RESUMO
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 ß 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 Gßγ, 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 ß subunit on ethanol sensitivity of α2 subunits. Specifically, we found a differential allosteric modulation in homomeric α2 GlyRs compared with the α2ß heteromeric conformation. Indeed, while α2 was insensitive, α2ß GlyRs were substantially potentiated by ethanol, GTP-γ-S, propofol, Zn2+ and trichloroethanol. Furthermore, a Gßγ scavenger (ct-GRK2) selectively attenuated the effects of ethanol on recombinant α2ß GlyRs. Mutations in an α2 GlyR co-expressed with the ß subunit (α2AAß) 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.
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Background: The beta-amyloid peptide (Aß) involved in Alzheimer's disease (AD) has been described to associate/aggregate on the cell surface disrupting the membrane through pore formation and breakage. However, molecular determinants involved for this interaction (e.g., some physicochemical properties of the cell membrane) are largely unknown. Since cholesterol is an important molecule for membrane structure and fluidity, we examined the effect of varying cholesterol content with the association and membrane perforation by Aß in cultured hippocampal neurons. Methods: To decrease or increase the levels of cholesterol in the membrane we used methyl-ß-cyclodextrin (MßCD) and MßCD/cholesterol, respectively. We analyzed if membrane fluidity was affected using generalized polarization (GP) imaging and the fluorescent dye di-4-ANEPPDHQ. Additionally membrane association and perforation was assessed using immunocytochemistry and electrophysiological techniques, respectively. Results: The results showed that cholesterol removal decreased the macroscopic association of Aß to neuronal membranes (fluorescent-puncta/20 µm: control = 18 ± 2 vs. MßCD = 10 ± 1, p < 0.05) and induced a facilitation of the membrane perforation by Aß with respect to control cells (half-time for maximal charge transferred: control = 7.2 vs. MßCD = 4.4). Under this condition, we found an increase in membrane fluidity (46 ± 3.3% decrease in GP value, p < 0.001). On the contrary, increasing cholesterol levels incremented membrane rigidity (38 ± 2.7% increase in GP value, p < 0.001) and enhanced the association and clustering of Aß (fluorescent-puncta/20 µm: control = 18 ± 2 vs. MßCD = 10 ± 1, p < 0.01), but inhibited membrane disruption. Conclusion: Our results strongly support the significance of plasma membrane organization in the toxic effects of Aß in hippocampal neurons, since fluidity can regulate distribution and insertion of the Aß peptide in the neuronal membrane.
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Alzheimer's disease is a neurodegenerative disorder that affects mostly the elderly. The main histopathological markers are the senile plaques formed by amyloid-ß peptide (Aß) aggregates that can perforate the plasma membrane of cells, increasing the intracellular calcium levels and releasing synaptic vesicles that finally lead to a delayed synaptic failure. Several membrane proteins and lipids interact with Aß affecting its toxicity in neurons. Here, we focus on NMDA receptors (NMDARs) as proteins that could be modulating the association and neurotoxic perforation induced by Aß on the plasma membrane. In fact, our results showed that decreasing NMDARs, using enzymatic or siRNA approaches, increased the association of Aß to the neurons. Furthermore, overexpression of NMDARs also resulted in an enhanced association between NMDA and Aß. Functionally, the reduction in membrane NMDARs augmented the process of membrane perforation. On the other hand, overexpressing NMDARs had a protective effect because Aß was now unable to cause membrane perforation, suggesting a complex relationship between Aß and NMDARs. Because previous studies have recognized that Aß oligomers are able to increase membrane permeability and produce amyloid pores, the present study supports the conclusion that NMDARs play a critical protective role on Aß actions in hippocampal neurons. These results could explain the lack of correlation between brain Aß burden and clinically observed dementia.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Membrana Celular/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Células HEK293 , Hipocampo/citologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Proteínas Associadas aos Microtúbulos/metabolismo , N-Metilaspartato/farmacologia , Gravidez , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
The mechanism by which amyloid-ß (Aß) produces brain dysfunction in patients with Alzheimer's disease is largely unknown. According to previous studies, Aß might share perforating properties with gramicidin, a well-accepted membrane-disrupting peptide. Therefore, we hypothesize that the key steps leading to synaptotoxicity by Aß and gramicidin involve peptide aggregation, pore formation, and calcium dysregulation. Here, we show that Aß and gramicidin form aggregates enriched in ß-sheet structures using electron microscopy, and Thioflavin and Congo Red staining techniques. Also, we found that Aß and gramicidin display fairly similar actions in hippocampal cell membranes, i.e. inducing Ca(2+) entry and synaptoxicity characterized by the loss of synaptic proteins and a decrease in neuronal viability. These effects were not observed in a Ca(2+) free solution, indicating that both Aß and gramicidin induce neurotoxicity by a Ca(2+)-dependent mechanism. Using combined perforated patch clamp and imaging recordings, we found that only Aß produced a perforation that progressed from a small (Cl(-)-selective pore) to a larger perforation that allowed the entry of fluorescent molecules. Therefore, based on these results, we propose that the perforation at the plasma membrane by Aß is a dynamic process that is critical in producing neurotoxicity similar to that found in the brains of AD patients.
Assuntos
Doença de Alzheimer/etiologia , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Gramicidina/metabolismo , Gramicidina/toxicidade , Hipocampo/citologia , Neurônios/citologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/patologia , Membrana Celular/ultraestrutura , Células HEK293 , Hipocampo/metabolismo , Humanos , Potenciais da Membrana/efeitos dos fármacos , Microscopia Eletrônica de Transmissão e Varredura , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-DawleyRESUMO
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 α1ß2 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 α1ß2 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.
Assuntos
Anestésicos Intravenosos/farmacologia , Fenilalanina/química , Fenilalanina/genética , Propofol/farmacologia , Receptores de GABA-A/química , Receptores de GABA-A/efeitos dos fármacos , Receptores de Glicina/química , Receptores de Glicina/efeitos dos fármacos , Alanina/química , Alanina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , DNA/genética , Fenômenos Eletrofisiológicos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Células HEK293 , Humanos , Cinética , Dados de Sequência Molecular , Mutação/fisiologia , Técnicas de Patch-Clamp , Receptores de GABA-A/genética , Receptores de Glicina/genética , TransfecçãoRESUMO
The mechanisms that induce Alzheimer's disease (AD) are largely unknown thereby deterring the development of disease-modifying therapies. One working hypothesis of AD is that Abeta excess disrupts membranes causing pore formation leading to alterations in ionic homeostasis. However, it is largely unknown if this also occurs in native brain neuronal membranes. Here we show that similar to other pore forming toxins, Abeta induces perforation of neuronal membranes causing an increase in membrane conductance, intracellular calcium and ethidium bromide influx. These data reveal that the target of Abeta is not another membrane protein, but that Abeta itself is the cellular target thereby explaining the failure of current therapies to interfere with the course of AD. We propose that this novel effect of Abeta could be useful for the discovery of anti AD drugs capable of blocking these "Abeta perforates". In addition, we demonstrate that peptides that block Abeta neurotoxicity also slow or prevent the membrane-perforating action of Abeta.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
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 Gßγ modulation. Even though both isoforms were able to physically interact with Gßγ, only the α(1) GlyR was functionally modulated by Gßγ 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 Gßγ 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 Gßγ modulation and provide a new mechanistic framework for allosteric modulations of abuse drugs.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Receptores de Glicina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Receptores de Glicina/genéticaRESUMO
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.
Assuntos
Etanol/farmacologia , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Subunidades gama da Proteína de Ligação ao GTP/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Humanos , Ligantes , Receptores de Glicina/agonistasRESUMO
Glycine receptors (GlyRs), together with GABA(A) and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein betagamma dimer (Gbetagamma). We found that the amplitude of the glycine-activated Cl- current was enhanced after application of purified Gbetagamma or after activation of a G protein-coupled receptor. Overexpression of three distinct G protein alpha subunits (Galpha), as well as the Gbetagamma scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gbetagamma increased the GlyR open probability (nP(o)). Our results indicate that this interaction of Gbetagamma with GlyRs regulates both motor and sensory functions in the central nervous system.
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Glicina/farmacologia , Proteínas Heterotriméricas de Ligação ao GTP/farmacologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Animais , Células Cultivadas , Canais de Cloreto/fisiologia , Condutividade Elétrica , Eletrofisiologia , Proteínas de Ligação ao GTP/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Peptídeos/farmacologia , Receptores de Glicina/efeitos dos fármacos , Receptores de Glicina/metabolismo , Receptores de Glicina/fisiologia , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
Despite the fact that ethanol is one of the most widely used psychoactive agents, the mechanisms and sites of action by which it modifies brain functions are only now being elucidated. Studies over the last decade have shown that ethanol can specifically alter the function of several ligand-activated ion channels including N-methyl-D-aspartate (NMDA), serotonin (5-HT(3)), glycine and GABA(A) receptors. After several years of extensive research in this field, the resolution of what, where and how ethanol modifies GABA(A) receptors continues to be controversial. For example, after demonstrating that ethanol was able to alter Cl(-) flux in synaptoneurosomes and cultured neurons, several electrophysiological studies were unable to show enhancement of the GABA(A) receptor current in single neurons. The lack of positive results with low ethanol concentrations was interpreted as being due to receptor heterogeneity and differences in intracellular modulation by protein kinases and calcium. The existence of high receptor heterogeneity with respect to ethanol sensitivity has been supported by studies done in a variety of cell types which showed that ethanol potentiated some, but not other neurons. Adding to this complexity, it was shown that while some hippocampal GABA(A) receptors can be affected by ethanol concentrations between 1 and 100 mM, others are only sensitive to concentrations above 200 mM. The curve of the relationship between low ethanol concentrations and current enhancement suggests a high degree of complexity in the molecular interaction because of its steepness and "inverted" U shape. Similarly, the effects of ethanol on GABA(A) receptors seems much more complex than those of benzodiazepines, barbiturates and neurosteroids. The major problem encountered in advancing understanding of the mechanism of ethanol action in native neuronal receptors has been the large variability detected in ethanol sensitivity. For example, several studies have shown that only some groups of neurons are sensitive to pharmacologically relevant concentrations of ethanol (1-100 mM). This receptor sensitivity variability has not been resolved using recombinant expression systems. For example, studies performed in recombinant receptors, although important for elucidating molecular requirements, have shown that they are less sensitive to ethanol suggesting that neuronal substrates are important for ethanol actions. In this review, we discuss the possibility that ethanol's action on the GABA(A) receptor may not be due solely to a direct interaction with the receptor protein, but that its effects could also be modulated by intracellular regulation, and that this latter effect is the more physiologically relevant one. Data in cortical and hippocampal neurons suggest that ethanol action on the receptor is labile, and that it also depends on repetitive stimulation and neuron integrity. In addition, the action of ethanol can be modified by activation of protein kinases and neuronal development. Finally, we discuss that the best approach for studying the interaction between the receptor and ethanol is through the combined use of recombinant receptors and overexpression in neurons.