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Multiple Binding Sites for the General Anesthetic Isoflurane Identified in the Nicotinic Acetylcholine Receptor Transmembrane Domain

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Multiple Binding Sites for the General Anesthetic Isoflurane Identified in the Nicotinic Acetylcholine Receptor Transmembrane Domain Multiple binding sites for the general anesthetic isoflurane identified in the nicotinic acetylcholine receptor transmembrane domain Grace Brannigana,1, David N. LeBarda, Jérôme Héninb, Roderic G. Eckenhoffc, and Michael L. Kleina,1 aInstitute for Computational and Molecular Science, Temple University, Philadelphia, PA 19122; bLaboratoire d’Ingénierie des Systèmes Macromoléculaires, Centre National de la Recherche Scientifique—Aix-Marseille Université, 13402 Marseille, France; and cDepartment of Anesthesiology and Critical Care, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Contributed by Michael L. Klein, June 24, 2010 (sent for review May 12, 2010) An extensive search for isoflurane binding sites in the nicotinic An intriguing difference between the nAChR structure acetylcholine receptor (nAChR) and the proton gated ion channel reported in 2BG9 and the prokaryotic structures are the large from Gloebacter violaceus (GLIC) has been carried out based on mo- gaps in protein density in the extracellular half of the nAChR lecular dynamics (MD) simulations in fully hydrated lipid membrane transmembrane domain (TMD). The high-resolution prokaryotic environments. Isoflurane introduced into the aqueous phase readily structures do not display such gaps (Fig. S1). Recently (14), we partitions into the lipid membrane and the membrane-bound pro- proposed that such gaps are occupied by cholesterol, which is tein. Specifically, isoflurane binds persistently to three classes of essential for nAChR function (15). Because cholesterol is not sites in the nAChR transmembrane domain: (i) An isoflurane dimer found in prokaryotic membranes, this hypothesis provides an occludes the pore, contacting residues identified by previous muta- alternate explanation to the source of differences between the genesis studies; analogous behavior is observed in GLIC. (ii) Several two structures. Furthermore, as we demonstrate in the present nAChR subunit interfaces are also occupied, in a site suggested paper, even with docked cholesterol, there is ample space in by photoaffinity labeling and thought to positively modulate the the nAChR TMD for binding of multiple isoflurane molecules. iii receptor; these sites are not occupied in GLIC. ( ) Isoflurane binds The collapse of the nAChRTMD observed in simulations of a cho- BIOPHYSICS AND to the subunit centers of both nAChR α chains and one of the GLIC lesterol-free model (14) results in a structure that presumably COMPUTATIONAL BIOLOGY chains, in a site that has had little experimental targeting. Inter- would not offer as many binding sites for anesthetics as preted in the context of existing structural and physiological data, observed here. Such a collapse, however, is inconsistent with struc- the present MD results support a multisite model for the mechanism tural information obtained on nAChR in native membranes (13), of receptor-channel modulation by anesthetics. and consequently no cholesterol-free nAChR models were consid- ered in this study. anesthesia ∣ cys-loop receptor ∣ ligand-gated ion channel In the absence of detailed structural information, most experi- mental efforts to determine binding sites for anesthetics in espite efforts reaching back over a century, the molecular Cys-loop receptors have depended on techniques such as electro- Dmechanism through which certain small molecules (general physiology, mutagenesis, and photolabeling of various anesthetics anesthetics) cause reversible immobilization and amnesia re- and alcohols to the GABAA, glycine, and nACh receptors. At mains unclear. Known general anesthetics fall into several diverse clinical concentrations, most volatile anesthetics positively modu- classes, but the dominant effects of nearly all general anesthetics late the GABAA receptor but negatively modulate the nAChR, are believed to reflect modulation of ion channels in the central indicating that some binding sites do not overlap (16). In general, nervous system (1–3). An understanding of the mechanisms by experiments suggest multiple binding sites (17, 18) for anesthetics which general anesthetics modulate such channels is therefore not only essential for medical progress, but can also serve to and alcohols on both the nAChR and GABAA receptor: Potential sites have been identified in the TMD, at subunit interfaces (1, 3, illuminate underlying behavior of ion channels and their larger – – role in the biological processes of mobilization and conscious- 16, 19 24) in the nAChR pore, (16, 25 29), and at various ness. Particular attention has focused on the anesthetic-sensitive positions in the agonist-binding domain (22). The multitude of Cys-loop superfamily of ligand-gated ion channels, including potential sites and mechanisms has particularly complicated inter- cation channels such as the nicotinic acetylcholine receptor pretation of ion current measurements, because of the possibility (nAChR) and serotonin receptors, as well as anion channels such of competing effects. Mutagenesis and photolabeling studies as the γ-aminobutyric acid class A (GABAA) receptor and the provide an incomplete picture of anesthetic binding sites, because glycine receptor. Recently a prokaryotic cation channel of this the choice of mutations or selective reactivity of the photolabel superfamily, the proton gated ion channel from Gloebacter viola- prevent the whole receptor from being explored, and the hydro- ceus (GLIC), has demonstrated sensitivity to both intravenous phobic regions to which anesthetics bind are difficult to isolate. and inhaled anesthetics at subclinical concentrations (4). In addition, such methods typically identify regions of the amino High-resolution crystal structures have demonstrated that acid sequence, from which spatial location of binding sites is indir- anesthetics do bind directly to proteins (5–8), whereas more ectly inferred. If multiple residues are identified, it is often not indirect means such as mutagenesis and photolabeling have indicated that general anesthetics bind to Cys-loop receptors – Author contributions: G.B., D.N.L., J.H., R.G.E., and M.L.K. designed research; G.B. and in particular (1 3). Obtaining high-resolution structures of Cys- D.N.L. performed research; G.B. and D.N.L. analyzed data; and G.B., D.N.L., J.H., R.G.E., loop receptors even in the absence of anesthetic has proven to be and M.L.K. wrote the paper. a challenge, however, and structures have only been solved for The authors declare no conflict of interest. – prokaryotic pentameric ion channels (9 11), including GLIC in Freely available online through the PNAS open access option. a putatively open state (3EHZ, 3EAM). For the most part, these 1To whom correspondence may be addressed. E-mail: [email protected] or gbrannigan@ crystal structures reveal a family of proteins that is consistent with temple.edu. Torpedo the earlier 4-Å cryo-EM structure of nAChR from solved This article contains supporting information online at www.pnas.org/lookup/suppl/ by Unwin and coworkers (2BG9) (12, 13). doi:10.1073/pnas.1008534107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1008534107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 27, 2021 clear whether they form multiple binding sites, or simply represent water, which is shown in ref. 8 to be an essential component of multiple sides of the same binding site. some anesthetic binding sites. The membrane lipid environment Complementing experimental approaches, computational can also be included, along with the cholesterol that we proposed methods can serve to directly illuminate microscopic features of (14) is embedded in the nAChR TM-gaps. Furthermore, the MD anesthetic-ion channel interactions. Structure-based docking is approach accounts for interactions between anesthetic molecules, a common technique used to find ligand binding sites on a protein and can therefore identify multiply occupied sites. of known structure. Tang and coworkers (30) reported several Flooding of a protein by anesthetics has been reported pre- mostly superficial sites for halothane detected using structure- viously by members of our group (31, 32); here we present based docking to their model of the α4β2 nAChR in an open MD simulations on a much increased scale involving isoflurane conformation; binding free energy calculations revealed that ha- partitioning into two nearly complete and fully solvated penta- lothane bound with low affinity to most sites, with the exception of meric ligand-gated ion channels: nAChR and GLIC. In order a deeper TM site suggested by experiments. Molecular dynamics to achieve full partitioning of isoflurane into deeply buried (MD) computation-based “flooding” of the receptor (in which a protein sites, this method requires substantially longer computa- high concentration of anesthetic is placed in the surrounding water tion times than those reached by previous simulations of Cys-loop and allowed to partition into lipid and protein binding sites over receptors. Such simulations typically involve about 200,000 the course of an MD simulation trajectory) is a more expensive atoms. The two systems presented here were simulated for alternative to structure-based docking which holds several advan- 0.4 μs each. The nAChR from Torpedo was used for the simula- tages for investigating volatile anesthetics and Cys-loop receptors. tions for the most direct comparison with experimental data and In the MD approach, nearly all protein degrees of freedom are to reduce errors caused by homology modeling. unrestrained, resulting
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