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7 Nicotinic Acetylcholine ReceptorStructural Bioinformatics and Computational Biochemistry Unit, Department of Biochemistry (S.A., R.V., M.S.P.S., P.C.B.) and Department of Physiology, Anatomy, and Genetics (A.K.J., D.B.S.), University of Oxford, Oxford, United Kingdom; Department of Applied Biological Chemistry, School of Agriculture, Kinki University, Nara, Japan (M.S., H.N., K.M.); and Graduate School of Agriculture, Kyoto University, Kyoto, Japan (M.A.)
Received September 7, 2007; accepted March 13, 2008
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Abstract |
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7 agonist-binding domain. The initial models and a preliminary docking study suggested that position Leu118 may play an important role in determining agonist actions on 7. A prediction from these in silico studies, that L118E and L118D would retain binding to acetylcholine but L118K and L118R would not, was confirmed in electrophysiological studies on functional recombinant mutant receptors expressed in Xenopus laevis oocytes. The functional studies also demonstrated that residues at position 118 have a dramatic effect on the actions of imidacloprid (a partial agonist of wild-type 7 receptors) and its des-nitro derivative. Molecular dynamics simulations confirmed that Leu118 can strongly influence agonist binding and that the model was robust in terms of its prediction for acetylcholine binding. Together, the results indicate a role for Leu118 in influencing agonist actions on 7 nAChRs.
; Karlin, 2002). The nAChR molecules are pentamers composed of identical or highly homologous subunits, each with four transmembrane regions (1-4) and an extracellular N-terminal domain containing six loops (A-F) that make up the ACh binding site (Corringer et al., 2000). Subunits possessing two adjacent cysteines in loop C of the ACh binding site are designated subunits, whereas those lacking this cysteine pair are denoted non- subunits.
Neuronal nicotinic receptor subunits (2-10; β2-4), which contribute to a variety of receptor subtypes depending on subunit composition (Millar, 2003), are important targets for new drugs (Arneric et al., 2007). These include drugs being developed as analgesics and drugs for ameliorating the symptoms of Alzheimer's disease (Prendergast et al., 1998; Papke et al., 2000), as well as drugs used to treat Parkinson's disease and schizophrenia (Lloyd and Williams, 2000). Congenital myasthenias (Ohno and Engel, 2002) result from mutations in muscle nAChR subunits and autosomal nocturnal frontal lobe epilepsy results from mutations in neuronal 4 and β2 nAChR subunits (Changeux and Edelstein, 2001; Steinlein, 2001). In addition, autoantibodies directed against nAChRs underlie several diseases, such as myasthenia gravis (muscle nAChRs; Lang and Vincent, 2003), Rasmussen's encephalitis (7; Watson et al., 2005), and autonomic neuropathy (3; Vernino and Lennon, 2003). The nAChRs of insects are the targets for imidacloprid, a neonicotinoid insecticide extensively used worldwide (Matsuda et al., 2001b). In addition, nematode nAChRs are important targets for anthelmintic drugs such as levamisole, pyrantel, and morantel (Harrow and Gration, 1985; Jones et al., 2005).
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, 2003; Unwin, 2005). Models based on these coordinates indicate that the extracellular region, containing the ligand-binding site, consists of twisted β-sheets, and loop regions form the binding sites. Brejc et al. (2001) first crystallized a glial-derived acetylcholine binding protein (AChBP) from the freshwater snail Lymnaea stagnalis. This 210-amino acid polypeptide forms a stable homopentamer with homology to the N-terminal extracellular region of nAChRs. ACh binding sites are located at each of the five subunit interfaces and the six binding loops (A-F) that can be recognized in nAChRs are also present. Several AChBP crystal structures have now been published (Brejc et al., 2001, 2002; Celie et al., 2004, 2005a,b; Hansen et al., 2005). In view of the important structural homology and sequence similarity (24% identity), between the AChBP and the ligand binding domain of neuronal 7, homology modeling can offer a useful tool to investigate ligand binding. The structure of the chicken 7 homopentamer has been constructed based on both the X-ray structure of the L. stagnalis AChBP and the electron microscopy-derived structure of the transmembrane region of the T. marmorata nicotinic receptor (Amiri et al., 2005). Le Novère et al. (2002) built a three-dimensional model of the N-terminal domain of a homopentameric chicken 7 nAChR based on AChBP, which was then used to analyze the docking of ACh, epibatidine, and nicotine (Le Novère et al., 2002). Plausible modes of binding were then suggested for these ligands. In a separate modeling study (Schapira et al., 2002), the binding affinities were related to different receptor isotypes. While the manuscript for this article was being written, the crystal structure of the extracellular domain of nAChR 1 subunit bound to -bungarotoxin at 1.94-Å resolution was solved (Dellisanti et al., 2007). Comparisons between the 1 and AChBP structures suggest that AChBP is indeed a good model for the extracellular ligand-binding domain (Dellisanti et al., 2007).
Photoaffinity labeling studies on the molluscan AChBP showed that Tyr195 in loop C and Met116 of loop E interact with the agonist azidoepibatidine (Tomizawa et al., 2007). Mutagenesis studies have previously shown that the equivalent residue in loop E of vertebrate muscle non- subunits is involved in ligand binding (Sine, 1997). We have generated homology models of the chicken 7 homopentameric nAChR (7)5 and show how in silico methods combined with site-directed mutagenesis yield further evidence that the corresponding loop E residue of 7, Leu118, controls ligand access to the agonist-binding site. We also show that this residue is important in the actions on 7 of the insecticide imidacloprid and its derivative desnitro-imidacloprid.
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Materials and Methods |
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7 and AChBP (PDB code 1UX2, which has HEPES buffer bound) was generated on the basis of multiple sequence alignments extracted from the pfam database (Bateman et al., 2002) and improved by manual adjustment. The alignment (Fig. 1A) was used as input for the program MODELLER (Sali and Blundell, 1993) to generate 100 initial pentameric models of the chicken 7 ligand binding domain. The quality of the five lowest energy structures was checked with PROCHECK version 3.5.4 (Laskowski et al., 1993) and the WHAT-IF (http://www.cmbi.kun.nl/gv/servers/WIWWWI) server. The model with the lowest percentage of residues in the disallowed region of the Ramachandran plot was selected for docking studies. Only one binding site from two adjacent subunits was used (see Fig. 1B). In silico site-directed mutagenesis was performed using PyMOL (DeLano, 2004). These structures were deployed as potential docking targets for ACh and imidacloprid using the program Autodock (Morris et al., 1998). Autodock was run with default parameters except that atom types were not set for the ligand but rather according to charges calculated as below. The active site was defined as a radius of (default) 15.0 Å from carbon 6 (IUPAC/IUBMB numbering) of the side chain of Trp148, a residue located centrally in the ACh binding pocket. The charges on ACh were assigned according to a previous report (Segall et al., 1998). Charges for imidacloprid were calculated with the 6-31G* basis set using Spartan (Wavefunction Inc., Irvine, CA). All docking results were visualized with VMD (Humphrey et al., 1996) and UCSF Chimera (Pettersen et al., 2004).
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) using the GROMOS96 (van Gunsteren et al., 1996) force field. Each system was energy-minimized until convergence using a steepest descents algorithm. Molecular dynamics with position restraints for 100 ps was performed followed by equilibration for 1 ns and finally the production run of 10 ns. During the equilibration phase, the temperature and pressure were coupled using the Berendsen methods (Berendsen et al., 1984). During the production runs, the Paranillo-Rahman method (Parinello and Rahman, 1981) was used for pressure coupling, and the temperature was coupled using the Nosé-Hoover (Nose, 1984) method at 310 K. Electrostatics were calculated with the particle mesh Ewald method (Darden et al., 1993). The LINCS algorithm (Hess et al., 1997) was used to constrain bond lengths, and a time step of 2 fs was used throughout. All calculations were performed on an Intel Pentium III personal computer running Linux kernel 2.4.19smp.
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7 Receptors. Xenopus laevis oocytes were prepared and injected with cDNA (Gallus gallus 7 cDNA in pMT3) as described previously (Shimomura et al., 2003). Membrane currents were recorded by the two-electrode voltage-clamp method using 2.0 M KCl-filled electrodes (resistances, 0.5-5.0 M
) and a GeneClamp 500B amplifier (Molecular Devices, Sunnyvale, CA). The oocyte membrane was clamped at -100 mV. Oocytes secured in a poly(methyl 2-methyl-propenoate) (Perspex) recording chamber (80-µl volume) were perfused continuously at 7 to 10 ml/min by a gravity-fed system with standard oocyte saline composed of 100 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, and 5.0 mM HEPES, pH 7.6. All the test compounds were dissolved in standard oocyte saline and bath-applied to oocytes at intervals of 3 to 5 min to minimize the effects of desensitization. Only oocytes that gave stable responses to two or more successive applications of 200 or 400 µM ACh were used. Concentration-response data were obtained by applying increasing concentrations of agonist to the oocytes. The maximum amplitude of the current recorded in response to each application was normalized to the response to 1 mM ACh. As the concentration-response curves for ACh and imidacloprid were changed by the mutations L118D and L118E, data from mutants were normalized using the current response to 3 mM ACh, whereas data from L118K and L118R mutants were normalized using the response to 3 mM imidacloprid. Using Prism software (GraphPad Software, San Diego, CA), normalized data were fitted as described previously (Shimomura et al., 2003).
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7 Receptors. The reaction mixtures (50 µl) containing membrane fraction (2 µg), [3H]ACh (31.25-750 µM), atropine (0.5 µM), and paraoxon (10 µM) were incubated for 1 h at room temperature before the reaction was stopped by filtering through a glass filter GF/C (Whatman International Ltd, Maidstone, England) prewetted with sodium phosphate buffer (10 mM, pH 7.4) containing sodium chloride (50 mM) and polyethylenimine [0.1% (v/v)]. The glass filters were immediately washed with 1.5 ml of sodium phosphate buffer (10 mM, pH 7.4) containing sodium chloride (50 mM), followed by two washes with 5 ml of the same buffer. Each glass filter was then transferred to 3 ml of xylene-based LSCocktail "Aquasol 2" (PerkinElmer Life and Analytical Sciences, Waltham, MA) in a glass vial to measure radioactivity using a liquid scintillation counter (LSC-5100; Aloka Co, Ltd, Tokyo, Japan). Specific binding was defined as the difference of the [3H]ACh binding to the membrane fractions from oocytes expressing recombinant nAChRs and those from vector-injected oocytes. Ligand binding assays were performed in at least triplicate (n = 3-11). The Kd values were calculated from the saturation curves using GraphPad Prism. |
Results |
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7 Model. We generated three-dimensional models of the wild-type chicken 7 nAChR. We found that Leu118, which is located in loop E (Corringer et al., 2000), is situated close to the ligand binding site (Fig. 1B) and consequently may influence ligand-protein interactions. We performed docking of ACh, imidacloprid (IMI), and desnitro-imidacloprid (DN-IMI) to the wild-type model. We found that the ACh solutions were tightly clustered (only four clusters) in the binding pocket (Fig. 2A) and spanned an energy range of 0.3 kcal/mol, but for IMI there were several binding modes in a several clusters (Fig. 2B), not all of which were in the immediate binding pocket (7 of 13 clusters were within an energy cut-off of 1.5 kcal/mol of the lowest energy dock). DN-IMI docking also found the binding pocket but several binding modes were again possible with 6 of 9 clusters within an energy cut-off of 1.5 kcal/mol of the lowest energy dock (Fig. 2C). The energies of the IMI and DN-IMI docks were comparable, but all were approximately 3 kcal/mol less than the ACh docks. A comparison of the ACh docking with the crystal structure of carbamylcholine with AChBP (Fig. 3A) revealed that the mode of binding is very similar and thus gave us confidence that the procedure could be used further to predict the interactions of ACh with receptor mutants. However, because the solutions found for IMI and DN-IMI were numerous and there is currently no structural information to confirm the docking solutions, we did not have confidence that the procedure could make reliable predictions for these compounds. Furthermore, there is evidence that the receptor can undergo substantial movement of residues in the binding pocket (Henchman et al., 2003) and, given the size of IMI (and DN-IMI), we reasoned that the pocket may have to undergo substantial movement to accommodate these molecules. We therefore examined ACh docking to mutant receptors and performed the following in silico mutagenesis on this position; L118E, L118D, L118K, and L118R. The results for ACh docking are shown in Fig. 3; for both the L118D mutation (Fig. 3B) and the L118E mutation (Fig. 3C), the mode of docking resembles that seen for the wild-type receptor (Fig. 3A). There is a slight shift in the position of ACh toward what would be the surface of the membrane in the nAChR molecule. This presumably stems from the increased negative charge at Leu118, which pulls the quaternary nitrogen moiety further downward compared with wild-type. When we tried to dock ACh to the L118K and L118R mutants, we found that ACh would generally not dock into the binding pocket. In the case of L118K, three clusters positioned ACh near the binding pocket but in completely the wrong orientation. The effect was even more marked for the L118R mutation, where not one ACh docking was even in the binding pocket. These studies suggested that negatively charged mutations at this position would retain ACh binding, but positively charged mutations would impair ACh binding.
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7 subunits were therefore performed. A common method for analyzing the relative stability of protein simulations is to analyze the root-mean-square fluctuations (RMSF) of the C atoms. Analysis of the RMSF for AChBP molecules revealed that the mutant structures showed a higher mean RMSF (Fig. 4, A-D), indicating that this residue has a large influence on the dynamics of the protein. In particular, the regions corresponding to loop E (residues 112-119) appear to exhibit larger fluctuations compared with wild-type. In the case of L118D and L118E, there are also increased fluctuations for loop C.
Because the result of an MD simulation is to produce a series of coordinates versus time, this presents a means to test the model and the sensitivity of the docking of ACh with respect to local fluctuations of the model. To do this, we took 100 snapshots from the simulation, one every 100 ps. We then docked ACh back into these snapshots to assess how well the binding pocket retained its shape and ability to accommodate ACh. This analysis showed that the results observed for the starting model were maintained throughout the simulation (see typical snapshots taken from frames at 5 ns in Fig. 5, A-C), suggesting that small, local residue fluctuations were not critical in determining the pattern of binding. Docking of ACh to snapshots from the wild-type, L118D, and L118E resulted in solutions that were both in the binding pocket and had an orientation consistent with carbamylcholine bound to AChBP (Celie et al., 2004). Conversely, L118K and L118R mutant models failed to produce docking solutions with ACh in the binding pocket. Thus, results from the in silico study suggested that the negatively charged mutations (L118D and L118E) would retain the ability to bind ACh, but the positively charged mutations (L118K and L118R) would not.
Electrophysiological Studies on Wild-Type and Leu118-Mutated 7 nAChRss. We investigated the effects of L118D, L118E, L118K, and L118R substitutions on the functional 7 nAChR expressed in X. laevis oocytes. Control experiments on wild-type 7 show that imidacloprid is a partial agonist (Fig. 6A) consistent with earlier experiments (Matsuda et al., 2000). Figure 6 shows that after the L118D and L118E mutations, responses of the 7 receptor to imidacloprid were abolished (P < 0.01 using a one-way ANOVA Dunnett's multiple comparison test). However, the 7 mutants still responded to ACh (Fig. 6, B and C). The EC50 values of ACh were increased by these two mutations (Table 1). In complete contrast to the findings for the L118D and L118E mutations, L118K and L118R mutations blocked the responses to ACh, whereas responses to imidacloprid were observed (Fig. 6, D and E). In addition to these striking effects on the responses to ACh and imidacloprid, the mutations of Leu118 slowed the desensitization of the responses.
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7 nAChR expressed in X. laevis oocytes was slightly greater than the response to ACh (Fig. 7A), resembling results of a previous study (Ihara et al., 2003). The L118D and L118E mutations did not significantly affect the maximum response of 7 to DN-IMI (Fig. 7, B and C) (Table 1). However, the concentration-response curve of DN-IMI was shifted to the right by the L118D and L118E mutations (Table 1). In contrast to the effects on the maximum response to imidacloprid, the L118K and L118R mutations abolished the nicotinic receptor response to DN-IMI (Fig. 7, D and E). Thus the pEC50 values for DN-IMI could not be determined for these two mutant receptors. The concentration-response curves of ACh, imidacloprid and DN-IMI for the wild-type (A) and L118D (B), L118E (C), L118K (D), and L118E (E) mutants are shown in Fig. 8. It could be argued that, in certain mutants, imidacloprid may antagonize the response to ACh and ACh may antagonize the actions of imidacloprid. To begin to address this, we have examined whether the response to imidacloprid of the L118R mutant and, similarly, the response to ACh of the L118D mutant are suppressed by coapplication with ACh and imidacloprid, respectively. It was found that the response to 1 mM imidaclolprid in loop E was suppressed by coapplication with 1 mM ACh to 0.306 ± 0.099 (n = 2) compared with the response (= 1.00) to 1 mM imidaclolprid alone. On the other hand, the response to 3 mM ACh of the L118D mutant was suppressed by coapplication with 1 mM imidacloprid to 0.721 ± 0.125 (n = 2) compared with the response (= 1.00) to 3 mM ACh alone.
Radioligand Binding Studies on Wild-Type and Mutant 7 nAChRs. Binding experiments were also conducted to evaluate the affinity of [3H]ACh for the oocyte membrane fractions including the recombinant wild type and mutant 7 nAChRs. [3H]ACh showed saturable binding to the membrane fractions (Fig. 9). The Kd values (mean ± S.E.M.) of ACh for the wild type, L118R, and L118D were 211.4 ± 56.9, 538.6 ± 285.1, and 109.5 ± 64.1 µM, respectively. The Kd value of [3H]imidaclolprid could not be determined because of its low binding affinity (data not shown).
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Discussion |
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7 is supported by the in vitro experiments, suggesting that this model is capable of capturing the effects of ACh interacting with homomeric 7 nAChRs. A previous report also highlights the involvement of the corresponding residue in ligand binding, where L119C, L119C, and L121C mutants of the vertebrate muscle nAChR subunits 
, 
, and 
, respectively, treated with aminoethyl methanethiosulfonate showed reduced affinity for dimethyl-d-tubocurarine and -conotoxin M1 (Sine, 1997). Similar results were observed for the L119K, L119K, and L121K mutants. In addition, using photoaffinity labeling, a methionine (Met116) at the equivalent position in loop E of another molluscan AChBP from Aplysia californica was shown to be involved in imidacloprid binding (Tomizawa et al., 2007). All of these findings indicate that the pyridine ring of imidacloprid is likely to interact with loop E, placing its nitro group so as to interact with other residues facing the agonist binding domain.
Several studies have shown that electrostatic forces are important for imidacloprid interaction with nAChRs (Sattelle et al., 2005; Ihara et al., 2007). Here we show that substitutions of Leu118 in loop E by acidic or basic residues strikingly influence the responses of 7 to ACh. It should be noted that the antagonist action of ACh on the response of the L118D mutant to 1 mM imidacloprid was not sufficiently potent to block the response completely. A possible simple explanation for the effects of such mutations is that if Leu118 were mutated to a positive side-chain, the interaction with the charged quaternary nitrogen might be strong enough to prevent binding to the pocket; as a result, the channel fails to open. We have confidence that our model of (7)5 is reasonable because an orientation of ACh similar to that found for carbamylcholine in AChBP (Celie et al., 2004) was predicted. Furthermore, our model seems robust to small local changes in residue conformation as exemplified by the molecular dynamics (Fig. 4). In addition, the recent structure of the 1 nAChR subunit ligand-binding domain (Dellisanti et al., 2007) suggests that AChBP provides a suitable template for modeling the extracellular domain in this way.
Our initial model did not allow us to make confident predictions about the interactions of imidacloprid with the (7)5 model. This is perhaps not too surprising given that our model is based upon AChBP with HEPES bound. If the receptor conforms to an induced-fit model in terms of its interactions with ligands, then the model will suffer from being biased away from a conformation that would bind imidacloprid. Furthermore, recent experiments on AChBP (Gao et al., 2005) and on homology models of 7 (Henchman et al., 2003, 2005) suggest that there can be substantial changes in the shape of the binding pocket, in particular the conformation of loop C.
However, taking all these factors into consideration along with our present findings, we propose a model that accounts for the data shown in Figs. 6 and 7. Imidacloprid has a negatively charged nitro group, whereas DN-IMI possesses a positive charge at the guanidine moiety. It is interesting to note that the effects of mutations on DN-IMI are quite similar to those seen for ACh. Thus it could be that the positions of their positive charges in the binding pocket are similar. However, that would presumably require a different conformation of loop C to allow the DN-IMI to adopt a sterically favorable position within the binding pocket. Imidacloprid itself fails to generate currents in L118D and L118E mutants, but can effect channel opening in the case of L118K and L118R mutants. However, imidacloprid modulated the ACh-induced response of the L118D mutant, suggesting that the electrostatic interaction between the nitro group and this residue is critical in determining whether it will permit channel opening in response to agonist binding. A similar explanation is possible for the action of ACh on the mutant receptors. The Leu118 mutations influenced the desensitization of the imidacloprid- and ACh-induced responses, reflecting the role for Leu118 in the channel gating mechanism.
Electrostatic interactions are not the only forces determining agonist interactions with the 7 nAChR. The L118D and L118E mutations shifted the ACh concentration-response curve to higher concentrations and slightly reduced the maximum current amplitudes observed in response to ACh (Fig. 8), suggesting that such mutations in loop E may lead indirectly to a conformational change in another region of the receptor playing a key role in interactions with agonists. Thus, it will be necessary to further improve the modeling to gain a more complete understanding of neonicotinoid-nAChR interactions. Nevertheless, electrostatic interactions can explain the changes in concentration-response curves of the imidacloprid derivative DM-IMI lacking the nitro group. Unlike imidacloprid, DN-IMI has a positive charge at the guanidine moiety, thereby mimicking ACh. Consistent with this, the L118K and L118R mutations abolished responses of the 7 nAChR to DN-IMI, whereas L118D and L118E mutations permitted the agonist actions of this ligand.
Leu118 is not highly conserved across the nAChR family. Thus, the residue at this location may participate in determining subunit-specific responses to agonists. In the Drosophila melanogaster Dβ2 subunit, the residue corresponding to Leu118 is glutamic acid (Table 2). Because we found that the L118E mutation in 7 abolished imidacloprid action but retained sensitivity to ACh, it is predicable that imidacloprid will not be efficacious on insect nAChRs containing Dβ2 subunit in opening the channel gate. Conversely, several other insect nAChR subunits, such as D. melanogaster D1, D3, and D4, as well as Myzus persicae 2 and 3, possess either an arginine or lysine at the position corresponding to Leu118 (Table 2), which in our site-directed mutagenesis experiments increased the efficacy of imidacloprid (Table 1). It is noteworthy that studies have indicated that these subunits play a role in determining sensitivity to the insecticide, imidacloprid (Huang et al., 1999; Lansdell and Millar, 2000; Matsuda et al., 2001a; Shimomura et al., 2002, 2003, 2004, 2006). In heteromeric nAChRs consisting of and β subunits, it is believed that loop E from the β subunit contributes to the agonist binding site (Corringer et al., 2000b); however, it is important to note that the subunit composition and stoichiometry of native insect nAChRs have yet to be determined. Such information will prove instructive in assessing the relevance of loops A to F of either or β subunits in agonist binding and hence neonicotinoid sensitivity. In addition, mutations at Leu118 may provide a route by which target-site resistance could develop. If it is the case that in several insect nAChR subunits, the arginine or lysine at the residue corresponding to Leu118 contributes to imidacloprid sensitivity, a simple negative charge mutation here would be enough to maintain responses to the natural agonist, ACh, while abolishing the agonist actions of imidacloprid. Thus in searching field strains resistant to imidacloprid, it may be of interest to look for changes in loop E as well as the important loop B mutations (Liu et al., 2005) already known to be associated with imidacloprid resistance.
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In conclusion, we have used molecular modeling to predict that Leu118 of the 7 nAChR subunit contributes to agonist binding. Site-directed mutagenesis and functional expression of wild-type and mutant nAChRs subsequently confirmed this prediction. These results, taken together with our previous studies on loops C, D, and F (Shimomura et al., 2002, 2003, 2004, 2006), enhance our understanding of the binding to nAChRs of commercially important nicotinic agonists.
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Acknowledgements |
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7 cDNA. |
Footnotes |
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S.A. and M.S. contributed equally to this work.
ABBREVIATIONS: nAChR, Nicotinic acetylcholine receptor; ACh, acetylcholine; AChBP, acetylcholine binding protein; IMI, imidacloprid; DN-IMI, desnitro-imidacloprid; RMSF, root-mean-square fluctuations.
Address correspondence to: David B. Sattelle, University of Oxford, South Parks Road, Oxford OX1 3QX, UK. E-mail: david.sattelle{at}anat.ox.ac.uk
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