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Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC

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Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biophysical Journal Volume 99 September 2010 1801–1809 1801 Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC Qiang Chen,†6 Mary Hongying Cheng,‡6 Yan Xu,†§{ and Pei Tang†§k* † ‡ § { k Departments of Anesthesiology, Chemistry, Pharmacology and Chemical Biology, Structural Biology, and Computational Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania ABSTRACT Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryp- tophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (KD) of 2.3 5 0.1 mM and 0.10 5 0.01 mM were derived from the fluorescence quenching data of halo- thane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins. INTRODUCTION Molecular mechanisms of general anesthesia remain a comprehensive understanding of interactions between unsolved. Although there is an ample amount of evidence anesthetics and cys-loop receptors. for anesthetic modulation on functions of various ion chan- Gloeobacter violaceus pentameric ligand-gated ion nels in the central nervous system (1), the knowledge channel (GLIC) is a bacterial homolog of open-channel regarding where anesthetic sites of action are located and nAChRs. Its x-ray crystal structures have been solved with how anesthetics alter the functions of these channels is still resolutions of 2.9 A˚ (6) and 3.1 A˚ (7). A recent electrophys- limited. Cys-loop receptors, a superfamily of ligand-gated iology study validated the relevance of GLIC to anesthetic ion channels (LGICs), have been identified as potential action (8). Similar to the findings in nAChRs, the channel anesthetic targets (1,2). Members of the LGIC family current of GLIC could be inhibited by inhaled and intrave- include glycine and GABA receptors with anion-selective nous general anesthetics. The low Hill number (0.3) impli- channels and serotonin 5HT3 receptors and nicotinic acetyl- cated multiple binding sites for some anesthetics, but did choline receptors (nAChRs) with cation-selective channels. not offer clues as to where the binding sites are and why The receptors share considerable structural similarity. Each binding induces channel inhibition. Collectively, the homol- of them is assembled by five subunits, forming a homo- or ogous nature of GLIC to nAChRs and the preceding exper- heteropentameric ion channel, and each subunit consists imental findings on GLIC provide several advantages for of an extracellular (EC) domain, four transmembrane mapping anesthetic interaction sites in GLIC. The high- segments (TM1–TM4), and an intracellular domain. resolution structure of GLIC allows us to identify anesthetic Binding of agonists to the EC domain activates the channel binding sites with less ambiguity. The established knowl- and produces ion current. The activities of LGICs could be edge of anesthetic inhibition on GLIC channel function altered by volatile and intravenous anesthetics. It is believed defines the biological relevance of GLIC for exploring the that anesthetics modulate the function of LGICs through molecular basis of anesthetic action. The availability of direct binding to these receptors (1,2). Experimental proof a relatively large quantity of GLIC through protein expres- of direct anesthetic binding to cys-loop receptors, however, sion makes the investigation of anesthetic effects on struc- has been presented only in a few examples (3–5). More ture and dynamics of GLIC more feasible. investigations with atomic resolution are required for The anesthetic binding sites have been exploited via various biophysical methods. NMR can sensitively detect the anesthetic binding sites in proteins with atomic resolution Submitted January 21, 2010, and accepted for publication July 15, 2010. (9–15). In addition, NMR can also probe the structural and 6Qiang Chen and Mary Hongying Cheng contributed equally to this work. dynamical changes of proteins upon anesthetic binding *Correspondence: [email protected] (9–11,14). However, some technical challenges need to be Editor: Benoit Roux. Ó 2010 by the Biophysical Society 0006-3495/10/09/1801/9 $2.00 doi: 10.1016/j.bpj.2010.07.023 1802 Chen et al. overcome to acquire high-resolution information of anes- thetic binding on integral protein complexes, such as GLIC. X-ray structures of anesthetic-protein complexes revealed anesthetic binding sites with atomic resolution, but the success has been limited, so far, in soluble proteins (16–19). Photoaffinity labeling identified the binding sites for halo- thane, [3H]azietomidate, and TDBzl-etomidate in nAChR 3 (3,4,20) and [ H]azietomidate in the GABAA receptor (21). The application of this method for mapping binding sites is often limited due to a small number of anesthetics that are equipped with photolabeling probes (22). Tryptophan (TRP) fluorescence of proteins can be quenched effectively by anesthetic binding near the TRP residues of the proteins (23–25). Thus, steady-state fluorescence measurements are effective for mapping anesthetic binding sites and affinities (23–26). In the case of GLIC, steady-state fluorescence experiments are particularly suitable to search for anesthetic sites. Five intrinsic TRPs are located in three sites in the EC domain, transmembrane (TM) domain, and EC-TM interface of GLIC (Fig. 1). They can serve as natural probes for iden- tifying anesthetic binding sites. In this study, steady-state fluorescence quenching experi- ments were fused with computational predictions for exploring binding information of the volatile anesthetic halo- thane and intravenous anesthetic thiopental in GLIC. The fluo- FIGURE 1 Three tryptophan-associated sites in the wild-type GLIC: rescence experiments suggested the binding of halothane and W47 and W72 in the EC domain (termed as Site-TrpEC), W160 at the thiopental at four sites, including three intrinsic TRP-associ- EC-TM interfacial region (termed as Site-TrpINT), and W213 and W217 Ô ated sites and one additional site at the EC-TM interface pre- in the TM domain (termed as Site-Trp ). dicted by computer docking. The docking analysis provided not only predictions for potential anesthetic binding sites, Site-directed mutagenesis but also details of anesthetic binding sites that the experiments Five tryptophan residues in a wild-type GLIC are situated at three different could not reveal. The subsequent multiple molecular dynamics sites, as shown in Fig. 1. We generated three mutants to dissect the (MD) simulations of halothane binding to those experimen- anesthetic binding information at each site. Each mutant contained trypto- tally predicted sites further explored the underlying cause phan(s) at only one site; all other tryptophans were mutated to tyrosines. Thus, the mutant GLICEC has W47 and W72 in the extracellular domain, for inhibition of GLIC channel current. Taken together, GLICINT has only W160 at the interface of extracellular and transmem- through experimental corroboration of computational predic- brane domains, and GLICÔ has W213 and W217 in the transmembrane tion, our study offered what to our knowledge are the first domain. N200 was predicted computationally as being involved in anes- insights into specific interactions between anesthetics and thetic binding. GLICN200W was made with replacing all natural tryptophans GLIC that facilitate the understanding of anesthetic modula- to tyrosines. All site-directed mutagenesis were done using the QuikChange Multi Site-Directed Mutagenesis Kit (Qiagen, Valencia, CA). The plasmid tion on functions of GLIC and homologous cys-loop receptors. DNAs were collected and purified using the QIAGEN miniprep kit, and confirmed by sequencing. The mutants were expressed and purified using MATERIALS AND METHODS the same protocol as for wild-type GLIC. GLIC expression and purification Fluorescence quenching experiments Wild-type GLIC plasmid was generously provided by Professor Raimund Dutzler’s group of the University of Zu¨rich, Zu¨rich, Switzerland. The Anesthetic halothane or thiopental binding to GLIC was determined by expression
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