The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Jonathan B Cohen - One of the best experts on this subject based on the ideXlab platform.
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contributions of torpedo nicotinic acetylcholine receptor γtrp 55 and δtrp 57 to agonist and Competitive Antagonist function
Journal of Biological Chemistry, 2001Co-Authors: Yu Xie, Jonathan B CohenAbstract:Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the alpha-gamma and alpha-delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the Competitive Antagonist d-[(3)H]tubocurarine (dTC) identified two tryptophans, gammaTrp-55 and deltaTrp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gammaTrp-55 and deltaTrp-57 to the interactions of agonists and Antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the deltaW57L and gammaW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gammaW55L, and deltaW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gammaW55L mutant, dTC binding at the alpha-gamma site acts not as a Competitive Antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.
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contributions of torpedo nicotinic acetylcholine receptor γtrp 55 and δtrp 57 to agonist and Competitive Antagonist function
Journal of Biological Chemistry, 2001Co-Authors: Yu Xie, Jonathan B CohenAbstract:Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the α-γ and α-δ subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the Competitive Antagonistd-[3H]tubocurarine (dTC) identified two tryptophans, γTrp-55 and δTrp-57, as the primary sites of photolabeling in the non-α subunits. To characterize the importance of γTrp-55 and δTrp-57 to the interactions of agonists and Antagonists, Torpedo nAChRs were expressed inXenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the δW57L and γW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, γW55L, and δW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the γW55L mutant, dTC binding at the α-γ site acts not as a Competitive Antagonist but as a coactivator or partial agonist. These results establish that interactions with γ Trp-55 of theTorpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.
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probing the structure of the nicotinic acetylcholine receptor with 4 benzoylbenzoylcholine a novel photoaffinity Competitive Antagonist
Journal of Biological Chemistry, 2000Co-Authors: Dong Wang, Yu Xie, David C Chiara, Jonathan B CohenAbstract:Abstract [3H]4-Benzoylbenzoylcholine (Bz2choline) was synthesized as a photoaffinity probe for the Torpedo nicotinic acetylcholine receptor (nAChR). [3H]Bz2choline acts as an nAChR Competitive Antagonist and binds at equilibrium with the same affinity (K D = 1.4 μm) to both agonist sites. Irradiation at 320 nm of nAChR-rich membranes equilibrated with [3H]Bz2choline results in the covalent incorporation of [3H]Bz2choline into the nAChR γ- and δ-subunits that is inhibitable by agonist, with little specific incorporation in the α-subunits. To identify the sites of photoincorporation, γ- and δ-subunits, isolated from nAChR-rich membranes photolabeled with [3H]Bz2choline, were digested enzymatically, and the labeled fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. For the γ-subunit, Staphylococcus aureus V8 protease produced a specifically labeled peptide beginning at γVal-102, whereas for the δ-subunit, endoproteinase Asp-N produced a labeled peptide beginning at δAsp-99. Amino-terminal sequence analysis identified the homologous residues γLeu-109 and δLeu-111 as the primary sites of [3H]Bz2choline photoincorporation. This is the first identification by affinity labeling of non-reactive amino acids within the acetylcholine-binding sites, and these results establish that when choline esters of benzoic acid are bound to the nAChR agonist sites, the para substituent is selectively oriented toward and in proximity to amino acids γLeu-109/δLeu-111.
Yu Xie - One of the best experts on this subject based on the ideXlab platform.
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contributions of torpedo nicotinic acetylcholine receptor γtrp 55 and δtrp 57 to agonist and Competitive Antagonist function
Journal of Biological Chemistry, 2001Co-Authors: Yu Xie, Jonathan B CohenAbstract:Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the alpha-gamma and alpha-delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the Competitive Antagonist d-[(3)H]tubocurarine (dTC) identified two tryptophans, gammaTrp-55 and deltaTrp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gammaTrp-55 and deltaTrp-57 to the interactions of agonists and Antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the deltaW57L and gammaW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gammaW55L, and deltaW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gammaW55L mutant, dTC binding at the alpha-gamma site acts not as a Competitive Antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.
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contributions of torpedo nicotinic acetylcholine receptor γtrp 55 and δtrp 57 to agonist and Competitive Antagonist function
Journal of Biological Chemistry, 2001Co-Authors: Yu Xie, Jonathan B CohenAbstract:Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the α-γ and α-δ subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the Competitive Antagonistd-[3H]tubocurarine (dTC) identified two tryptophans, γTrp-55 and δTrp-57, as the primary sites of photolabeling in the non-α subunits. To characterize the importance of γTrp-55 and δTrp-57 to the interactions of agonists and Antagonists, Torpedo nAChRs were expressed inXenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the δW57L and γW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, γW55L, and δW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the γW55L mutant, dTC binding at the α-γ site acts not as a Competitive Antagonist but as a coactivator or partial agonist. These results establish that interactions with γ Trp-55 of theTorpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.
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probing the structure of the nicotinic acetylcholine receptor with 4 benzoylbenzoylcholine a novel photoaffinity Competitive Antagonist
Journal of Biological Chemistry, 2000Co-Authors: Dong Wang, Yu Xie, David C Chiara, Jonathan B CohenAbstract:Abstract [3H]4-Benzoylbenzoylcholine (Bz2choline) was synthesized as a photoaffinity probe for the Torpedo nicotinic acetylcholine receptor (nAChR). [3H]Bz2choline acts as an nAChR Competitive Antagonist and binds at equilibrium with the same affinity (K D = 1.4 μm) to both agonist sites. Irradiation at 320 nm of nAChR-rich membranes equilibrated with [3H]Bz2choline results in the covalent incorporation of [3H]Bz2choline into the nAChR γ- and δ-subunits that is inhibitable by agonist, with little specific incorporation in the α-subunits. To identify the sites of photoincorporation, γ- and δ-subunits, isolated from nAChR-rich membranes photolabeled with [3H]Bz2choline, were digested enzymatically, and the labeled fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. For the γ-subunit, Staphylococcus aureus V8 protease produced a specifically labeled peptide beginning at γVal-102, whereas for the δ-subunit, endoproteinase Asp-N produced a labeled peptide beginning at δAsp-99. Amino-terminal sequence analysis identified the homologous residues γLeu-109 and δLeu-111 as the primary sites of [3H]Bz2choline photoincorporation. This is the first identification by affinity labeling of non-reactive amino acids within the acetylcholine-binding sites, and these results establish that when choline esters of benzoic acid are bound to the nAChR agonist sites, the para substituent is selectively oriented toward and in proximity to amino acids γLeu-109/δLeu-111.
Edson X Albuquerque - One of the best experts on this subject based on the ideXlab platform.
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strychnine a potent Competitive Antagonist of alpha bungarotoxin sensitive nicotinic acetylcholine receptors in rat hippocampal neurons
Journal of Pharmacology and Experimental Therapeutics, 1998Co-Authors: Hiroaki Matsubayashi, Edna F R Pereira, Manickavasagom Alkondon, K L Swanson, Edson X AlbuquerqueAbstract:In our study, evidence is provided that strychnine, a Competitive Antagonist at glycine-gated Cl- channels, is also a potent Competitive Antagonist at native alpha-7-containing, alpha-bungarotoxin-sensitive nicotinic acetylcholine receptor (nAChRs). To address the effects of strychnine on two types of nicotinic responses, the whole-cell mode of the patch-clamp technique was applied to rat hippocampal neurons in culture. Type IA and type II nicotinic currents evoked by acetylcholine (ACh) were inhibited by strychnine in a concentration-dependent manner with IC50S of 1.2 and 38 microM, respectively. Strychnine (2 microM) decreased the peak amplitude of the alpha-bungarotoxin-sensitive type IA current in a voltage-independent manner and prolonged the decay phase of this current. The concentration-response curve for ACh in evoking type IA current showed a parallel shift to the right in the presence of strychnine (2 microM); the EC50 for ACh was increased from 0.4 to 0.8 mM. These findings suggest that strychnine acts as a Competitive Antagonist of ACh at the alpha 7 nAChRs that subserve type IA current. In contrast, the inhibition by strychnine of type II current was strongly voltage dependent, and the decay phase of this current was markedly accelerated by the toxin, suggesting an open-channel blockade by strychnine of the alpha 4 beta 2 nAChRs subserving type II currents. Preexposure of the neurons to strychnine enhanced its ability to decrease the peak amplitude of type II currents, indicating that the toxin may also act on alpha 4 beta 2 nAChR channels that are not open. It is concluded that strychnine is a potent Competitive Antagonist of ACh at neuronal alpha 7 nAChRs and a nonCompetitive Antagonist at the alpha 4 beta 2 nAChR.
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alpha conotoxin imi a Competitive Antagonist at alpha bungarotoxin sensitive neuronal nicotinic receptors in hippocampal neurons
Journal of Pharmacology and Experimental Therapeutics, 1996Co-Authors: Edna F R Pereira, Manickavasagom Alkondon, J M Mcintosh, Edson X AlbuquerqueAbstract:In the present study, the patch-clamp technique was applied to rat hippocampal neurons or myoballs in culture to study the actions of alpha-conotoxin-ImI on the native alpha-bungarotoxin-sensitive, presumably alpha 7-bearing, neuronal nicotinic receptor and on other ligand-gated channels. Preexposure of the neurons for 5 min to alpha-conotoxin-ImI decreased the peak amplitude of alpha-BGT-sensitive currents (referred to as type IA currents) in a concentration-dependent fashion. Several lines of evidence revealed that the inhibitory effect of alpha-conotoxin-ImI was Competitive with respect to the agonist (IC50 approximately 85 nM) and reversible by washing. At 300 nM, alpha-conotoxin-ImI decreased by only 15% the peak amplitude of ACh-evoked currents in rat myoballs, did not affect the activation of currents gated by gamma-aminobutyric acid, glycine, N-methyl-D-aspartate, kainate, or quisqualate in hippocampal neurons, but reduced to approximately 60% the peak amplitude and shortened the decay phase of curare-sensitive, serotonin-gated currents in these neurons. The Competitive and reversible nature of the alpha-conotoxin-ImI-induced inhibition of native alpha 7-bearing neuronal nicotinic receptors makes this peptide a valuable new tool for the functional and structural characterization of these receptors in the central nervous system.
Douglas E Raines - One of the best experts on this subject based on the ideXlab platform.
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the effects of a Competitive Antagonist on gaba evoked currents in the presence of sedative hypnotic agents
Pharmacological Reports, 2020Co-Authors: Megan Mcgrath, Mansi Tolia, Douglas E RainesAbstract:Background Many sedative-hypnotic agents are thought to act by positively modulating γ-aminobutyric acid type A (GABAA) receptors. However, for many agents, the location(s) of the binding site(s) responsible for such receptor modulation is uncertain. We previously developed a low efficacy ligand (naphthalene-etomidate) that binds within a homologous set of hydrophobic cavities located at GABAA receptor subunit interfaces in the transmembrane domain, and thus acts as a Competitive Antagonist for higher efficacy sedative-hypnotics that also bind to these sites. In this report, we describe studies using this compound as a pharmacological screening tool to test whether sedative-hypnotics representing a range of chemical classes can modulate GABAA receptors by binding within these receptor cavities. Methods The impact of naphthalene-etomidate on GABA-evoked currents that were mediated by oocyte-expressed α1β3γ2L GABAA receptors and potentiated by muscimol, alphaxalone, 2,2,2-trichloroethanol, isoflurane, AA29504, loreclezole, or diazepam was quantified using electrophysiological techniques. Results Naphthalene-etomidate (300 µM) significantly reduced GABAA receptor currents potentiated by alphaxalone (by 22 ± 11%), 2,2,2-trichloroethanol (by 23 ± 6%), isoflurane (by 32 ± 10%), AA29504 (by 41 ± 6%), loreclezole (by 43 ± 9%), but significantly increased those potentiated by muscimol (by 26 ± 11%). Naphthalene-etomidate significantly increased currents potentiated by a low (1 µM) diazepam concentration (by 56 ± 14%) while reducing those potentiated by a high (100 µM) diazepam concentration (by 11 ± 7%). Conclusions Our results suggest that many (but not all) sedative-hypnotics are capable of positively modulating the GABAA receptor by binding within a common set of hydrophobic cavities.
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the effects of a Competitive Antagonist on gaba evoked currents in the presence of sedative hypnotic agents
Pharmacological Reports, 2020Co-Authors: Megan Mcgrath, Mansi Tolia, Douglas E RainesAbstract:Many sedative-hypnotic agents are thought to act by positively modulating γ-aminobutyric acid type A (GABAA) receptors. However, for many agents, the location(s) of the binding site(s) responsible for such receptor modulation is uncertain. We previously developed a low efficacy ligand (naphthalene-etomidate) that binds within a homologous set of hydrophobic cavities located at GABAA receptor subunit interfaces in the transmembrane domain, and thus acts as a Competitive Antagonist for higher efficacy sedative-hypnotics that also bind to these sites. In this report, we describe studies using this compound as a pharmacological screening tool to test whether sedative-hypnotics representing a range of chemical classes can modulate GABAA receptors by binding within these receptor cavities. The impact of naphthalene-etomidate on GABA-evoked currents that were mediated by oocyte-expressed α1β3γ2L GABAA receptors and potentiated by muscimol, alphaxalone, 2,2,2-trichloroethanol, isoflurane, AA29504, loreclezole, or diazepam was quantified using electrophysiological techniques. Naphthalene-etomidate (300 µM) significantly reduced GABAA receptor currents potentiated by alphaxalone (by 22 ± 11%), 2,2,2-trichloroethanol (by 23 ± 6%), isoflurane (by 32 ± 10%), AA29504 (by 41 ± 6%), loreclezole (by 43 ± 9%), but significantly increased those potentiated by muscimol (by 26 ± 11%). Naphthalene-etomidate significantly increased currents potentiated by a low (1 µM) diazepam concentration (by 56 ± 14%) while reducing those potentiated by a high (100 µM) diazepam concentration (by 11 ± 7%). Our results suggest that many (but not all) sedative-hypnotics are capable of positively modulating the GABAA receptor by binding within a common set of hydrophobic cavities.
Jesper L Kristensen - One of the best experts on this subject based on the ideXlab platform.
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tying up nicotine new selective Competitive Antagonist of the neuronal nicotinic acetylcholine receptors
ACS Medicinal Chemistry Letters, 2015Co-Authors: Ida Nymann Petersen, Francois Crestey, Anders A Jensen, Dinesh C Indurthi, Henrik Pedersen, Jesper T Andreasen, Thomas Balle, Jesper L KristensenAbstract:Conformational restriction of the pyrrolidine nitrogen in nicotine by the introduction of an ethylene bridge provided a potent and selective Antagonist of the α4β2-subtype of the nicotinic acetylcholine receptors. Resolution by chiral SFC, pharmacological characterization of the two enantiomers, and determination of absolute configuration via enantioselective synthesis showed that the pharmacological activity resided almost exclusively in the (R)-enantiomer.
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synthesis and pharmacological evaluation of dhβe analogues as neuronal nicotinic acetylcholine receptor Antagonists
ACS Medicinal Chemistry Letters, 2014Co-Authors: Tue Heesgaard Jepsen, Anders A Jensen, Mads Lund, Emil Glibstrup, Jesper L KristensenAbstract:Dihydro-β-erythroidine (DHβE) is a member of the Erythrina family of alkaloids and a potent Competitive Antagonist of the α4β2-subtype of the nicotinic acetylcholine receptors (nAChRs). Guided by an X-ray structure of DHβE in complex with an ACh binding protein, we detail the design, synthesis, and pharmacological characterization of a series of DHβE analogues in which two of the four rings in the natural product has been excluded. We found that the direct analogue of DHβE maintains affinity for the α4β2-subtype, but further modifications of the simplified analogues were detrimental to their activities on the nAChRs.
