| 1. |
- Brömstrup, Torben, et al.
(författare)
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Inhibition versus Potentiation of Ligand-Gated Ion Channels Can Be Altered by a Single Mutation that Moves Ligands between Intra- and Intersubunit Sites
- 2013
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 21:8, s. 1307-1316
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Tidskriftsartikel (refereegranskat)abstract
- Pentameric ligand-gated ion channels (pLGICs) are similar in structure but either inhibited or potentiated by alcohols and anesthetics. This dual modulation has previously not been understood, but the determination of X-ray structures of prokaryotic GLIC provides an ideal model system. Here, we show that a single-site mutation at the F14' site in the GLIC transmembrane domain turns desflurane and chloroform from inhibitors to potentiators, and that this is explained by competing allosteric sites. The F14'A mutation opens an intersubunit site lined by N239 (15'), 1240 (16'), and Y263. Free energy calculations confirm this site is the preferred binding location for desflurane and chloroform in GLIC F14'A. In contrast, both anesthetics prefer an intrasubunit site in wild-type GLIC. Modulation is therefore the net effect of competitive binding between the intersubunit potentiating site and an intrasubunit inhibitory site. This provides direct evidence for a dual-site model of allosteric regulation of pLGICs.
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| 4. |
- Howard, Rebecca J, et al.
(författare)
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Structural basis for alcohol modulation of a pentameric ligand-gated ion channel
- 2011
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:29, s. 12149-54
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Tidskriftsartikel (refereegranskat)abstract
- Despite its long history of use and abuse in human culture, the molecular basis for alcohol action in the brain is poorly understood. The recent determination of the atomic-scale structure of GLIC, a prokaryotic member of the pentameric ligand-gated ion channel (pLGIC) family, provides a unique opportunity to characterize the structural basis for modulation of these channels, many of which are alcohol targets in brain. We observed that GLIC recapitulates bimodal modulation by n-alcohols, similar to some eukaryotic pLGICs: methanol and ethanol weakly potentiated proton-activated currents in GLIC, whereas n-alcohols larger than ethanol inhibited them. Mapping of residues important to alcohol modulation of ionotropic receptors for glycine, γ-aminobutyric acid, and acetylcholine onto GLIC revealed their proximity to transmembrane cavities that may accommodate one or more alcohol molecules. Site-directed mutations in the pore-lining M2 helix allowed the identification of four residues that influence alcohol potentiation, with the direction of their effects reflecting α-helical structure. At one of the potentiation-enhancing residues, decreased side chain volume converted GLIC into a highly ethanol-sensitive channel, comparable to its eukaryotic relatives. Covalent labeling of M2 positions with an alcohol analog, a methanethiosulfonate reagent, further implicated residues at the extracellular end of the helix in alcohol binding. Molecular dynamics simulations elucidated the structural consequences of a potentiation-enhancing mutation and suggested a structural mechanism for alcohol potentiation via interaction with a transmembrane cavity previously termed the "linking tunnel." These results provide a unique structural model for independent potentiating and inhibitory interactions of n-alcohols with a pLGIC family member.
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| 6. |
- Ökvist, Anna, et al.
(författare)
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Neuroadaptations in human chronic alcoholics : dysregulation of the NF-κB system
- 2007
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 2:9, s. e930-
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Alcohol dependence and associated cognitive impairments apparently result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. Here we investigated whether transcription factors of Nuclear Factor-kappaB (NF-kappaB) family, controlling neuronal plasticity and neurodegeneration, are involved in these adaptations in human chronic alcoholics. METHODS AND FINDINGS: Analysis of DNA-binding of NF-kappaB (p65/p50 heterodimer) and the p50 homodimer as well as NF-kappaB proteins and mRNAs was performed in postmortem human brain samples from 15 chronic alcoholics and 15 control subjects. The prefrontal cortex involved in alcohol dependence and cognition was analyzed and the motor cortex was studied for comparison. The p50 homodimer was identified as dominant kappaB binding factor in analyzed tissues. NF-kappaB and p50 homodimer DNA-binding was downregulated, levels of p65 (RELA) mRNA were attenuated, and the stoichiometry of p65/p50 proteins and respective mRNAs was altered in the prefrontal cortex of alcoholics. Comparison of a number of p50 homodimer/NF-kappaB target DNA sites, kappaB elements in 479 genes, down- or upregulated in alcoholics demonstrated that genes with kappaB elements were generally upregulated in alcoholics. No significant differences between alcoholics and controls were observed in the motor cortex. CONCLUSIONS: We suggest that cycles of alcohol intoxication/withdrawal, which may initially activate NF-kappaB, when repeated over years downregulate RELA expression and NF-kappaB and p50 homodimer DNA-binding. Downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently resulted in derepression of kappaB regulated genes. Alterations in expression of p50 homodimer/NF-kappaB regulated genes may contribute to neuroplastic adaptation underlying alcoholism.
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| 8. |
- Heusser, Stephanie A., et al.
(författare)
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Functional Validation of Virtual Screening for Novel Agents with General Anesthetic Action at Ligand-Gated Ion Channelss
- 2013
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Ingår i: Molecular Pharmacology. - : American Society for Pharmacology & Experimental Therapeutics (ASPET). - 0026-895X .- 1521-0111. ; 84:5, s. 670-678
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Tidskriftsartikel (refereegranskat)abstract
- GABA(A) receptors play a crucial role in the actions of general anesthetics. The recently published crystal structure of the general anesthetic propofol bound to Gloeobacter violaceus ligand-gated ion channel (GLIC), a bacterial homolog of GABA(A) receptors, provided an opportunity to explore structure-based ligand discovery for pentameric ligand-gated ion channels (pLGICs). We used molecular docking of 153,000 commercially available compounds to identify molecules that interact with the propofol binding site in GLIC. In total, 29 compounds were selected for functional testing on recombinant GLIC, and 16 of these compounds modulated GLIC function. Active compounds were also tested on recombinant GABA(A) receptors, and point mutations around the presumed binding pocket were introduced into GLIC and GABA(A) receptors to test for binding specificity. The potency of active compounds was only weakly correlated with properties such as lipophilicity or molecular weight. One compound was found to mimic the actions of propofol on GLIC and GABA(A), and to be sensitive to mutations that reduce the action of propofol in both receptors. Mutant receptors also provided insight about the position of the binding sites and the relevance of the receptor's conformation for anesthetic actions. Overall, the findings support the feasibility of the use of virtual screening to discover allosteric modulators of pLGICs, and suggest that GLIC is a valid model system to identify novel GABA(A) receptor ligands.
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