Control of Ethanol Sensitivity of the Glycine Receptor <i>α</i>3 Subunit by Transmembrane 2, the Intracellular Splice Cassette and C-Terminal Domain

Sánchez, Alexandra; Yévenes, Gonzalo E.; Martín, Loreto San; Burgos, Carlos F.; Moraga-Cid, Gustavo; Harvey, Robert J.; Aguayo, Luis G.

doi:10.1124/jpet.114.221143

Cited by 16 publications

(30 citation statements)

References 56 publications

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“…These receptors were attractive targets since they are expressed on macrophages, have been shown to alter IL-1β production, and are modified by ethanol. However, there are many other receptors on macrophages, not already associated with inflammasome activity, with which ethanol may interact (59, 60). It is possible that one of these may be the relevant target of the alcohol to prevent inflammasome activation.…”

Section: Discussionmentioning

confidence: 99%

Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation

Hoyt

Ather

Randall

et al. 2016

The Journal of Immunology

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Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the pro-inflammatory cytokines IL-1β and IL-18, can be inhibited by ethanol and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as diminished ASC speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of GABAA receptor activation or NMDA receptor inhibition, but was associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, while administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1β secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate-induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC, were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other short-chain alcohols.

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Section: Discussionmentioning

confidence: 99%

Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation

Hoyt

Ather

Randall

et al. 2016

The Journal of Immunology

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“…For example, recent electrophysiological studies in α1/3 GlyR chimeras (Sánchez et al, 2015) indicate that a complete understanding of subtype-specific alcohol modulation involves both TM and intracellular mechanisms of action. However, the dominant influence of the GlyR TM domain suggests that the intracellular loop, which is absent in both GLIC and the GLIC/GlyR chimera ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors

Horani

Stater

Corringer

et al. 2015

Alcoholism Clin & Exp Res

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Background Mutagenesis and labeling studies have identified amino acids from the human α1 glycine receptor (GlyR) extracellular, transmembrane (TM), and intracellular domains in mediating ethanol potentiation. However, limited high-resolution structural data for physiologically relevant receptors in this Cys-loop receptor superfamily have made pinpointing the critical amino acids difficult. Homologous ion channels from lower organisms provide conserved models for structural and functional properties of Cys-loop receptors. We previously demonstrated that a single amino acid variant of the Gloeobacter violaceus ligand-gated ion channel (GLIC) produced ethanol and anesthetic sensitivity similar to that of GlyRs and provided crystallographic evidence for ethanol binding to GLIC. Methods We directly compared ethanol modulation of the α1 GlyR and GLIC to a chimera containing the transmembrane domain from human α1 GlyRs and the ligand-binding domain of GLIC using two-electrode voltage clamp electrophysiology of receptors expressed in Xenopus laevis oocytes. Results Ethanol potentiated α1 GlyRs in a concentration-dependent manner in the presence of zinc-chelating agents, but did not potentiate GLIC at pharmacologically relevant concentrations. The GLIC/GlyR chimera recapitulated the ethanol potentiation of GlyRs, without apparent sensitivity to zinc chelation. For chimera expression in oocytes, it was essential to suppress leakage current by adding 50 μM picrotoxin to the media, a technique that may have applications in expression of other ion channels. Conclusions Our results are consistent with a transmembrane mechanism of ethanol modulation in Cys-loop receptors. This work highlights the relevance of bacterial homologs as valuable model systems for studying ion channel function of human receptors and demonstrates the modularity of these channels across species.

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“…For instance, α3 GlyRs differ in their modulation by ethanol with regards to α1 GlyRs. The α3 GlyRs expressed in HEK293 cells were not potentiated by low concentrations of ethanol (≤100 mM) or by Gβγ [162]. The structural elements of ethanol insensitivity present in α3 subunits revealed interesting information about the mechanisms involved in the modulation of GlyR by ethanol.…”

Section: Mechanisms Of Ethanol Modulation On Glyrsmentioning

confidence: 99%

“…Thus, insertion of the intracellular α3L splice cassette into the α1 GlyR inhibited the potentiation of the glycine evoked-current in the presence of 100 mM ethanol or after the dialysis of GTP-γ-S. This indicates that the 15 amino acids encoded by this splice cassette act as a negative regulator in the response to ethanol, likely associated to the formation of an α-helix and change in flexibility of the intracellular loop domain [162,163]. Meanwhile, the A254G mutation and the addition of the C-terminus of α1 into the α3 subunit converted the receptor into one capable of being potentiated by ethanol, which indicates that the absence of these elements along with the splice cassette are responsible for the α3 GlyR insensitivity to ethanol and Gβγ [162].…”

Section: Mechanisms Of Ethanol Modulation On Glyrsmentioning

confidence: 99%

“…This indicates that the 15 amino acids encoded by this splice cassette act as a negative regulator in the response to ethanol, likely associated to the formation of an α-helix and change in flexibility of the intracellular loop domain [162,163]. Meanwhile, the A254G mutation and the addition of the C-terminus of α1 into the α3 subunit converted the receptor into one capable of being potentiated by ethanol, which indicates that the absence of these elements along with the splice cassette are responsible for the α3 GlyR insensitivity to ethanol and Gβγ [162]. Additionally, based on the full conservation of amino acids between α3 and α1 subunits that generate a binding pocket in the transmembrane domains for ethanol, the loss of sensitivity for this alcohol in α3 GlyRs is not related to this particular region [162].…”

Section: Mechanisms Of Ethanol Modulation On Glyrsmentioning

confidence: 99%

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Ethanol effects on glycinergic transmission: From molecular pharmacology to behavior responses

Burgos

Muñoz

Guzmán

et al. 2015

Pharmacological Research

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It is well accepted that ethanol is able to produce major health and economic problems associated to its abuse. Because of its intoxicating and addictive properties, it is necessary to analyze its effect in the central nervous system. However, we are only now learning about the mechanisms controlling the modification of important membrane proteins such as ligand-activated ion channels by ethanol. Furthermore, only recently are these effects being correlated to behavioral changes. Current studies show that the glycine receptor (GlyR) is a susceptible target for low concentrations of ethanol (5 to 100 mM). GlyRs are relevant for the effects of ethanol because they are found in the spinal cord and brain stem where they primarily express the α1 subunit. More recently, the presence of GlyRs was described in higher regions, such as the hippocampus and nucleus accumbens, with a prevalence of α2/α3 subunits. Here, we review data on the following aspects of ethanol effects on GlyRs: 1) direct interaction of ethanol with amino acids in the extracellular or transmembrane domains, and indirect mechanisms through the activation of signal transduction pathways; 2) analysis of α2 and α3 subunits having different sensitivities to ethanol which allows the identification of structural requirements for ethanol modulation present in the intracellular domain and C-terminal region; 3) Genetically modified knock-in mice for α1 GlyRs that have an impaired interaction with G protein and demonstrate reduced ethanol sensitivity without changes in glycinergic transmission; and 4) GlyRs as potential therapeutic targets.

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Control of Ethanol Sensitivity of the Glycine Receptor α3 Subunit by Transmembrane 2, the Intracellular Splice Cassette and C-Terminal Domain

Cited by 16 publications

References 56 publications

Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation

Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation

Ethanol Modulation is Quantitatively Determined by the Transmembrane Domain of Human α1 Glycine Receptors

Ethanol effects on glycinergic transmission: From molecular pharmacology to behavior responses

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