER-mitochondria crosstalk, calcium homeostasis and synaptic function in Alzheimer's disease.
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Panes-Fernandez J, Godoy PA, Gavilan J, Ramírez-Molina O, Burgos CF, Marileo A, Flores-Núñez O, Castro PA, Moraga-Cid G, Yévenes GE, Muñoz-Montesino C, and Fuentealba J
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- Mice, Animals, Calcium metabolism, Mitochondria metabolism, Endoplasmic Reticulum metabolism, Homeostasis, Amyloid beta-Peptides metabolism, Alzheimer Disease metabolism
- Abstract
Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by cognitive impairment that increasingly affects the elderly. AD's main features have been related to cellular and molecular events, including the aberrant aggregation of the amyloid beta peptide (Aβ), Ca
2+ dyshomeostasis, and increased mitochondria-associated membranes (MAMs). Transglutaminase type 2 (TG2) is a ubiquitous enzyme whose primary role is the Ca2+ -dependent proteins transamidation, including the Aβ peptide. TG2 activity has been closely related to cellular damage and death. We detected increased TG2 levels in neuronal cells treated with Aβ oligomers (AβOs) and hippocampal slices from J20 mice using cellular and molecular approaches. In this work, we characterized the capacity of TG2 to interact and promote Aβ toxic aggregates (AβTG2). AβTG2 induced an acute increase in intracellular Ca2+ , miniature currents, and hiperexcitability, consistent with an increased mitochondrial Ca2+ overload, IP3R-VDAC tethering, and mitochondria-endoplasmic reticulum contacts (MERCs). AβTG2 also decreased neuronal viability and excitatory postsynaptic currents, reinforcing the idea of synaptic failure associated with MAMs dysregulation mediated by TG2. Z-DON treatment, TG2 inhibitor, reduced calcium overload, mitochondrial membrane potential loss, and synaptic failure, indicating an involvement of TG2 in a toxic cycle which increases Aβ aggregation, Ca2+ overload, and MAMs upregulation. These data provide novel information regarding the role TG2 plays in synaptic function and contribute additional evidence to support the further development of TG2 inhibitors as a disease-modifying strategy for AD., Competing Interests: Conflict of interest statement None., (Copyright © 2023. Published by Elsevier Masson SAS.) more...- Published
- 2023
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8. Understanding the Role of ATP Release through Connexins Hemichannels during Neurulation.
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Tovar LM, Burgos CF, Yévenes GE, Moraga-Cid G, Fuentealba J, Coddou C, Bascunan-Godoy L, Catrupay C, Torres A, and Castro PA
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- Animals, Neurulation, Gap Junctions metabolism, Neural Tube metabolism, Adenosine Triphosphate metabolism, Connexins metabolism, Neural Tube Defects metabolism
- Abstract
Neurulation is a crucial process in the formation of the central nervous system (CNS), which begins with the folding and fusion of the neural plate, leading to the generation of the neural tube and subsequent development of the brain and spinal cord. Environmental and genetic factors that interfere with the neurulation process promote neural tube defects (NTDs). Connexins (Cxs) are transmembrane proteins that form gap junctions (GJs) and hemichannels (HCs) in vertebrates, allowing cell-cell (GJ) or paracrine (HCs) communication through the release of ATP, glutamate, and NAD
+ ; regulating processes such as cell migration and synaptic transmission. Changes in the state of phosphorylation and/or the intracellular redox potential activate the opening of HCs in different cell types. Cxs such as Cx43 and Cx32 have been associated with proliferation and migration at different stages of CNS development. Here, using molecular and cellular biology techniques (permeability), we demonstrate the expression and functionality of HCs-Cxs, including Cx46 and Cx32, which are associated with the release of ATP during the neurulation process in Xenopus laevis . Furthermore, applications of FGF2 and/or changes in intracellular redox potentials (DTT), well known HCs-Cxs modulators, transiently regulated the ATP release in our model. Importantly, the blockade of HCs-Cxs by carbenoxolone (CBX) and enoxolone (ENX) reduced ATP release with a concomitant formation of NTDs. We propose two possible and highly conserved binding sites (N and E) in Cx46 that may mediate the pharmacological effect of CBX and ENX on the formation of NTDs. In summary, our results highlight the importance of ATP release mediated by HCs-Cxs during neurulation. more...- Published
- 2023
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9. Modulation of GABA A receptors and of GABAergic synapses by the natural alkaloid gelsemine.
- Author
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Marileo AM, Gavilán J, San Martín VP, Lara CO, Sazo A, Muñoz-Montesino C, Castro PA, Burgos CF, Leiva-Salcedo E, Aguayo LG, Moraga-Cid G, Fuentealba J, and Yévenes GE
- Abstract
The Gelsemium elegans plant preparations have shown beneficial activity against common diseases, including chronic pain and anxiety. Nevertheless, their clinical uses are limited by their toxicity. Gelsemine, one of the most abundant alkaloids in the Gelsemium plants, have replicated these therapeutic and toxic actions in experimental behavioral models. However, the molecular targets underlying these biological effects remain unclear. The behavioral activity profile of gelsemine suggests the involvement of GABA
A receptors (GABAA Rs), which are the main biological targets of benzodiazepines (BDZs), a group of drugs with anxiolytic, hypnotic, and analgesic properties. Here, we aim to define the modulation of GABAA Rs by gelsemine, with a special focus on the subtypes involved in the BDZ actions. The gelsemine actions were determined by electrophysiological recordings of recombinant GABAA Rs expressed in HEK293 cells, and of native receptors in cortical neurons. Gelsemine inhibited the agonist-evoked currents of recombinant and native receptors. The functional inhibition was not associated with the BDZ binding site. We determined in addition that gelsemine diminished the frequency of GABAergic synaptic events, likely through a presynaptic modulation. Our findings establish gelsemine as a negative modulator of GABAA Rs and of GABAergic synaptic function. These pharmacological features discard direct anxiolytic or analgesic actions of gelsemine through GABAA Rs but support a role of GABAA Rs on the alkaloid induced toxicity. On the other hand, the presynaptic effects of the alkaloid provide an additional mechanism to explain their beneficial effects. Collectively, our results contribute novel information to improve understanding of gelsemine actions in the mammalian nervous system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Marileo, Gavilán, San Martín, Lara, Sazo, Muñoz-Montesino, Castro, Burgos, Leiva-Salcedo, Aguayo, Moraga-Cid, Fuentealba and Yévenes.) more...- Published
- 2023
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10. Antiseizure medication in early nervous system development. Ion channels and synaptic proteins as principal targets.
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Castro PA, Pinto-Borguero I, Yévenes GE, Moraga-Cid G, and Fuentealba J
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The main strategy for the treatment of epilepsy is the use of pharmacological agents known as antiseizure medication (ASM). These drugs control the seizure onset and improves the life expectancy and quality of life of patients. Several ASMs are contraindicated during pregnancy, due to a potential teratogen risk. For this reason, the pharmacological treatments of the pregnant Women with Epilepsy (WWE) need comprehensive analyses to reduce fetal risk during the first trimester of pregnancy. The mechanisms by which ASM are teratogens are still under study and scientists in the field, propose different hypotheses. One of them, which will be addressed in this review, corresponds to the potential alteration of ASM on ion channels and proteins involved in relevant signaling and cellular responses (i.e., migration, differentiation) during embryonic development. The actual information related to the action of ASM and its possible targets it is poorly understood. In this review, we will focus on describing the eventual presence of some ion channels and synaptic proteins of the neurotransmitter signaling pathways present during early neural development, which could potentially interacting as targets of ASM. This information leads to elucidate whether these drugs would have the ability to affect critical signaling during periods of neural development that in turn could explain the fetal malformations observed by the use of ASM during pregnancy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Castro, Pinto-Borguero, Yévenes, Moraga-Cid and Fuentealba.) more...
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- 2022
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11. Glycine Receptor Subtypes and Their Roles in Nociception and Chronic Pain.
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San Martín VP, Sazo A, Utreras E, Moraga-Cid G, and Yévenes GE
- Abstract
Disruption of the inhibitory control provided by the glycinergic system is one of the major mechanisms underlying chronic pain. In line with this concept, recent studies have provided robust proof that pharmacological intervention of glycine receptors (GlyRs) restores the inhibitory function and exerts anti-nociceptive effects on preclinical models of chronic pain. A targeted regulation of the glycinergic system requires the identification of the GlyR subtypes involved in chronic pain states. Nevertheless, the roles of individual GlyR subunits in nociception and in chronic pain are yet not well defined. This review aims to provide a systematic outline on the contribution of GlyR subtypes in chronic pain mechanisms, with a particular focus on molecular pathways of spinal glycinergic dis-inhibition mediated by post-translational modifications at the receptor level. The current experimental evidence has shown that phosphorylation of synaptic α1β and α3β GlyRs are involved in processes of spinal glycinergic dis-inhibition triggered by chronic inflammatory pain. On the other hand, the participation of α2-containing GlyRs and of β subunits in pain signaling have been less studied and remain undefined. Although many questions in the field are still unresolved, future progress in GlyR research may soon open new exciting avenues into understanding and controlling chronic pain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 San Martín, Sazo, Utreras, Moraga-Cid and Yévenes.) more...
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- 2022
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12. PrP C as a Transducer of Physiological and Pathological Signals.
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Panes JD, Saavedra P, Pineda B, Escobar K, Cuevas ME, Moraga-Cid G, Fuentealba J, Rivas CI, Rezaei H, and Muñoz-Montesino C
- Abstract
After the discovery of prion phenomenon, the physiological role of the cellular prion protein (PrP
C ) remained elusive. In the past decades, molecular and cellular analysis has shed some light regarding interactions and functions of PrPC in health and disease. PrPC , which is located mainly at the plasma membrane of neuronal cells attached by a glycosylphosphatidylinositol (GPI) anchor, can act as a receptor or transducer from external signaling. Although the precise role of PrPC remains elusive, a variety of functions have been proposed for this protein, namely, neuronal excitability and viability. Although many issues must be solved to clearly define the role of PrPC , its connection to the central nervous system (CNS) and to several misfolding-associated diseases makes PrPC an interesting pharmacological target. In a physiological context, several reports have proposed that PrPC modulates synaptic transmission, interacting with various proteins, namely, ion pumps, channels, and metabotropic receptors. PrPC has also been implicated in the pathophysiological cell signaling induced by β-amyloid peptide that leads to synaptic dysfunction in the context of Alzheimer's disease (AD), as a mediator of Aβ-induced cell toxicity. Additionally, it has been implicated in other proteinopathies as well. In this review, we aimed to analyze the role of PrPC as a transducer of physiological and pathological signaling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Panes, Saavedra, Pineda, Escobar, Cuevas, Moraga-Cid, Fuentealba, Rivas, Rezaei and Muñoz-Montesino.) more...- Published
- 2021
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13. Modulatory Actions of the Glycine Receptor β Subunit on the Positive Allosteric Modulation of Ethanol in α2 Containing Receptors.
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Muñoz B, Mariqueo T, Murath P, Peters C, Yevenes GE, Moraga-Cid G, Peoples RW, and Aguayo LG
- Abstract
Alpha1-containing glycine receptors (GlyRs) are major mediators of synaptic inhibition in the spinal cord and brain stem. Recent studies reported the presence of α2-containing GlyRs in other brain regions, such as nucleus accumbens and cerebral cortex. GlyR activation decreases neuronal excitability associated with sensorial information, motor control, and respiratory functions; all of which are significantly altered during ethanol intoxication. We evaluated the role of β GlyR subunits and of two basic amino acid residues, K389 and R390, located in the large intracellular loop (IL) of the α2 GlyR subunit, which are important for binding and functional modulation by Gβγ, the dimer of the trimeric G protein conformation, using HEK-293 transfected cells combined with patch clamp electrophysiology. We demonstrate a new modulatory role of the β subunit on ethanol sensitivity of α2 subunits. Specifically, we found a differential allosteric modulation in homomeric α2 GlyRs compared with the α2β heteromeric conformation. Indeed, while α2 was insensitive, α2β GlyRs were substantially potentiated by ethanol, GTP-γ-S, propofol, Zn
2+ and trichloroethanol. Furthermore, a Gβγ scavenger (ct-GRK2) selectively attenuated the effects of ethanol on recombinant α2β GlyRs. Mutations in an α2 GlyR co-expressed with the β subunit (α2AAβ) specifically blocked ethanol sensitivity, but not propofol potentiation. These results show a selective mechanism for low ethanol concentration effects on homomeric and heteromeric conformations of α2 GlyRs and provide a new mechanism for ethanol pharmacology, which is relevant to upper brain regions where α2 GlyRs are abundantly expressed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Muñoz, Mariqueo, Murath, Peters, Yevenes, Moraga-Cid, Peoples and Aguayo.) more...- Published
- 2021
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14. Stereospecific Inhibition of Ethanol Potentiation on Glycine Receptor by M554 Stereoisomers.
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Vásquez PA, San Martín L, Argel Y, Riquelme C, Torres J, Vidal F, Cayuman F, Castro P, Fuentealba J, Moraga-Cid G, Yevenes G, Jin C, Jiménez VA, and Guzmán L
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- Stereoisomerism, Ethanol, Receptors, Glycine
- Abstract
Blocking the interaction between the Gβγ protein and the glycine receptor (GlyR) has emerged as a promising pharmacological strategy to treat acute alcohol intoxication by inhibiting ethanol potentiation on GlyR. M554 is a recently discovered small molecule capable of binding to Gβγ with potent in vitro and in vivo inhibitory activity. This compound has been tested as a mixture of diastereomers, and no information is available concerning the stereospecific activity of each species, which is critical to pursue efforts on lead optimization and drug development. In this work, we explored the differential activity of four M554 stereoisomers by in silico molecular dynamics simulations and electrophysiological experiments. Our results revealed that the ( R , R )-M554 stereoisomer is a promising lead compound that inhibits ethanol potentiation of GlyR. more...
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- 2020
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15. Inhibition of the Glycine Receptor alpha 3 Function by Colchicine.
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Muñoz-Montesino C, Burgos CF, Lara CO, Riquelme CR, Flaig D, San Martin VP, Aguayo LG, Fuentealba J, Castro PA, Guzmán L, Yévenes GE, and Moraga-Cid G
- Abstract
Colchicine is a plant alkaloid that is widely used as a therapeutic agent. It is widely accepted that colchicine reduces the production of inflammatory mediators mainly by altering cytoskeleton dynamics due to its microtubule polymerization inhibitory activity. However, other lines of evidence have shown that colchicine exerts direct actions on the function of ion channels, which are independent of cytoskeleton alterations. Colchicine is able to modify the function of several pentameric ligand-gated ion channels, including glycine receptors (GlyRs). Previous electrophysiological studies have shown that colchicine act as an antagonist of GlyRs composed by the α
1 subunit. In addition, it was recently demonstrated that colchicine directly bind to the α3 subunit of GlyRs. Interestingly, other studies have shown a main role of α3 GlyRs on chronic inflammatory pain. Nevertheless, the functional effects of colchicine on the α3 GlyR function are still unknown. Here, by using electrophysiological techniques and bioinformatics, we show that colchicine inhibited the function of the α3 GlyRs. Colchicine elicited concentration-dependent inhibitory effects on α3 GlyRs at micromolar range and decreased the apparent affinity for glycine. Single-channel recordings show that the colchicine inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that colchicine preferentially bind to the orthosteric site in the closed state of the ion channel. Altogether, our results suggest that colchicine is a competitive antagonist of the α3 GlyRs., (Copyright © 2020 Muñoz-Montesino, Burgos, Lara, Riquelme, Flaig, San Martin, Aguayo, Fuentealba, Castro, Guzmán, Yévenes and Moraga-Cid.) more...- Published
- 2020
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16. Correction: Canonical Wnt3a modulates intracellular calcium and enhances excitatory neurotransmission in hippocampal neurons.
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Avila ME, Sepúlveda FJ, Burgos CF, Moraga-Cid G, Parodi J, Moon RT, Aguayo LG, Opazo C, and De Ferrari GV
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- 2020
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17. Altered Glutaminase 1 Activity During Neurulation and Its Potential Implications in Neural Tube Defects.
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Benavides-Rivas C, Tovar LM, Zúñiga N, Pinto-Borguero I, Retamal C, Yévenes GE, Moraga-Cid G, Fuentealba J, Guzmán L, Coddou C, Bascuñán-Godoy L, and Castro PA
- Abstract
The neurulation process is regulated by a large amount of genetic and environmental factors that determine the establishment, folding, and fusion of the neural plate to form the neural tube, which develops into the main structure of the central nervous system. A recently described factor involved in this process is glutamate. Through NMDA ionotropic receptor, glutamate modifies intracellular Ca
2+ dynamics allowing the oriented cell migration and proliferation, essentials processes in neurulation. Glutamate synthesis depends on the mitochondrial enzyme known as glutaminase 1 (GLS1) that is widely expressed in brain and kidney. The participation of GLS 1 in prenatal neurogenic processes and in the adult brain has been experimentally established, however, its participation in early stages of embryonic development has not been described. The present investigation describes for the first time the presence and functionality of GLS1 in Xenopus laevis embryos during neurulation. Although protein expression levels remains constant, the catalytic activity of GLS1 increases significantly (~66%) between early (stage 12) and middle to late (stages 14-19) neurulation process. Additionally, the use of 6-diazo-5-oxo-L-norleucine (L-DON, competitive inhibitor of glutamine-depend enzymes), reduced significantly the GLS1 specific activity during neurulation (~36%) and induce the occurrence of neural tube defects involving its possible participation in the neural tube closure in Xenopus laevis embryos., (Copyright © 2020 Benavides-Rivas, Tovar, Zúñiga, Pinto-Borguero, Retamal, Yévenes, Moraga-Cid, Fuentealba, Guzmán, Coddou, Bascuñán-Godoy and Castro.) more...- Published
- 2020
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18. Changes in PGC-1α/SIRT1 Signaling Impact on Mitochondrial Homeostasis in Amyloid-Beta Peptide Toxicity Model.
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Panes JD, Godoy PA, Silva-Grecchi T, Celis MT, Ramirez-Molina O, Gavilan J, Muñoz-Montecino C, Castro PA, Moraga-Cid G, Yévenes GE, Guzmán L, Salisbury JL, Trushina E, and Fuentealba J
- Abstract
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive impairment that increasingly afflicts the elderly population. Soluble oligomers (AβOs) has been implicated in AD pathogenesis: however, the molecular events underlying a role for Aβ are not well understood. We studied the effects of AβOs on mitochondrial function and on key proteins that regulate mitochondrial dynamics and biogenesis in hippocampal neurons and PC-12 cells. We find that AβOs treatment caused a reduction in total Mfn1 after a 2 h exposure (42 ± 11%); while DRP1 increased at 1 and 2 h (205 ± 22% and 198 ± 27%, respectively), correlating to changes in mitochondrial morphology. We also observed that SIRT1 levels were reduced after acute and chronic AβOs treatment (68 ± 7% and 77 ± 6%, respectively); while PGC-1α levels were reduced with the same time treatments (68 ± 8% and 67 ± 7%, respectively). Interestingly, we found that chronic treatment with AβOs increased the levels of pSIRT1 (24 h: 157 ± 18%), and we observed changes in the PGC-1α and p-SIRT1 nucleus/cytosol ratio and SIRT1-PGC-1α interaction pattern after chronic exposure to AβOs. Our data suggest that AβOs induce important changes in the level and localization of mitochondrial proteins related with the loss of mitochondrial function that are mediated by a fast and sustained SIRT1/PGC-1α complex disruption promoting a "non-return point" to an irreversible synaptic failure and neuronal network disconnection., (Copyright © 2020 Panes, Godoy, Silva-Grecchi, Celis, Ramirez-Molina, Gavilan, Muñoz-Montecino, Castro, Moraga-Cid, Yévenes, Guzmán, Salisbury, Trushina and Fuentealba.) more...
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- 2020
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19. Modulation of glycine receptor single-channel conductance by intracellular phosphorylation.
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Moraga-Cid G, San Martín VP, Lara CO, Muñoz B, Marileo AM, Sazo A, Muñoz-Montesino C, Fuentealba J, Castro PA, Guzmán L, Burgos CF, Zeilhofer HU, Aguayo LG, Corringer PJ, and Yévenes GE
- Subjects
- Amino Acid Substitution, Cyclic AMP-Dependent Protein Kinases metabolism, Humans, Intracellular Space metabolism, Phosphorylation, Protein Domains, Protein Processing, Post-Translational, Receptors, Glycine chemistry, Receptors, Prostaglandin E, EP2 Subtype, Signal Transduction, Excitatory Postsynaptic Potentials, Receptors, Glycine metabolism, Receptors, Glycine physiology
- Abstract
Glycine receptors (GlyRs) are anion-permeable pentameric ligand-gated ion channels (pLGICs). The GlyR activation is critical for the control of key neurophysiological functions, such as motor coordination, respiratory control, muscle tone and pain processing. The relevance of the GlyR function is further highlighted by the presence of abnormal glycinergic inhibition in many pathophysiological states, such as hyperekplexia, epilepsy, autism and chronic pain. In this context, previous studies have shown that the functional inhibition of GlyRs containing the α3 subunit is a pivotal mechanism of pain hypersensitivity. This pathway involves the activation of EP2 receptors and the subsequent PKA-dependent phosphorylation of α3GlyRs within the intracellular domain (ICD), which decrease the GlyR-associated currents and enhance neuronal excitability. Despite the importance of this mechanism of glycinergic dis-inhibition associated with dysfunctional α3GlyRs, our current understanding of the molecular events involved is limited. Here, we report that the activation of PKA signaling pathway decreases the unitary conductance of α3GlyRs. We show in addition that the substitution of the PKA-targeted serine with a negatively charged residue within the ICD of α3GlyRs and of chimeric receptors combining bacterial GLIC and α3GlyR was sufficient to generate receptors with reduced conductance. Thus, our findings reveal a potential biophysical mechanism of glycinergic dis-inhibition and suggest that post-translational modifications of the ICD, such as phosphorylation, may shape the conductance of other pLGICs. more...
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- 2020
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20. Therapeutic Use of Vitamin C in Cancer: Physiological Considerations.
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Roa FJ, Peña E, Gatica M, Escobar-Acuña K, Saavedra P, Maldonado M, Cuevas ME, Moraga-Cid G, Rivas CI, and Muñoz-Montesino C
- Abstract
Since the early studies of William J. McCormick in the 1950s, vitamin C has been proposed as a candidate for the treatment of cancer. A number of reports have shown that pharmacological concentrations of vitamin C selectively kill cancer cells in vitro and decrease the growth rates of a number of human tumor xenografts in immunodeficient mice. However, up to the date there is still doubt regarding this possible therapeutic role of vitamin C in cancer, mainly because high dose administration in cancer patients has not showed a clear antitumor activity. These apparent controversial findings highlight the fact that we lack information on the interactions that occurs between cancer cells and vitamin C, and if these transformed cells can uptake, metabolize and compartmentalize vitamin C like normal human cells do. The role of SVCTs and GLUTs transporters, which uptake the reduced form and the oxidized form of vitamin C, respectively, has been recently highlighted in the context of cancer showing that the relationship between vitamin C and cancer might be more complex than previously thought. In this review, we analyze the state of art of the effect of vitamin C on cancer cells in vitro and in vivo , and relate it to the capacity of cancer cells in acquiring, metabolize and compartmentalize this nutrient, with its implications on the potential therapeutic role of vitamin C in cancer., (Copyright © 2020 Roa, Peña, Gatica, Escobar-Acuña, Saavedra, Maldonado, Cuevas, Moraga-Cid, Rivas and Muñoz-Montesino.) more...
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- 2020
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21. Pentameric Ligand-Gated Ion Channels as Pharmacological Targets Against Chronic Pain.
- Author
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Lara CO, Burgos CF, Moraga-Cid G, Carrasco MA, and Yévenes GE
- Abstract
Chronic pain is a common detrimental condition that affects around 20% of the world population. The current drugs to treat chronic pain states, especially neuropathic pain, have a limited clinical efficiency and present significant adverse effects that complicates their regular use. Recent studies have proposed new therapeutic strategies focused on the pharmacological modulation of G-protein-coupled receptors, transporters, enzymes, and ion channels expressed on the nociceptive pathways. The present work intends to summarize recent advances on the pharmacological modulation of pentameric ligand-gated ion channels, which plays a key role in pain processing. Experimental data have shown that novel allosteric modulators targeting the excitatory nicotinic acetylcholine receptor, as well as the inhibitory GABA
A and glycine receptors, reverse chronic pain-related behaviors in preclinical assays. Collectively, these evidences strongly suggest the pharmacological modulation of pentameric ligand-gated ion channels is a promising strategy towards the development of novel therapeutics to treat chronic pain states in humans., (Copyright © 2020 Lara, Burgos, Moraga-Cid, Carrasco and Yévenes.) more...- Published
- 2020
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22. Large Intracellular Domain-Dependent Effects of Positive Allosteric Modulators on Glycine Receptors.
- Author
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Lara CO, Burgos CF, Silva-Grecchi T, Muñoz-Montesino C, Aguayo LG, Fuentealba J, Castro PA, Guzmán JL, Corringer PJ, Yévenes GE, and Moraga-Cid G
- Subjects
- Allosteric Regulation drug effects, Cells, Cultured, Central Nervous System Depressants pharmacology, Chimera, Cyanobacteria, Ethanol pharmacology, Extracellular Space physiology, Humans, Hydrogen-Ion Concentration, Intracellular Membranes physiology, Isoflurane pharmacology, Ligand-Gated Ion Channels physiology, Membrane Potentials drug effects, Allosteric Regulation physiology, Receptors, Glycine physiology
- Abstract
Glycine receptors (GlyRs) are members of the pentameric ligand-gated ionic channel family (pLGICs) and mediate fast inhibitory neurotransmission in the brain stem and spinal cord. The function of GlyRs can be modulated by positive allosteric modulators (PAMs). So far, it is largely accepted that both the extracellular (ECD) and transmembrane (TMD) domains constitute the primary target for many of these PAMs. On the other hand, the contribution of the intracellular domain (ICD) to the PAM effects on GlyRs remains poorly understood. To gain insight about the role of the ICD in the pharmacology of GlyRs, we examined the contribution of each domain using a chimeric receptor. Two chimeras were generated, one consisting of the ECD of the prokaryotic homologue Gloeobacter violaceus ligand-gated ion channel (GLIC) fused to the TMD of the human α
1 GlyR lacking the ICD (Lily) and a second with the ICD (Lily-ICD). The sensitivity to PAMs of both chimeric receptors was studied using electrophysiological techniques. The Lily receptor showed a significant decrease in the sensitivity to four recognized PAMs. Remarkably, the incorporation of the ICD into the Lily background was sufficient to restore the wild-type α1 GlyR sensitivity to these PAMs. Based on these data, we can suggest that the ICD is necessary to form a pLGIC having full sensitivity to positive allosteric modulators. more...- Published
- 2019
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23. Data on SVCT2 transporter expression and localization in cancer cell lines and tissues.
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Roa FJ, Peña E, Inostroza E, Sotomayor K, González M, Gutierrez-Castro FA, Maurin M, Sweet K, Labrousse C, Gatica M, Aylwin CF, Mendoza P, Maldonado M, Delgado C, Madariaga J, Panes J, Silva-Grecchi T, Concha II, Moraga-Cid G, Reyes AM, Muñoz-Montesino C, Vera JC, and Rivas CI more...
- Abstract
The data presented in this article are related to the research paper entitled "Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer", available in Free Radical Biology and Medicine Journal [1]. In this article, we examined the SVCT2 transporter expression in various breast cancer cell lines using RT-PCR and Western blot assays. In addition, we analyzed the subcellular localization of SVCT2 by immunofluorescence colocalization assays and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented. more...
- Published
- 2019
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24. Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer.
- Author
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Peña E, Roa FJ, Inostroza E, Sotomayor K, González M, Gutierrez-Castro FA, Maurin M, Sweet K, Labrousse C, Gatica M, Aylwin CF, Mendoza P, Maldonado M, Delgado C, Madariaga J, Panes J, Silva-Grecchi T, Concha II, Moraga-Cid G, Reyes AM, Muñoz-Montesino C, Vera JC, and Rivas CI more...
- Subjects
- Ascorbic Acid metabolism, Breast Neoplasms pathology, Bystander Effect, Female, Gene Expression Regulation, Neoplastic genetics, Humans, MCF-7 Cells, Mitochondria pathology, Oxidation-Reduction, Reactive Oxygen Species metabolism, Sodium metabolism, Breast Neoplasms genetics, Mitochondria genetics, Mitochondrial Membrane Transport Proteins genetics, Sodium-Coupled Vitamin C Transporters genetics
- Abstract
The potential role of vitamin C in cancer prevention and treatment remains controversial. While normal human cells obtain vitamin C as ascorbic acid, the prevalent form of vitamin C in vivo, the uptake mechanisms by which cancer cells acquire vitamin C has remained unclear. The aim of this study is to characterize how breast cancer cells acquire vitamin C. For this, we determined the expression of vitamin C transporters in normal and breast cancer tissue samples, and in ZR-75, MCF-7, MDA-231 and MDA-468 breast cancer cell lines. At the same time, reduced (AA) and oxidized (DHA) forms of vitamin C uptake experiments were performed in all cell lines. We show here that human breast cancer tissues differentially express a form of SVCT2 transporter, that is systematically absent in normal breast tissues and it is increased in breast tumors. In fact, estrogen receptor negative breast cancer tissue, exhibit the most elevated SVCT2 expression levels. Despite this, our analysis in breast cancer cell lines showed that these cells are not able to uptake ascorbic acid and depend on glucose transporter for the acquisition of vitamin C by a bystander effect. This is consistent with our observations that this form of SVCT2 is completely absent from the plasma membrane and is overexpressed in mitochondria of breast cancer cells, where it mediates ascorbic acid transport. This work shows that breast cancer cells acquire vitamin C in its oxidized form and are capable of accumulated high concentrations of the reduced form. Augmented expression of an SVCT2 mitochondrial form appears to be a common hallmark across all human cancers and might have implications in cancer cells survival capacity against pro-oxidant environments., (Copyright © 2019. Published by Elsevier Inc.) more...
- Published
- 2019
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25. Inhibitory Actions of Tropeines on the α3 Glycine Receptor Function.
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San Martín VP, Burgos CF, Marileo AM, Lara CO, Sazo A, Fuentealba J, Guzmán L, Castro PA, Aguayo LG, Moraga-Cid G, and Yévenes GE
- Abstract
Glycine receptors (GlyRs) are chloride-permeable pentameric ligand-gated ion channels. The inhibitory activity of GlyRs is essential for many physiological processes, such as motor control and respiration. In addition, several pathological states, such as hyperekplexia, epilepsy, and chronic pain, are associated with abnormal glycinergic inhibition. Recent studies have pointed out that positive allosteric modulators targeting the GlyR α3 subunit (α3GlyR) displayed beneficial effects in chronic pain models. Interestingly, previous electrophysiological studies have shown that tropeines, which are a family of synthetic antagonists of the serotonin type 3 receptors (5-HT
3 Rs), potentiate the activity of GlyRs conformed by α1 subunits. However, despite its importance as a pharmacological target in chronic pain, it is currently unknown whether the α3GlyR function is modulated by tropeines. Using electrophysiological techniques and molecular docking simulations, here we show that tropeines are inhibitors of the α3GlyR function. Tropisetron, a prototypical tropeine, exerted concentration-dependent inhibitory effects on α3GlyRs at the low micromolar range. In addition, three other tropeines showed similar effects. Single-channel recordings show that tropisetron inhibition is associated with a decrease in the open probability of the ion channel. Molecular docking assays suggest that tropeines preferentially bind to an agonist-free, closed state of the ion channel. The tropeine binding occurs in a discrete pocket around the vicinity of the orthosteric site within the extracellular domain of α3GlyR. Thus, our results describe the pharmacological modulation of tropeines on α3GlyRs. These findings may contribute to the development of GlyR-selective tropeine derivatives for basic and/or clinical applications. more...- Published
- 2019
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26. 17 Oxo Sparteine and Lupanine, Obtained from Cytisus scoparius, Exert a Neuroprotection against Soluble Oligomers of Amyloid-β Toxicity by Nicotinic Acetylcholine Receptors.
- Author
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Gavilan J, Mennickent D, Ramirez-Molina O, Triviño S, Perez C, Silva-Grecchi T, Godoy PA, Becerra J, Aguayo LG, Moraga-Cid G, Martin VS, Yevenes GE, Castro PA, Guzman L, and Fuentealba J
- Subjects
- Animals, Calcium Signaling drug effects, HEK293 Cells, Hippocampus pathology, Humans, Mice, Inbred C57BL, Nerve Net drug effects, Neurons pathology, Oncogene Protein v-akt metabolism, PC12 Cells, Rats, Sparteine chemistry, Sparteine isolation & purification, Synapses drug effects, Amyloid beta-Peptides antagonists & inhibitors, Amyloid beta-Peptides toxicity, Cytisus chemistry, Neuroprotective Agents pharmacology, Receptors, Nicotinic drug effects, Sparteine analogs & derivatives, Sparteine pharmacology
- Abstract
Alzheimer's disease (AD) is a neurodegenerative pathology, which is characterized by progressive and irreversible cognitive impairment. Most of the neuronal perturbations described in AD can be associated with soluble amyloid- β oligomers (SO-Aβ). There is a large amount of evidence demonstrating the neuroprotective effect of Nicotine neurotransmission in AD, mainly through nicotinic acetylcholine receptor (nAChR) activation and antiapoptotic PI3K/Akt/Bcl-2 pathway signaling. Using HPLC and GC/MS, we isolated and characterized two alkaloids obtained from C. scoparius, Lupanine (Lup), and 17- oxo-sparteine (17- ox), and examined their neuroprotective properties in a cellular model of SO-Aβ toxicity. Our results showed that Lup and 17- ox (both at 0.03μM) prevented SO-Aβ-induced toxicity in PC12 cells (Lup: 64±7%; 17- ox: 57±6%). Similar results were seen in hippocampal neurons where these alkaloids prevented SO-Aβ neurotoxicity (Lup: 57±2%; 17- ox: 52±3%) and increased the frequency of spontaneous calcium transients (Lup: 60±4%; 17- Ox: 40±3%), suggesting an enhancing effect on neural network activity and synaptic activity potentiation. All of the neuroprotective effects elicited by both alkaloids were completely blocked by α-bungarotoxin. Additionally, we observed that the presence of both Lup and 17- ox increased Akt phosphorylation levels (52±4% and 35±7%, respectively) in cells treated with SO-Aβ (3 h). Taken together, our results suggest that the activation of nAChR by Lup and 17- ox induces neuroprotection in different cellular models, and appears to be an interesting target for the development of new pharmacological tools and strategies against AD. more...
- Published
- 2019
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27. Phosphorylation state-dependent modulation of spinal glycine receptors alleviates inflammatory pain.
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Acuña MA, Yévenes GE, Ralvenius WT, Benke D, Di Lio A, Lara CO, Muñoz B, Burgos CF, Moraga-Cid G, Corringer PJ, and Zeilhofer HU
- Subjects
- Allosteric Site, Animals, Female, HEK293 Cells, Humans, Inflammation Mediators metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Models, Molecular, Neurons, Pain, Phenols chemistry, Phenylalanine chemistry, Phosphorylation, Protein Conformation, Recombinant Proteins chemistry, Hyperalgesia metabolism, Inflammation metabolism, Pain Management methods, Receptors, Glycine metabolism, Spinal Cord metabolism
- Abstract
Diminished inhibitory neurotransmission in the superficial dorsal horn of the spinal cord is thought to contribute to chronic pain. In inflammatory pain, reductions in synaptic inhibition occur partially through prostaglandin E2- (PGE2-) and PKA-dependent phosphorylation of a specific subtype of glycine receptors (GlyRs) that contain α3 subunits. Here, we demonstrated that 2,6-di-tert-butylphenol (2,6-DTBP), a nonanesthetic propofol derivative, reverses inflammation-mediated disinhibition through a specific interaction with heteromeric αβGlyRs containing phosphorylated α3 subunits. We expressed mutant GlyRs in HEK293T cells, and electrophysiological analyses of these receptors showed that 2,6-DTBP interacted with a conserved phenylalanine residue in the membrane-associated stretch between transmembrane regions 3 and 4 of the GlyR α3 subunit. In native murine spinal cord tissue, 2,6-DTBP modulated synaptic, presumably αβ heteromeric, GlyRs only after priming with PGE2. This observation is consistent with results obtained from molecular modeling of the α-β subunit interface and suggests that in α3βGlyRs, the binding site is accessible to 2,6-DTBP only after PKA-dependent phosphorylation. In murine models of inflammatory pain, 2,6-DTBP reduced inflammatory hyperalgesia in an α3GlyR-dependent manner. Together, our data thus establish that selective potentiation of GlyR function is a promising strategy against chronic inflammatory pain and that, to our knowledge, 2,6-DTBP has a unique pharmacological profile that favors an interaction with GlyRs that have been primed by peripheral inflammation. more...
- Published
- 2016
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28. Potentiation of Gamma Aminobutyric Acid Receptors (GABAAR) by Ethanol: How Are Inhibitory Receptors Affected?
- Author
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Förstera B, Castro PA, Moraga-Cid G, and Aguayo LG
- Abstract
In recent years there has been an increase in the understanding of ethanol actions on the type A γ-aminobutyric acid chloride channel (GABAAR), a member of the pentameric ligand gated ion channels (pLGICs). However, the mechanism by which ethanol potentiates the complex is still not fully understood and a number of publications have shown contradictory results. Thus many questions still remain unresolved requiring further studies for a better comprehension of this effect. The present review concentrates on the involvement of GABAAR in the acute actions of ethanol and specifically focuses on the immediate, direct or indirect, synaptic and extra-synaptic modulatory effects. To elaborate on the immediate, direct modulation of GABAAR by acute ethanol exposure, electrophysiological studies investigating the importance of different subunits, and data from receptor mutants will be examined. We will also discuss the nature of the putative binding sites for ethanol based on structural data obtained from other members of the pLGICs family. Finally, we will briefly highlight the glycine gated chloride channel (GlyR), another member of the pLGIC family, as a suitable target for the development of new pharmacological tools. more...
- Published
- 2016
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29. Control of ethanol sensitivity of the glycine receptor α3 subunit by transmembrane 2, the intracellular splice cassette and C-terminal domains.
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Sánchez A, Yévenes GE, San Martin L, Burgos CF, Moraga-Cid G, Harvey RJ, and Aguayo LG
- Subjects
- Alternative Splicing, Amino Acid Sequence, Animals, GTP-Binding Protein beta Subunits metabolism, GTP-Binding Protein gamma Subunits metabolism, HEK293 Cells, Humans, Models, Molecular, Molecular Sequence Data, Mutation, Protein Structure, Tertiary, Rats, Receptors, Glycine genetics, Ethanol pharmacology, Receptors, Glycine metabolism
- Abstract
Previous studies have shown that the effect of ethanol onglycine receptors (GlyRs) containing the a1 subunit is affected by interaction with heterotrimeric G proteins (Gβγ). GlyRs containing the α3 subunit are involved in inflammatory pain sensitization and rhythmic breathing and have received much recent attention. For example, it is unknown whether ethanol affects the function of this important GlyR subtype. Electrophysiologic experiments showed that GlyR α3 subunits were not potentiated by pharmacologic concentrations of ethanol or by Gβγ. Thus, we studied GlyR α1–α3 chimeras and mutants to determine the molecular properties that confer ethanol insensitivity. Mutation of corresponding glycine 254 in transmembrane domain 2 (TM2) found in a1 in the α3(A254G) –α1 chimera induced a glycine-evoked current that displayed potentiation during application of ethanol (46 ± 5%, 100 mM) and Gβγ activation (80 ± 17%). Interestingly,insertion of the intracellular α3L splice cassette into GlyR α1 abolished the enhancement of the glycine-activated current by ethanol (5 ± 6%) and activation by Gβγ (21 6 7%). In corporation of the GlyR α1 C terminus into the ethanol-resistant α3S(A254G) mutant produced a construct that displayed potentiation of the glycine-activated current with 100 mM ethanol (40 ± 6%)together with a current enhancement after G protein activation (68 ± 25%). Taken together, these data demonstrate that GlyRα3 subunits are not modulated by ethanol. Residue A254 in TM2, the α3L splice cassette, and the C-terminal domain of α3GlyRs are determinants for low ethanol sensitivity and form the molecular basis of subtype-selective modulation of GlyRs by alcohol. more...
- Published
- 2015
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30. Allosteric and hyperekplexic mutant phenotypes investigated on an α1 glycine receptor transmembrane structure.
- Author
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Moraga-Cid G, Sauguet L, Huon C, Malherbe L, Girard-Blanc C, Petres S, Murail S, Taly A, Baaden M, Delarue M, and Corringer PJ
- Subjects
- Allosteric Regulation physiology, Animals, Crystallography, X-Ray, Drosophila melanogaster, Humans, Protein Structure, Tertiary, Receptors, Glycine genetics, Receptors, Glycine metabolism, Structure-Activity Relationship, Receptors, Glycine chemistry
- Abstract
The glycine receptor (GlyR) is a pentameric ligand-gated ion channel (pLGIC) mediating inhibitory transmission in the nervous system. Its transmembrane domain (TMD) is the target of allosteric modulators such as general anesthetics and ethanol and is a major locus for hyperekplexic congenital mutations altering the allosteric transitions of activation or desensitization. We previously showed that the TMD of the human α1GlyR could be fused to the extracellular domain of GLIC, a bacterial pLGIC, to form a functional chimera called Lily. Here, we overexpress Lily in Schneider 2 insect cells and solve its structure by X-ray crystallography at 3.5 Å resolution. The TMD of the α1GlyR adopts a closed-channel conformation involving a single ring of hydrophobic residues at the center of the pore. Electrophysiological recordings show that the phenotypes of key allosteric mutations of the α1GlyR, scattered all along the pore, are qualitatively preserved in this chimera, including those that confer decreased sensitivity to agonists, constitutive activity, decreased activation kinetics, or increased desensitization kinetics. Combined structural and functional data indicate a pore-opening mechanism for the α1GlyR, suggesting a structural explanation for the effect of some key hyperekplexic allosteric mutations. The first X-ray structure of the TMD of the α1GlyR solved here using GLIC as a scaffold paves the way for mechanistic investigation and design of allosteric modulators of a human receptor. more...
- Published
- 2015
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31. Intermediate closed state for glycine receptor function revealed by cysteine cross-linking.
- Author
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Prevost MS, Moraga-Cid G, Van Renterghem C, Edelstein SJ, Changeux JP, and Corringer PJ
- Subjects
- Allosteric Regulation drug effects, Allosteric Regulation physiology, Anesthetics, Intravenous chemistry, Anesthetics, Intravenous pharmacology, Animals, Crystallography, X-Ray, HEK293 Cells, Humans, Ion Channel Gating drug effects, Ion Transport physiology, Propofol chemistry, Propofol pharmacology, Protein Structure, Secondary, Protein Structure, Tertiary, Receptors, Glycine agonists, Receptors, Glycine genetics, Receptors, Glycine metabolism, Xenopus laevis, Ion Channel Gating physiology, Models, Molecular, Receptors, Glycine chemistry
- Abstract
Pentameric ligand-gated ion channels (pLGICs) mediate signal transmission by coupling the binding of extracellular ligands to the opening of their ion channel. Agonist binding elicits activation and desensitization of pLGICs, through several conformational states, that are, thus far, incompletely characterized at the structural level. We previously reported for GLIC, a prokaryotic pLGIC, that cross-linking of a pair of cysteines at both sides of the extracellular and transmembrane domain interface stabilizes a locally closed (LC) X-ray structure. Here, we introduced the homologous pair of cysteines on the human α1 glycine receptor. We show by electrophysiology that cysteine cross-linking produces a gain-of-function phenotype characterized by concomitant constitutive openings, increased agonist potency, and equalization of efficacies of full and partial agonists. However, it also produces a reduction of maximal currents at saturating agonist concentrations without change of the unitary channel conductance, an effect reversed by the positive allosteric modulator propofol. The cross-linking thus favors a unique closed state distinct from the resting and longest-lived desensitized states. Fitting the data according to a three-state allosteric model suggests that it could correspond to a LC conformation. Its plausible assignment to a gating intermediate or a fast-desensitized state is discussed. Overall, our data show that relative movement of two loops at the extracellular-transmembrane interface accompanies orthosteric agonist-mediated gating. more...
- Published
- 2013
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32. Structural basis for ion permeation mechanism in pentameric ligand-gated ion channels.
- Author
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Sauguet L, Poitevin F, Murail S, Van Renterghem C, Moraga-Cid G, Malherbe L, Thompson AW, Koehl P, Corringer PJ, Baaden M, and Delarue M
- Subjects
- Animals, Crystallography, X-Ray, Electrophysiology, Female, Hydrophobic and Hydrophilic Interactions, Ligand-Gated Ion Channels genetics, Ligand-Gated Ion Channels metabolism, Ligands, Models, Chemical, Models, Molecular, Molecular Dynamics Simulation, Mutation genetics, Protein Structure, Quaternary, Serine chemistry, Serine genetics, Serine metabolism, Sodium chemistry, Threonine chemistry, Threonine genetics, Threonine metabolism, Xenopus laevis growth & development, Xenopus laevis metabolism, Ion Channel Gating, Ligand-Gated Ion Channels chemistry, Oocytes metabolism, Sodium metabolism, Water chemistry
- Abstract
To understand the molecular mechanism of ion permeation in pentameric ligand-gated ion channels (pLGIC), we solved the structure of an open form of GLIC, a prokaryotic pLGIC, at 2.4 Å. Anomalous diffraction data were used to place bound anions and cations. This reveals ordered water molecules at the level of two rings of hydroxylated residues (named Ser6' and Thr2') that contribute to the ion selectivity filter. Two water pentagons are observed, a self-stabilized ice-like water pentagon and a second wider water pentagon, with one sodium ion between them. Single-channel electrophysiology shows that the side-chain hydroxyl of Ser6' is crucial for ion translocation. Simulations and electrostatics calculations complemented the description of hydration in the pore and suggest that the water pentagons observed in the crystal are important for the ion to cross hydrophobic constriction barriers. Simulations that pull a cation through the pore reveal that residue Ser6' actively contributes to ion translocation by reorienting its side chain when the ion is going through the pore. Generalization of these findings to the pLGIC family is proposed. more...
- Published
- 2013
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33. Activated G protein α s subunits increase the ethanol sensitivity of human glycine receptors.
- Author
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Yévenes GE, Moraga-Cid G, Romo X, and Aguayo LG
- Subjects
- Cyclic AMP biosynthesis, Cyclic AMP-Dependent Protein Kinases metabolism, Fluorescent Antibody Technique, HEK293 Cells, Humans, Ligand-Gated Ion Channels, Patch-Clamp Techniques, Phosphorylation, Receptors, Glycine genetics, Signal Transduction, Central Nervous System Depressants pharmacology, Ethanol pharmacology, GTP-Binding Protein alpha Subunits, Gs metabolism, GTP-Binding Proteins metabolism, Receptors, Glycine metabolism
- Abstract
It is well known that ethanol modulates the function of the Cys loop ligand-gated ion channels, which include the inhibitory glycine receptors (GlyRs). Previous studies have consistently shown that transmembrane and extracellular sites are essential for ethanol actions in GlyRs. In addition, recent evidence has shown that the ethanol modulation of GlyRs is also affected by G protein activation through Gβγ subunits. However, more specific roles of G protein α subunits on ethanol actions are unknown. Here, we show that the allosteric effect of ethanol on the human α(1) GlyR is selectively enhanced by the expression of Gα(s) Q-L. For example, constitutively active Gα(s), but not Gα(q) or Gα(i), was able to displace the alcohol sensitivity of GlyRs toward low millimolar concentrations (17 ± 4 versus 48 ± 5% at 100 mM). Experiments under conditions that increased cAMP and protein kinase A (PKA)-mediated signaling, on the contrary, did not produce the same enhancement in sensitivity, suggesting that the Gα(s) Q-L effect was not dependent on cAMP/PKA-dependent signaling. On the other hand, the effect of Gα(s) Q-L was blocked by a Gβγ scavenger (9 ± 3% of control). Furthermore, two mutant receptors previously shown to have impaired interactions with Gβγ were not affected by Gα(s) Q-L, suggesting that Gβγ is needed for enhancing ethanol sensitivity. These results support the conclusion that activated Gα(s) can facilitate the Gβγ interaction with GlyRs in presence of ethanol, independent of increases in cAMP signaling. Thus, these data indicate that the activated form of Gα(s) is able to positively influence the effect of ethanol on a type of inhibitory receptor important for motor control, pain, and respiration. more...
- Published
- 2011
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34. Functional prokaryotic-eukaryotic chimera from the pentameric ligand-gated ion channel family.
- Author
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Duret G, Van Renterghem C, Weng Y, Prevost M, Moraga-Cid G, Huon C, Sonner JM, and Corringer PJ
- Subjects
- Alcohols, Amino Acid Sequence, Anesthetics, General, Animals, Bacterial Proteins genetics, Base Sequence, Cell Line, Cloning, Molecular, Cricetinae, DNA, Complementary genetics, Electrophysiology, Ivermectin, Ligand-Gated Ion Channels genetics, Molecular Sequence Data, Oocytes metabolism, Patch-Clamp Techniques, Propofol, Protein Structure, Tertiary genetics, Receptors, Glycine genetics, Recombinant Fusion Proteins genetics, Sequence Analysis, DNA, Xenopus, Bacterial Proteins metabolism, Ligand-Gated Ion Channels metabolism, Models, Molecular, Protein Structure, Tertiary physiology, Receptors, Glycine metabolism, Recombinant Fusion Proteins metabolism
- Abstract
Pentameric ligand-gated ion channels (pLGICs), which mediate chemo-electric signal transduction in animals, have been recently found in bacteria. Despite clear sequence and 3D structure homology, the phylogenetic distance between prokaryotic and eukaryotic homologs suggests significant structural divergences, especially at the interface between the extracellular (ECD) and the transmembrane (TMD) domains. To challenge this possibility, we constructed a chimera in which the ECD of the bacterial protein GLIC is fused to the TMD of the human α1 glycine receptor (α1GlyR). Electrophysiology in Xenopus oocytes shows that it functions as a proton-gated ion channel, thereby locating the proton activation site(s) of GLIC in its ECD. Patch-clamp experiments in BHK cells show that the ion channel displays an anionic selectivity with a unitary conductance identical to that of the α1GlyR. In addition, pharmacological investigations result in transmembrane allosteric modulation similar to the one observed on α1GlyR. Indeed, the clinically active drugs propofol, four volatile general anesthetics, alcohols, and ivermectin all potentiate the chimera while they inhibit GLIC. Collectively, this work shows the compatibility between GLIC and α1GlyR domains and points to conservation of the ion channel and transmembrane allosteric regulatory sites in the chimera. This provides evidence that GLIC and α1GlyR share a highly homologous 3D structure. GLIC is thus a relevant model of eukaryotic pLGICs, at least from the anionic type. In addition, the chimera is a good candidate for mass production in Escherichia coli, opening the way for investigations of "druggable" eukaryotic allosteric sites by X-ray crystallography. more...
- Published
- 2011
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35. Potentiation and inhibition of glycine receptors by tutin.
- Author
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Fuentealba J, Muñoz B, Yévenes G, Moraga-Cid G, Pérez C, Guzmán L, Rigo JM, and Aguayo LG
- Subjects
- Dose-Response Relationship, Drug, HEK293 Cells, Humans, Picrotoxin isolation & purification, Picrotoxin metabolism, Picrotoxin pharmacology, Plant Extracts isolation & purification, Plant Extracts metabolism, Protein Binding physiology, Receptors, Glycine metabolism, Sesquiterpenes isolation & purification, Sesquiterpenes metabolism, Picrotoxin analogs & derivatives, Plant Extracts pharmacology, Plant Leaves, Receptors, Glycine agonists, Receptors, Glycine antagonists & inhibitors, Sesquiterpenes pharmacology
- Abstract
In the present study we characterized the effects of the South American neurotoxin tutin on recombinant glycine receptors (GlyR) expressed in HEK 293 cells using whole-cell patch-clamp techniques. Tutin induced a concentration-dependent inhibition of α(1) and α(2) homomeric GlyRs, with IC(50)s of 35 ± 1 and 15 ± 3 μM, respectively. The co-expression of αβ subunits reduced the potency of tutin, thus increasing the IC(50) to 51 ± 4 and 41 ± 8 μM for α(1)β and α(2)β GlyRs, respectively. The inhibitory effect of tutin was competitive, independent of membrane potential and reversible suggesting a pore independent site. On the other hand, low tutin concentrations enhanced the current, which was not synergic with Zn(2+) or ethanol. A mutation in Lys385 altered ethanol but not tutin sensitivity, suggesting different sites for modulation of α1-containing GlyRs. Our results suggest that tutin affects the GlyR by a mechanism distinct to that of picrotoxin and ethanol, and that the pharmacological profile of tutin exhibits a "Zn-like" behaviour. In conclusion, these results provide information on molecular mechanisms important for understanding the toxic effects of a recently discovered South American neurotoxin., (Copyright © 2010 Elsevier Ltd. All rights reserved.) more...
- Published
- 2011
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36. Molecular requirements for ethanol differential allosteric modulation of glycine receptors based on selective Gbetagamma modulation.
- Author
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Yevenes GE, Moraga-Cid G, Avila A, Guzmán L, Figueroa M, Peoples RW, and Aguayo LG
- Subjects
- Allosteric Regulation drug effects, Dose-Response Relationship, Drug, Humans, Receptors, Glycine genetics, Central Nervous System Depressants pharmacology, Ethanol pharmacology, Receptors, Glycine metabolism
- Abstract
It is now believed that the allosteric modulation produced by ethanol in glycine receptors (GlyRs) depends on alcohol binding to discrete sites within the protein structure. Thus, the differential ethanol sensitivity of diverse GlyR isoforms and mutants was explained by the presence of specific residues in putative alcohol pockets. Here, we demonstrate that ethanol sensitivity in two ligand-gated ion receptor members, the GlyR adult α(1) and embryonic α(2) subunits, can be modified through selective mutations that rescued or impaired Gβγ modulation. Even though both isoforms were able to physically interact with Gβγ, only the α(1) GlyR was functionally modulated by Gβγ and pharmacological ethanol concentrations. Remarkably, the simultaneous switching of two transmembrane and a single extracellular residue in α(2) GlyRs was enough to generate GlyRs modulated by Gβγ and low ethanol concentrations. Interestingly, although we found that these TM residues were different to those in the alcohol binding site, the extracellular residue was recently implicated in conformational changes important to generate a pre-open-activated state that precedes ion channel gating. Thus, these results support the idea that the differential ethanol sensitivity of these two GlyR isoforms rests on conformational changes in transmembrane and extracellular residues within the ion channel structure rather than in differences in alcohol binding pockets. Our results describe the molecular basis for the differential ethanol sensitivity of two ligand-gated ion receptor members based on selective Gβγ modulation and provide a new mechanistic framework for allosteric modulations of abuse drugs. more...
- Published
- 2010
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37. Canonical Wnt3a modulates intracellular calcium and enhances excitatory neurotransmission in hippocampal neurons.
- Author
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Avila ME, Sepúlveda FJ, Burgos CF, Moraga-Cid G, Parodi J, Moon RT, Aguayo LG, Opazo C, and De Ferrari GV
- Subjects
- Animals, Electrophysiology methods, Exocytosis, Immunohistochemistry, Ligands, Low Density Lipoprotein Receptor-Related Protein-6, Models, Biological, Neurons metabolism, Protein Processing, Post-Translational, Rats, Rats, Sprague-Dawley, Receptors, LDL metabolism, Signal Transduction, Wnt3 Protein, Calcium metabolism, Hippocampus metabolism, Wnt Proteins metabolism
- Abstract
A role for Wnt signal transduction in the development and maintenance of brain structures is widely acknowledged. Recent studies have suggested that Wnt signaling may be essential for synaptic plasticity and neurotransmission. However, the direct effect of a Wnt protein on synaptic transmission had not been demonstrated. Here we show that nanomolar concentrations of purified Wnt3a protein rapidly increase the frequency of miniature excitatory synaptic currents in embryonic rat hippocampal neurons through a mechanism involving a fast influx of calcium from the extracellular space, induction of post-translational modifications on the machinery involved in vesicle exocytosis in the presynaptic terminal leading to spontaneous Ca(2+) transients. Our results identify the Wnt3a protein and a member of its complex receptor at the membrane, the low density lipoprotein receptor-related protein 6 (LRP6) coreceptor, as key molecules in neurotransmission modulation and suggest cross-talk between canonical and Wnt/Ca(2+) signaling in central neurons. more...
- Published
- 2010
- Full Text
- View/download PDF
38. Blockade of ethanol-induced potentiation of glycine receptors by a peptide that interferes with Gbetagamma binding.
- Author
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Guzman L, Moraga-Cid G, Avila A, Figueroa M, Yevenes GE, Fuentealba J, and Aguayo LG
- Subjects
- Amino Acid Sequence, Binding Sites, Cell Line, Electrophysiology, Escherichia coli genetics, G Protein-Coupled Inwardly-Rectifying Potassium Channels metabolism, Glutathione Transferase genetics, Glutathione Transferase metabolism, Humans, Models, Molecular, Molecular Sequence Data, Peptide Fragments chemistry, Protein Binding, Protein Conformation, Protein Subunits, Receptors, GABA-B genetics, Receptors, Glycine metabolism, Recombinant Fusion Proteins, Signal Transduction drug effects, Ethanol pharmacology, GTP-Binding Protein beta Subunits metabolism, GTP-Binding Protein gamma Subunits metabolism, Peptide Fragments pharmacology, Receptors, Glycine antagonists & inhibitors
- Abstract
The large intracellular loop (IL) of the glycine receptor (GlyR) interacts with various signaling proteins and plays a fundamental role in trafficking and regulation of several receptor properties, including a direct interaction with Gbetagamma. In the present study, we found that mutation of basic residues in the N-terminal region of the IL reduced the binding of Gbetagamma to 21 +/- 10% of control. Two basic residues in the C-terminal region, on the other hand, contributed to a smaller extent to Gbetagamma binding. Using docking analysis, we found that both basic regions of the IL bind in nearby regions to the Gbetagamma dimer, within an area of high density of amino acids having an electronegative character. Thereafter, we generated a 17-amino acid peptide with the N-terminal sequence of the wild-type IL (RQH) that was able to inhibit the in vitro binding of Gbetagamma to GlyRs to 57 +/- 5% of control in glutathione S-transferase pull-down assays using purified proteins. More interestingly, when the peptide was intracellularly applied to human embryonic kidney 293 cells, it inhibited the Gbetagamma-mediated modulations of G protein-coupled inwardly rectifying potassium channel by baclofen (24 +/- 14% of control) and attenuated the GlyR potentiation by ethanol (51 +/- 10% versus 10 +/- 3%). more...
- Published
- 2009
- Full Text
- View/download PDF
39. A selective G betagamma-linked intracellular mechanism for modulation of a ligand-gated ion channel by ethanol.
- Author
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Yevenes GE, Moraga-Cid G, Peoples RW, Schmalzing G, and Aguayo LG
- Subjects
- Humans, Ligands, Receptors, Glycine agonists, Ethanol pharmacology, GTP-Binding Protein beta Subunits physiology, GTP-Binding Protein gamma Subunits physiology, Ion Channel Gating drug effects, Ion Channels drug effects
- Abstract
The current understanding about ethanol effects on the ligand-gated ion channel (LGIC) superfamily has been restricted to identify potential binding sites within transmembrane (TM) domains in the Cys-loop family. Here, we demonstrate a key role of the TM3-4 intracellular loop and G betagamma signaling for potentiation of glycine receptors (GlyRs) by ethanol. We discovered 2 motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for the actions of pharmacological concentrations of ethanol. Significantly, the sites were ethanol-specific because they did not alter the sensitivity to general anesthetics, neurosteroids, or longer n-alcohols. Furthermore, G betagamma scavengers selectively attenuated the ethanol effects on recombinant and native neuronal GlyRs. These results show a selective mechanism for low-ethanol concentration effects on the GlyR and provide a mechanism on ethanol pharmacology, which may be applicable to other LGIC members. Moreover, these data provide an opportunity to develop new genetically modified animal models and novel drugs to treat alcohol-related medical concerns. more...
- Published
- 2008
- Full Text
- View/download PDF
40. Mechanistic insights and functional determinants of the transport cycle of the ascorbic acid transporter SVCT2. Activation by sodium and absolute dependence on bivalent cations.
- Author
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Godoy A, Ormazabal V, Moraga-Cid G, Zúñiga FA, Sotomayor P, Barra V, Vasquez O, Montecinos V, Mardones L, Guzmán C, Villagrán M, Aguayo LG, Oñate SA, Reyes AM, Cárcamo JG, Rivas CI, and Vera JC
- Subjects
- Amino Acid Sequence, Ascorbic Acid chemistry, Calcium chemistry, Cations, Cell Line, Tumor, Dose-Response Relationship, Drug, Humans, Kinetics, Magnesium chemistry, Melanoma metabolism, Models, Biological, Molecular Sequence Data, Organic Anion Transporters, Sodium-Dependent chemistry, Sequence Homology, Amino Acid, Sodium-Coupled Vitamin C Transporters, Symporters chemistry, Organic Anion Transporters, Sodium-Dependent physiology, Sodium chemistry, Symporters physiology
- Abstract
We characterized the human Na(+)-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na(+)-dependent transporter. The properties of SVCT2 are modulated by Ca(2+)/Mg(2+) and a reciprocal functional interaction between Na(+) and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na(+) increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport K(m) without affecting the V(max), thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na(+) cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na(+):ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na(+):ascorbic acid:Na(+). However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca(2+)/Mg(2+) for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport V(max) without affecting the transport K(m) or the Na(+) cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca(2+)/Mg(2+). At least three active states can be envisioned, including a low affinity conformation at Na(+) concentrations below 20 mM and two high affinity conformations at elevated Na(+) concentrations whose Na(+) cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca(2+)/Mg(2+)-dependent transporter. more...
- Published
- 2007
- Full Text
- View/download PDF
41. Molecular determinants for G protein betagamma modulation of ionotropic glycine receptors.
- Author
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Yevenes GE, Moraga-Cid G, Guzmán L, Haeger S, Oliveira L, Olate J, Schmalzing G, and Aguayo LG
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Cell Line, Electrophysiology, GTP-Binding Protein beta Subunits chemistry, GTP-Binding Protein gamma Subunits chemistry, GTP-Binding Proteins chemistry, Humans, Lipid Bilayers, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Structure, Secondary, Signal Transduction, GTP-Binding Protein beta Subunits physiology, GTP-Binding Protein gamma Subunits physiology, Receptors, Glycine chemistry, Receptors, Glycine physiology
- Abstract
The ligand-gated ion channel superfamily plays a critical role in neuronal excitability. The functions of glycine receptor (GlyR) and nicotinic acetylcholine receptor are modulated by G protein betagamma subunits. The molecular determinants for this functional modulation, however, are still unknown. Studying mutant receptors, we identified two basic amino acid motifs within the large intracellular loop of the GlyR alpha(1) subunit that are critical for binding and functional modulation by Gbetagamma. Mutations within these sequences demonstrated that all of the residues detected are important for Gbetagamma modulation, although both motifs are necessary for full binding. Molecular modeling predicts that these sites are alpha-helixes near transmembrane domains 3 and 4, near to the lipid bilayer and highly electropositive. Our results demonstrate for the first time the sites for G protein betagamma subunit modulation on GlyRs and provide a new framework regarding the ligand-gated ion channel superfamily regulation by intracellular signaling. more...
- Published
- 2006
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42. Effects of pulp and paper mill discharges on caged rainbow trout (Oncorhynchus mykiss): biomarker responses along a pollution gradient in the Biobio River, Chile.
- Author
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Orrego R, Burgos A, Moraga-Cid G, Inzunza B, Gonzalez M, Valenzuela A, Barra R, and Gavilán JF
- Subjects
- Acetylcholine metabolism, Animals, Chile, Cytochrome P-450 CYP1A1 metabolism, Female, Gonads, Industry, Reproduction drug effects, Sexual Maturation, Vitellogenins blood, Water Pollution, Chemical adverse effects, Biomarkers analysis, Environmental Monitoring methods, Industrial Waste adverse effects, Oncorhynchus mykiss physiology, Paper, Rivers, Water Pollutants, Chemical toxicity
- Abstract
Caging experiments were conducted using hatchery-reared, immature, female rainbow trout (Oncorhynchus mykiss) in three previously defined areas of the Biobio River (south central Chile) representing a pollution gradient from the pulp and paper mill discharges area: a pre-impact area (upstream area, reference location), an impact area (area directly influenced), and a postimpact area (downstream area, less influenced). No significant changes were observed in the physiological index as represented by condition factor (K) and liver somatic index during different sampling times (after 11, 21, and 30 d of exposure). Ethoxyresorufin-O-deethylase activities were significantly higher in trout caged at the impact and postimpact discharges areas (two- to fourfold) compared with the reference (pre-impact) area, and a strong inhibition of acetylcholinesterase activity, reaching 50%, was observed mainly in fish caged at the impact area. A significant endocrine-disrupting effect (reproductive level) was evidenced by significant increments in gonad somatic index and plasma vitellogenin levels combined with an induction of gonad maturation (presence of vitellogenic oocytes) in trout caged at the impact and postimpact areas. These results, generated by an in situ approach, confirmed our group's findings for trout exposed to sediment in the laboratory: discharges of pulp mill effluent in the Biobio River are associated with the effects evaluated at different biological levels. more...
- Published
- 2006
- Full Text
- View/download PDF
43. Reproductive, physiological, and biochemical responses in juvenile female rainbow trout (Oncorhynchus mykiss) exposed to sediment from pulp and paper mill industrial discharge areas.
- Author
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Orrego R, Moraga-Cid G, González M, Barra R, Valenzuela A, Burgos A, and Gavilán JF
- Subjects
- Animals, Cytochrome P-450 CYP1A1 metabolism, Female, Gonads drug effects, Male, Oncorhynchus mykiss metabolism, Vitellogenins blood, Industrial Waste, Oncorhynchus mykiss physiology, Reproduction drug effects, Textile Industry, Water Pollutants, Chemical toxicity
- Abstract
Four pulp and paper mills discharge their effluents in the same section of the Biobio River in central southern Chile. Pulp mill effluents are a very complex mixture with characteristics that depend on the type of raw material, the process technology, and the effluent treatment. To investigate the effect of pulp mill effluent discharges, immature Oncorhynchus mykiss were exposed to river sediments in the laboratory for 29 d. Three sampling areas were defined in a spatial gradient in the river: Preimpact, impact, and postimpact zones relative to the pulp and paper mill discharge areas. Ethoxyresorufin-O-deethylase activities were significantly higher in fish exposed to impact and postimpact sediments when compared to those exposed to preimpact sediments, and higher levels of vitellogenin were observed in the plasma of female fish exposed to impact and postimpact sediments. Histological analysis of the gonadal tissue showed an induction of gonadal maturation in fish exposed to sediment coming from the impact and postimpact zones (oocytes in a vitellogenic state). No site differences were observed in erythrocytes, although differences were noted in the leukocytes in the exposure areas. Finally, the biomarker approach showed evidence that the sediment associated with pulp mill effluent discharges produces some effects in fish under laboratory conditions. more...
- Published
- 2005
- Full Text
- View/download PDF
44. An RNA vaccine based on recombinant Semliki Forest virus particles expressing the Cu,Zn superoxide dismutase protein of Brucella abortus induces protective immunity in BALB/c mice.
- Author
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Oñate AA, Donoso G, Moraga-Cid G, Folch H, Céspedes S, and Andrews E
- Subjects
- Animals, Female, Mice, Mice, Inbred BALB C, Superoxide Dismutase genetics, T-Lymphocytes, Cytotoxic immunology, Vaccination, Brucella Vaccine immunology, Brucella abortus immunology, Brucellosis prevention & control, RNA immunology, Semliki forest virus genetics, Superoxide Dismutase immunology, Vaccines, Synthetic immunology, Virion genetics
- Abstract
We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections. more...
- Published
- 2005
- Full Text
- View/download PDF
45. Intraspleen delivery of a DNA vaccine coding for superoxide dismutase (SOD) of Brucella abortus induces SOD-specific CD4+ and CD8+ T cells.
- Author
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Muñoz-Montesino C, Andrews E, Rivers R, González-Smith A, Moraga-Cid G, Folch H, Céspedes S, and Oñate AA
- Subjects
- Animals, Bacterial Vaccines administration & dosage, Bacterial Vaccines immunology, Brucella abortus enzymology, Brucella abortus genetics, Brucella abortus pathogenicity, Brucellosis immunology, Brucellosis microbiology, Brucellosis prevention & control, Colony Count, Microbial, Female, Interferon-gamma biosynthesis, Lymphocyte Activation, Mice, Mice, Inbred BALB C, Superoxide Dismutase genetics, Vaccination, Vaccines, DNA administration & dosage, Brucella abortus immunology, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Spleen microbiology, Superoxide Dismutase immunology, Vaccines, DNA immunology
- Abstract
In the development of vaccines capable of providing immunity against brucellosis, Cu-Zn superoxide dismutase (SOD) has been demonstrated to be one of the protective immunogens of Brucella abortus. In an earlier study, we provided strong evidence that intramuscular injection with a plasmid DNA carrying the SOD gene (pcDNA-SOD) was able to induce a protective immune response. The present study was designed to characterize T-cell immune responses after an intraspleen (i.s.) vaccination of BALB/c mice with pcDNA-SOD. Animals vaccinated with pcDNA-SOD did not develop SOD-specific antibodies, at least until week 4 after immunization (the end of the experiment), and in vitro stimulation of their splenocytes with either recombinant Cu-Zn SOD or crude Brucella protein induced the secretion of gamma interferon (IFN-gamma), but not interleukin-4, and elicited the induction of cytotoxic-T-lymphocyte activity. Upon analyzing the SOD-specific T-cell responses, the pcDNA-SOD vaccination was found to be stimulating both CD4(+)- and CD8(+)-T-cell populations. However, only the CD4(+) population was able to produce IFN-gamma and only the CD8(+) population was able to induce cytotoxic activity. Nevertheless, although i.s. route vaccination induces a significant level of protection in BALB/c mice against challenge with the virulent B. abortus strain 2308, vaccination by the intramuscular route with a similar amount of plasmid DNA does not protect. Based on these results, we conclude that i.s. immunization with pcDNA-SOD vaccine efficiently induced a Th1 type of immune response and a protective response that could be related to IFN-gamma production and cytotoxic activity against infected cells by SOD-specific CD4(+) and CD8(+) T cells, respectively. more...
- Published
- 2004
- Full Text
- View/download PDF
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