NMR resolved multiple anesthetic binding sites in the TM domains of the ?4?2 nAChR.
ABSTRACT: The ?4?2 nicotinic acetylcholine receptor (nAChR) has significant roles in nervous system function and disease. It is also a molecular target of general anesthetics. Anesthetics inhibit the ?4?2 nAChR at clinically relevant concentrations, but their binding sites in ?4?2 remain unclear. The recently determined NMR structures of the ?4?2 nAChR transmembrane (TM) domains provide valuable frameworks for identifying the binding sites. In this study, we performed solution NMR experiments on the ?4?2 TM domains in the absence and presence of halothane and ketamine. Both anesthetics were found in an intra-subunit cavity near the extracellular end of the ?2 transmembrane helices, homologous to a common anesthetic binding site observed in X-ray structures of anesthetic-bound GLIC (Nury et al., ). Halothane, but not ketamine, was also found in cavities adjacent to the common anesthetic site at the interface of ?4 and ?2. In addition, both anesthetics bound to cavities near the ion selectivity filter at the intracellular end of the TM domains. Anesthetic binding induced profound changes in protein conformational exchanges. A number of residues, close to or remote from the binding sites, showed resonance signal splitting from single to double peaks, signifying that anesthetics decreased conformation exchange rates. It was also evident that anesthetics shifted population of two conformations. Altogether, the study comprehensively resolved anesthetic binding sites in the ?4?2 nAChR. Furthermore, the study provided compelling experimental evidence of anesthetic-induced changes in protein dynamics, especially near regions of the hydrophobic gate and ion selectivity filter that directly regulate channel functions.
Project description:The ?7 nicotinic acetylcholine receptor (nAChR), assembled as homomeric pentameric ligand-gated ion channels, is one of the most abundant nAChR subtypes in the brain. Despite its importance in memory, learning and cognition, no structure has been determined for the ?7 nAChR TM domain, a target for allosteric modulators. Using solution state NMR, we determined the structure of the human ?7 nAChR TM domain (PDB ID: 2MAW) and demonstrated that the ?7 TM domain formed functional channels in Xenopus oocytes. We identified the associated binding sites for the anesthetics halothane and ketamine; the former cannot sensitively inhibit ?7 function, but the latter can. The ?7 TM domain folds into the expected four-helical bundle motif, but the intra-subunit cavity at the extracellular end of the ?7 TM domain is smaller than the equivalent cavity in the ?4?2 nAChRs (PDB IDs: 2LLY; 2LM2). Neither drug binds to the extracellular end of the ?7 TM domain, but two halothane molecules or one ketamine molecule binds to the intracellular end of the ?7 TM domain. Halothane and ketamine binding sites are partially overlapped. Ketamine, but not halothane, perturbed the ?7 channel-gate residue L9'. Furthermore, halothane did not induce profound dynamics changes in the ?7 channel as observed in ?4?2. The study offers a novel high-resolution structure for the human ?7 nAChR TM domain that is invaluable for developing ?7-specific therapeutics. It also provides evidence to support the hypothesis: only when anesthetic binding perturbs the channel pore or alters the channel motion, can binding generate functional consequences.
Project description:The neuronal alpha4beta2 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, 113, 12581) were performed on the closed-channel alpha4beta2 nAChR to investigate the conformation-dependent 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 (approximately -6.0 kcal/mol) were identified at the interface of extracellular (EC) and transmembrane (TM) domains or at the interface between alpha4 and beta2 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 nonbonded interactions at the EC/TM interfaces suggested that the beta2 subunits were more prone than the alpha4 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 alpha4beta2 nAChR to halothane are conformation dependent. The effect of halothane on the global dynamics of the open-channel conformation might also account for the action of other inhaled general anesthetics.
Project description:Neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated as targets for general anesthetics, but the functional responses to anesthetic modulation vary considerably among different subtypes of nAChRs. Inhaled general anesthetics, such as halothane, could effectively inhibit the channel activity of the alpha4beta2 nAChR but not the homologous alpha7 nAChR. To understand why alpha7 is insensitive to inhaled general anesthetics, we performed multiple sets of 20 ns molecular dynamics (MD) simulations on the closed- and open-channel alpha7 in the absence and presence of halothane and critically compared the results with those from our studies on the alpha4beta2 nAChR (Liu et al. J. Phys. Chem. B 2009, 113, 12581 and Liu et al. J. Phys. Chem. B 2010, 114, 626). Several halothane binding sites with fairly high binding affinities were identified in both closed- and open-channel alpha7, suggesting that a lack of sensitive functional responses of the alpha7 nAChR to halothane in the previous experiments was unlikely due to a lack of halothane interaction with alpha7. The binding affinities of halothane in alpha7 seemed to be protein conformation-dependent. Overall, halothane affinity was higher in the closed-channel alpha7. Halothane binding to alpha7 did not induce profound changes in alpha7 structure and dynamics that could be related to the channel function. In contrast, correlated motion of the open-channel alpha4beta2 was reduced substantially in the presence of halothane, primarily due to the more susceptible nature of beta2 to anesthetic modulation. The amphiphilic extracellular and transmembrane domain interface of the beta2 subunit is attractive to halothane and is susceptible to halothane perturbation, which may be why alpha4beta2 is functionally more sensitive to halothane than alpha7.
Project description: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. Tryptophan 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 (K(D)) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane 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.
Project description:Water is an essential component for many biological processes. Pauling proposed that water might play a critical role in general anesthesia by forming water clathrates around anesthetic molecules. To examine potential involvement of water in general anesthesia, we analyzed water within alpha4beta2 nAChR, a putative protein target hypersensitive to volatile anesthetics. Experimental structure-derived closed- and open-channel nAChR systems in a fully hydrated lipid bilayer were examined using all-atom molecular dynamics simulations. At the majority of binding sites in alpha4beta2 nAChR, halothane replaced the slow-exchanging water molecules and caused a regional water population decrease. Only two binding sites had an increased quantity of water in the presence of halothane, where water arrangements resemble clathrate-like structures. The small number of such clathrate-like water clusters suggests that the formation of water clathrates is unlikely to be a primary cause for anesthesia. Despite the decrease in water population at most of the halothane binding sites, the number of sites that can be occupied transiently by water is actually increased in the presence of halothane. Many of these water sites were located between two subunits or in regions containing agonist binding sites or critical structural elements for transducing agonist binding to channel gating. Changes in water sites in the presence of halothane affected water-mediated protein-protein interactions and the protein dynamics, which can have direct impact on protein function. Collectively, water contributes to the action of anesthetics in proteins by mediating interactions between protein subunits and altering protein dynamics, instead of forming water clathrates around anesthetics.
Project description:The neuronal alpha4beta2 nicotinic acetylcholine receptor (nAChR) is a target for general anesthetics. Currently available experimental structural information is inadequate to understand where anesthetics bind and how they modulate the receptor motions essential to function. Using our published open-channel structure model of alpha4beta2 nAChR, we identified and evaluated six amphiphilic interaction sites for the volatile anesthetic halothane via flexible ligand docking and subsequent 20-ns molecular dynamics simulations. Halothane binding energies ranged from -6.8 to -2.4 kcal/mol. The primary binding sites were located at the interface of extracellular and transmembrane domains, where halothane perturbed conformations of, and widened the gap among, the Cys loop, the beta1-beta2 loop, and the TM2-TM3 linker. The halothane with the highest binding affinity at the interface between the alpha4 and beta2 subunits altered interactions between the protein and nearby lipids by competing for hydrogen bonds. Gaussian network model analyses of the alpha4beta2 nAChR structures at the end of 20-ns simulations in the absence or presence of halothane revealed profound changes in protein residue mobility. The concerted motions critical to protein function were also perturbed considerably. Halothane's effect on protein dynamics was not confined to the residues adjacent to the binding sites, indicating an action on a more global scale.
Project description:As a model of the protein targets for volatile anesthetics, the dimeric four-alpha-helix bundle, (Aalpha(2)-L1M/L38M)(2), was designed to contain a long hydrophobic core, enclosed by four amphipathic alpha-helices, for specific anesthetic binding. The structural and dynamical analyses of (Aalpha(2)-L1M/L38M)(2) in the absence of anesthetics (another study) showed a highly dynamic antiparallel dimer with an asymmetric arrangement of the four helices and a lateral accessing pathway from the aqueous phase to the hydrophobic core. In this study, we determined the high-resolution NMR structure of (Aalpha(2)-L1M/L38M)(2) in the presence of halothane, a clinically used volatile anesthetic. The high-solution NMR structure, with a backbone root mean-square deviation of 1.72 A (2JST), and the NMR binding measurements revealed that the primary halothane binding site is located between two side-chains of W15 from each monomer, different from the initially designed anesthetic binding sites. Hydrophobic interactions with residues A44 and L18 also contribute to stabilizing the bound halothane. Whereas halothane produces minor changes in the monomer structure, the quaternary arrangement of the dimer is shifted by about half a helical turn and twists relative to each other, which leads to the closure of the lateral access pathway to the hydrophobic core. Quantitative dynamics analyses, including Modelfree analysis of the relaxation data and the Carr-Purcell-Meiboom-Gill transverse relaxation dispersion measurements, suggest that the most profound anesthetic effect is the suppression of the conformational exchange both near and remote from the binding site. Our results revealed a novel mechanism of an induced fit between anesthetic molecule and its protein target, with the direct consequence of protein dynamics changing on a global rather than a local scale. This mechanism may be universal to anesthetic action on neuronal proteins.
Project description:Nicotinic acetylcholine receptors (nAChRs) are targets of general anesthetics, but functional sensitivity to anesthetic inhibition varies dramatically among different subtypes of nAChRs. Potential causes underlying different functional responses to anesthetics remain elusive. Here we show that in contrast to the ?7 nAChR, the ?7?2 nAChR is highly susceptible to inhibition by the volatile anesthetic isoflurane in electrophysiology measurements. Isoflurane-binding sites in ?2 and ?7 were found at the extracellular and intracellular end of their respective transmembrane domains using NMR. Functional relevance of the identified ?2 site was validated via point mutations and subsequent functional measurements. Consistent with their functional responses to isoflurane, ?2 but not ?7 showed pronounced dynamics changes, particularly for the channel gate residue Leu-249(9'). These results suggest that anesthetic binding alone is not sufficient to generate functional impact; only those sites that can modulate channel dynamics upon anesthetic binding will produce functional effects.
Project description:General anesthetics cause sedation, hypnosis, and immobilization via CNS mechanisms that remain incompletely understood; contributions of particular anesthetic targets in specific neural pathways remain largely unexplored. Among potential molecular targets for mediating anesthetic actions, members of the TASK subgroup [TASK-1 (K2P3.1) and TASK-3 (K2P9.1)] of background K(+) channels are appealing candidates since they are expressed in CNS sites relevant to anesthetic actions and activated by clinically relevant concentrations of inhaled anesthetics. Here, we used global and conditional TASK channel single and double subunit knock-out mice to demonstrate definitively that TASK channels account for motoneuronal, anesthetic-activated K(+) currents and to test their contributions to sedative, hypnotic, and immobilizing anesthetic actions. In motoneurons from all knock-out mice lines, TASK-like currents were reduced and cells were less sensitive to hyperpolarizing effects of halothane and isoflurane. In an immobilization assay, higher concentrations of both halothane and isoflurane were required to render TASK knock-out animals unresponsive to a tail pinch; in assays of sedation (loss of movement) and hypnosis (loss-of-righting reflex), TASK knock-out mice showed a modest decrease in sensitivity, and only for halothane. In conditional knock-out mice, with TASK channel deletion restricted to cholinergic neurons, immobilizing actions of the inhaled anesthetics and sedative effects of halothane were reduced to the same extent as in global knock-out lines. These data indicate that TASK channels in cholinergic neurons are molecular substrates for select actions of inhaled anesthetics; for immobilization, which is spinally mediated, these data implicate motoneurons as the likely neuronal substrates.
Project description:The physiological effects of anesthetics have been ascribed to their interaction with hydrophobic sites within functionally relevant CNS proteins. Studies have shown that volatile anesthetics compete for luciferin binding to the hydrophobic substrate binding site within firefly luciferase and inhibit its activity (Franks, N. P., and Lieb, W. R. (1984) Nature 310, 599-601). To assess whether anesthetics also compete for ligand binding to a mammalian signal transduction protein, we investigated the interaction of the volatile anesthetic, halothane, with the Rho GDP dissociation inhibitor (RhoGDIalpha), which binds the geranylgeranyl moiety of GDP-bound Rho GTPases. Consistent with the existence of a discrete halothane binding site, the intrinsic tryptophan fluorescence of RhoGDIalpha was quenched by halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in a saturable, concentration-dependent manner. Bromine quenching of tryptophan fluorescence is short-range and W192 and W194 of the RhoGDIalpha are located within the geranylgeranyl binding pocket, suggesting that halothane binds within this region. Supporting this, N-acetyl-geranylgeranyl cysteine reversed tryptophan quenching by halothane. Short chain n-alcohols ( n < 6) also reversed tryptophan quenching, suggesting that RhoGDIalpha may also bind n-alkanols. Consistent with this, E193 was photolabeled by 3-azibutanol. This residue is located in the vicinity of, but outside, the geranylgeranyl chain binding pocket, suggesting that the alcohol binding site is distinct from that occupied by halothane. Supporting this, N-acetyl-geranylgeranyl cysteine enhanced E193 photolabeling by 3-azibutanol. Overall, the results suggest that halothane binds to a site within the geranylgeranyl chain binding pocket of RhoGDIalpha, whereas alcohols bind to a distal site that interacts allosterically with this pocket.