General anesthetic modulation of neuronal nicotinic acetylcholine receptors

Yamashita, Megumi; Mori, Takashi; Zhao, Xilong; Nagata, Keiichi; Marszalec, William; Yeh, Jay Z.; Narahashi, Toshio

doi:10.1016/j.ics.2005.07.048

Cited by 4 publications

(4 citation statements)

References 16 publications

(25 reference statements)

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“…The neuronal α4β2 nAChR is one of the most abundant subtype nAChRs in the brain and was identified as a putative target for general anesthetics. – As with other nAChRs, the molecular details regarding the interaction between anesthetics and the α4β2 nAChR have not been experimentally determined. A mechanistic understanding of how anesthetics altered the function of the α4β2 nAChR is still lacking.…”

Section: Introductionmentioning

confidence: 99%

Higher Susceptibility to Halothane Modulation in Open- Than in Closed-Channel α4β2 nAChR Revealed by Molecular Dynamics Simulations

et al. 2009

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The neuronal α4β2 nicotinic acetylcholine receptor (nAChR) is a potential molecular target for general anesthetics. It is unclear, however, whether anesthetic action produces the same effect on the open and closed channels. Computations parallel to our previous open channel study (J. Phys. Chem. B, 2009) were performed on the closed-channel α4β2 nAChR to investigate the conformationdependent anesthetic effects on channel structures and dynamics. Flexible ligand docking and over 20-ns molecular dynamics simulations revealed similar halothane-binding sites in the closed and open channels. The sites with relatively high binding affinities (~−6.0 kcal/mol) were identified at the interface of extracellular (EC) and transmembrane (TM) domains or at the interface between α4 and β2 subunits. Despite similar sites for halothane binding, the closed-channel conformation showed much less sensitivity than the open channel to the structural and dynamical perturbations from halothane. Compared to the systems without anesthetics, the amount of water inside the pore decreased by 22% in the presence of halothane in the open channel, but only by 6% in the closed channel. Comparison of the non-bonded interactions at the EC/TM interfaces suggested that the β2 subunits were more prone than the α4 subunits to halothane binding. In addition, our data support the notion that halothane exerts its effect by disturbing the quaternary structure and dynamics of the channel. The study concludes that sensitivity and global dynamics responsiveness of α4β2 nAChR to halothane are conformation dependent. The effect of halothane on global dynamics of openchannel conformation might also account for the action of other inhaled general anesthetics.

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Section: Introductionmentioning

confidence: 99%

Higher Susceptibility to Halothane Modulation in Open- Than in Closed-Channel α4β2 nAChR Revealed by Molecular Dynamics Simulations

et al. 2009

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“…For example, alcohol potentiation of GABA-mediated tonic current from ␣␤␦ GABA A Rs is routinely observed in whole cell recordings of hippocampal granule cells and cerebellar granule cells (32)(33)(34), but it is not reproducibly observed in recombinant systems. Although some studies have reported that GABA-activated currents from recombinant ␣␤␦ GABA A Rs are potentiated by low concentrations of alcohol (1-30 mM) (13,14), others have reported that recombinant ␣␤␦ receptors are not responsive to alcohol (15,16). Numerous groups expressing recombinant ␣␤␦ GABA A Rs use a greater ratio of ␦ cDNA or cRNA to ensure adequate ␦ subunit expression and assembly (8,10,11,35).…”

Section: Discussionmentioning

confidence: 99%

“…The actions of these drugs are dependent on the GABA A R subunit isoforms present, 16 of which have been identified, including ␣1-6, ␤1-3, ␥1-3, ␦, ⑀, , and . Although the highly prevalent GABA A R subtype comprised of ␣, ␤, and ␥ subunits mediates the majority of fast synaptic inhibition in the brain, ␣␤␦ GABA A Rs also play a key role, mediating tonic inhibition and the molecular actions of endogenous neuroactive steroids, alcohol, and several anesthetic agents (1).…”

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confidence: 99%

“…If different ratios of cDNA/cRNA alter subunit stoichiometry of surface-expressed ␣␤␦ GABA A Rs, this may contribute to the discrepancies in the functional properties (e.g. alcohol sensitivity, GABA EC 50 values, maximal GABA currents) of expressed ␣␤␦ GABA A Rs reported in the literature (13)(14)(15)(16). In heteromeric neuronal nicotinic acetylcholine receptors, varying the transfection ratios of ␣ 4 and ␤ 2 subunits results in receptors with alternate stoichiometries of 3␣:2␤ or 2␣:3␤ (17).…”

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confidence: 99%

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Stoichiometry of Expressed α4β2δ γ-Aminobutyric Acid Type A Receptors Depends on the Ratio of Subunit cDNA Transfected

Wagoner

Czajkowski

2010

Journal of Biological Chemistry

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The ␥-aminobutyric acid type A receptor (GABA A R) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent ␣␤␥ GABA A R subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing ␦ subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of ␦-containing GABA A Rs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed ␦-containing GABA A Rs, we inserted the ␣-bungarotoxin binding site tag in the ␣ 4 , ␤ 2 , and ␦ subunit N termini. An enhanced green fluorescent protein tag was also inserted into the ␤ 2 subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged ␣ 4 ␤ 2 ␦ GABA A Rs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent ␣-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA A Rs. The results demonstrate that the subunit stoichiometry of ␣ 4 ␤ 2 ␦ GABA A Rs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of ␦ subunit cDNA transfected increased ␦ subunit incorporation into surface receptors with a concomitant decrease in ␤ 2 subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA A R pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed ␣ 4 ␤ 2 ␦ GABA A Rs. Successful bungarotoxin binding site tagging of GABA A R subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA A R trafficking in live cells.The ␥-aminobutyric acid type A receptor (GABA A R) 2 is the main inhibitory ligand-gated ion channel in the brain and is the target for a wide range of drugs, including benzodiazepines, anesthetics, neurosteroids, and barbiturates. The actions of these drugs are dependent on the GABA A R subunit isoforms present, 16 of which have been identified, including ␣1-6, ␤1-3, ␥1-3, ␦, ⑀, , and . Although the highly prevalent GABA A R subtype comprised of ␣, ␤, and ␥ subunits mediates the majority of fast synaptic inhibition in the brain, ␣␤␦ GABA A Rs also play a key role, mediating tonic inhibition and the molecular actions of endogenous neuroactive steroids, alcohol, and several anesthetic agents (1).Heterologous expression of GABA A R subtypes in cell lines (e.g. HEK293 and Chinese hamster ovary cells) and Xenopus laevis oocytes has been used extensively to study their structural, electrophysiological, and pharmacological properties. Although the subunit stoichiometry of ␣␤␥ GABA A Rs has been established as 2␣:2␤:1␥ using a variety of methods, including using a reporter mutation (2), quantifying antibody bound per subunit (3), fluorescence resonance energy transfer (4),...

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