Crystal Structures of a Cysteine-modified Mutant in Loop D of Acetylcholine-binding Protein
2010; Elsevier BV; Volume: 286; Issue: 6 Linguagem: Inglês
10.1074/jbc.m110.188730
ISSN1083-351X
AutoresMarijke Brams, Elaine A. Gay, José O. Colón‐Sáez, Albert Guskov, René van Elk, Roel C. van der Schors, Steve Peigneur, Jan Tytgat, Sergei V. Strelkov, August B. Smit, Jerrel L. Yakel, Chris Ulens,
Tópico(s)Insect and Pesticide Research
ResumoCovalent modification of α7 W55C nicotinic acetylcholine receptors (nAChR) with the cysteine-modifying reagent [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+) produces receptors that are unresponsive to acetylcholine, whereas methyl methanethiolsulfonate (MMTS) produces enhanced acetylcholine-gated currents. Here, we investigate structural changes that underlie the opposite effects of MTSET+ and MMTS using acetylcholine-binding protein (AChBP), a homolog of the extracellular domain of the nAChR. Crystal structures of Y53C AChBP show that MTSET+-modification stabilizes loop C in an extended conformation that resembles the antagonist-bound state, which parallels our observation that MTSET+ produces unresponsive W55C nAChRs. The MMTS-modified mutant in complex with acetylcholine is characterized by a contracted C-loop, similar to other agonist-bound complexes. Surprisingly, we find two acetylcholine molecules bound in the ligand-binding site, which might explain the potentiating effect of MMTS modification in W55C nAChRs. Unexpectedly, we observed in the MMTS-Y53C structure that ten phosphate ions arranged in two rings at adjacent sites are bound in the vestibule of AChBP. We mutated homologous residues in the vestibule of α1 GlyR and observed a reduction in the single channel conductance, suggesting a role of this site in ion permeation. Taken together, our results demonstrate that targeted modification of a conserved aromatic residue in loop D is sufficient for a conformational switch of AChBP and that a defined region in the vestibule of the extracellular domain contributes to ion conduction in anion-selective Cys-loop receptors. Covalent modification of α7 W55C nicotinic acetylcholine receptors (nAChR) with the cysteine-modifying reagent [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+) produces receptors that are unresponsive to acetylcholine, whereas methyl methanethiolsulfonate (MMTS) produces enhanced acetylcholine-gated currents. Here, we investigate structural changes that underlie the opposite effects of MTSET+ and MMTS using acetylcholine-binding protein (AChBP), a homolog of the extracellular domain of the nAChR. Crystal structures of Y53C AChBP show that MTSET+-modification stabilizes loop C in an extended conformation that resembles the antagonist-bound state, which parallels our observation that MTSET+ produces unresponsive W55C nAChRs. The MMTS-modified mutant in complex with acetylcholine is characterized by a contracted C-loop, similar to other agonist-bound complexes. Surprisingly, we find two acetylcholine molecules bound in the ligand-binding site, which might explain the potentiating effect of MMTS modification in W55C nAChRs. Unexpectedly, we observed in the MMTS-Y53C structure that ten phosphate ions arranged in two rings at adjacent sites are bound in the vestibule of AChBP. We mutated homologous residues in the vestibule of α1 GlyR and observed a reduction in the single channel conductance, suggesting a role of this site in ion permeation. Taken together, our results demonstrate that targeted modification of a conserved aromatic residue in loop D is sufficient for a conformational switch of AChBP and that a defined region in the vestibule of the extracellular domain contributes to ion conduction in anion-selective Cys-loop receptors. IntroductionCys-loop receptors (CLRs) 2The abbreviations used are: CLR, Cys-loop receptor; AChBP, acetylcholine-binding protein; nAChR, nicotinic acetylcholine receptor; MMTS, methyl methanethiolsulfonate; MTSET+, [2-(trimethylammonium)ethyl] methanethiosulfonate; RMSD, root mean square deviation from ideal geometry. belong to a class of ligand-gated ion channels that are involved in fast synaptic transmission in the central nervous system and the neuromuscular junction. This transmission can either be excitatory or inhibitory depending on the charge of ions that pass through the ion conduction pathway of the channel. Excitatory transmission is mediated by nicotinic acetylcholine receptors (nAChR) and 5-HT3 serotonin receptors, which selectively pass cations. On the other hand, inhibitory transmission is mediated by glycine receptors (GlyR) and γ-aminobutyric acid (GABAA and GABAC) receptors, which selectively conduct anions. In both types of receptors, the ion conduction pathway lies along the central axis formed by five channel subunits, which can either be identical for homomeric CLRs or non-identical for heteromeric CLRs. Opening and closing of the ion conduction pathway is controlled by a gate that is allosterically coupled to the extracellular ligand-binding domain (for a recent review see Ref. 1Taly A. Corringer P.J. Guedin D. Lestage P. Changeux J.P. Nat. Rev. Drug Discov. 2009; 8: 733-750Crossref PubMed Scopus (540) Google Scholar).Detailed structural data are still lacking for an intact eukaryotic CLR, but structural information has been obtained from 4 Å resolution electron microscopic images of the Torpedo nAChR (2Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1404) Google Scholar) and higher resolution x-ray crystal structures of AChBP, a molluscan homolog of the extracellular domain of nAChRs (3Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1571) Google Scholar, 4Smit A.B. Syed N.I. Schaap D. van Minnen J. Klumperman J. Kits K.S. Lodder H. van der Schors R.C. van Elk R. Sorgedrager B. Brejc K. Sixma T.K. Geraerts W.P. Nature. 2001; 411: 261-268Crossref PubMed Scopus (460) Google Scholar), the monomeric α1 nAChR subunit (5Dellisanti C.D. Yao Y. Stroud J.C. Wang Z.Z. Chen L. Nat. Neurosci. 2007; 10: 953-962Crossref PubMed Scopus (354) Google Scholar), and two prokaryotic homologs ELIC (6Hilf R.J. Dutzler R. Nature. 2008; 452: 375-379Crossref PubMed Scopus (574) Google Scholar) and GLIC (7Hilf R.J. Dutzler R. Nature. 2009; 457: 115-118Crossref PubMed Scopus (467) Google Scholar, 8Bocquet N. Nury H. Baaden M. Le Poupon C. Changeux J.P. Delarue M. Corringer P.J. Nature. 2009; 457: 111-114Crossref PubMed Scopus (586) Google Scholar), which presumably represent the closed and open state of a CLR. Despite their different pharmacological properties, these CLRs share a common architectural arrangement of aromatic residues in their ligand-binding site. The ligand-binding site for CLRs is found at the interface between two subunits and ligands interact with amino acids from both the principal face (containing loops A, B, and C) and complementary face (containing loops D, E, and F). We have focused our attention on an aromatic residue that lies on the complementary face of the binding pocket and is highly conserved among eukaryotic and prokaryotic CLRs.Recently, we found that the tryptophan residue at position 55 of the rat α7 nAChR (Trp-55) was the site where synthetic peptides derived from apolipoprotein E non-competitively inhibited α7 receptors through hydrophobic interactions (9Gay E.A. Bienstock R.J. Lamb P.W. Yakel J.L. Mol. Pharmacol. 2007; 72: 838-849Crossref PubMed Scopus (19) Google Scholar). In addition, when Trp-55 was mutated to alanine, the α7 W55A nAChR desensitized more slowly and recovered from desensitization more rapidly than wild-type receptor (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar). Mutating Trp-55 to other aromatic residues (Phe or Tyr) had no significant effect on the kinetics of desensitization, whereas mutation to various hydrophobic residues (Ala, Cys, or Val) significantly decreased the rate of onset and increased the rate of recovery from desensitization (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar). To gain insight into possible structural rearrangements during desensitization, we probed the accessibility of Trp-55 by mutating Trp-55 to cysteine (α7 W55C) and tested the ability of various sulfhydryl reagents to react with this cysteine (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar). Modification with several positively charged sulfhydryl reagents, including [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+), produced α7 W55C nAChRs that became unresponsive to acetylcholine, whereas a neutral sulfhydryl reagent methyl methanethiolsulfonate (MMTS) enhanced acetylcholine-activated currents by nearly 60% (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar). These data suggested that Trp-55 plays an important role in both the onset and recovery from desensitization in the rat α7 nAChR, and suggested that Trp-55 may be a potential target for modulatory agents operating via hydrophobic interactions.However, these data left unresolved how modification of loop D with MTSET+ or MMTS leads to two clearly distinct functional effects. MTSET+ modification renders α7 W55C receptors unresponsive to acetylcholine, whereas MMTS modification produces receptors with enhanced responses to acetylcholine. In this study, we used AChBP to determine x-ray crystal structures of the homologous Y53C mutant modified either with MTSET+ alone or MMTS in the presence of acetylcholine. We investigated whether structural changes of methanethiosulfonate (MTS)-modified AChBP correlate with the opposing functional effects of MTSET+ and MMTS observed on α7 W55C nAChRs.DISCUSSIONSubstituted cysteine accessibility scanning (SCAM) is a widely used method to study the relationship between structure and function of ion channels (24Karlin A. Akabas M.H. Methods Enzymol. 1998; 293: 123-145Crossref PubMed Scopus (542) Google Scholar). With the availability of an increasing number of x-ray crystal structures for ion channels, SCAM is now frequently employed to test the validity of three-dimensional models in the context of channel dynamics and their lipid membrane environment (25Gandhi C.S. Clark E. Loots E. Pralle A. Isacoff E.Y. Neuron. 2003; 40: 515-525Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 26Ahern C.A. Horn R. Neuron. 2005; 48: 25-29Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 27Broomand A. Elinder F. Neuron. 2008; 59: 770-777Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). SCAM has powerful advantages in that the method allows real-time monitoring of the reaction speed between the MTS reagent and the targeted cysteine residue for the closed, open, or desensitized state of the channel. However, this reaction is typically followed indirectly by measuring changes in the functional properties of ion channels such as ion conduction, voltage dependence of activation or ligand activation. Because of the indirect nature of this method, it is not always possible to establish a clear relationship between the observed functional effects of MTS modification and the underlying conformational changes of the channel.In this study, we report the first x-ray crystal structures of an MTS-modified cysteine mutant of the nAChR homolog AChBP. We used the homologous mutant of α7 W55C nAChRs, which is oppositely regulated by two different MTS reagents, namely MTSET+ and MMTS (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar), to investigate the correlation between structure and function. We previously demonstrated that modification of α7 W55C nAChRs with MTSET+ produces receptors that become unresponsive to saturating concentrations of acetylcholine (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar). The crystal structure of MTSET+-modified Y53C AChBP shows that loop C is stabilized in a conformational state that resembles the antagonist-bound state. This result suggests that the unresponsive behavior of MTSET+-modified α7 W55C nAChRs may arise from a similar stabilization of the receptor in an antagonist-bound state. This also suggests that the conformation of loop C is correlated with the activational state of the receptor. In contrast to MTSET+-modified α7 W55C nAChRs, we previously showed that modification of α7 W55C nAChRs with MMTS enhances acetylcholine-evoked currents by nearly 60% (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar). The crystal structure of MMTS-modified Y53C AChBP shows that loop C is strongly contracted, similar to other agonist-bound structures. We also observed that two acetylcholine molecules occupy the binding pocket, which possibly explains the potentiating effect of acetylcholine on MMTS-modified α7 W55C nAChRs. One of these acetylcholine molecules faces the principal binding site and occupies a position that overlaps with the binding mode observed in the related carbamylcholine-bound structure of AChBP. The second acetylcholine molecule interacts with residues of the complementary face, including the MMTS-modified side chain of Cys-53. Therefore, we propose that the potentiating effect of MMTS on α7 W55C nAChRs arises from a favorable interaction with the MMTS-modified side chain that stabilizes two acetylcholine molecules in the ligand-binding site. Together, both AChBP crystal structures parallel our observations from functional studies on α7 W55C nAChRs, and offer possible explanations for the opposing effects of MTSET+ and MMTS. Our results suggest that targeted modification of a single residue in loop D is sufficient to trigger conformational changes of AChBP.The functional importance of residues in loop D has been demonstrated by mutagenesis studies in different CLRs. Mutation of the homologous W55 residue in the GABAA-R γ2-subunit, F77C, abolished binding by [3H]Ro15–1788 and the benzodiazepine [3H]flunitrazepam. Mutation of neighboring residues in loop D, A79C and T81C, caused a 10-fold reduction in the affinity of the tranquilizers eszopiclone and zolpidem (28Hanson S.M. Morlock E.V. Satyshur K.A. Czajkowski C. J. Med. Chem. 2008; 51: 7243-7252Crossref PubMed Scopus (182) Google Scholar). In the GABAA-R α1-subunit, it was shown that for the homologous Trp-55 subtle changes were caused by unnatural amino acid mutations (29Padgett C.L. Hanek A.P. Lester H.A. Dougherty D.A. Lummis S.C. J. Neurosci. 2007; 27: 886-892Crossref PubMed Scopus (95) Google Scholar). In the muscle nAChR it was shown that mutation W57F in the δ-subunit and W55F in the ϵ-subunit have only minor effects on acetylcholine-sensitivity but mainly affect channel gating by reducing the channel opening rate (30Akk G. J. Physiol. 2002; 544: 695-705Crossref PubMed Scopus (36) Google Scholar). In insect nAChRs, it was shown that the basic Arg and Lys residues at positions +2 and +4 of the homologous W55 residue are responsible for the high affinity of the insecticide imidacloprid and related neonicotinoids (31Shimomura M. Yokota M. Ihara M. Akamatsu M. Sattelle D.B. Matsuda K. Mol. Pharmacol. 2006; 70: 1255-1263Crossref PubMed Scopus (105) Google Scholar). Mutation of loop D residues N55S and V56I in the human nAChR β4-subunit, which are at positions −2 and −1 of the homologous Trp-55 residue, abolishes sensitivity to TMAQ, a novel agonist for β4-containing nAChRs (32Young G.T. Broad L.M. Zwart R. Astles P.C. Bodkin M. Sher E. Millar N.S. Mol. Pharmacol. 2007; 71: 389-397Crossref PubMed Scopus (38) Google Scholar). In a related study it was shown that mutations N55S, V56I, T59K, and E63T in the human nAChR β2-subunit reduce the affinity of acetylcholine and nicotine, while having little to no effect on the affinity of epibatidine and dimethylphenylpiperazinium (DMPP) (33Parker M.J. Harvey S.C. Luetje C.W. J. Pharmacol. Exp. Ther. 2001; 299: 385-391PubMed Google Scholar).In addition to loop D, accumulating evidence implicates loop C as a structural component that is key to both ligand binding and subsequent conformational changes underlying CLR activation, inhibition, and desensitization (34Hibbs R.E. Johnson D.A. Shi J. Taylor P. J. Mol. Neurosci. 2006; 30: 73-74Crossref PubMed Scopus (4) Google Scholar). The important role of loop C derives from a large body of work using x-ray crystallography (11Ulens C. Hogg R.C. Celie P.H. Bertrand D. Tsetlin V. Smit A.B. Sixma T.K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 3615-3620Crossref PubMed Scopus (180) Google Scholar, 35Hansen S.B. Sulzenbacher G. Huxford T. Marchot P. Taylor P. Bourne Y. EMBO J. 2005; 24: 3635-3646Crossref PubMed Scopus (569) Google Scholar, 36Dutertre S. Lewis R.J. Biochem. Pharmacol. 2006; 72: 661-670Crossref PubMed Scopus (56) Google Scholar, 37Hibbs R.E. Sulzenbacher G. Shi J. Talley T.T. Conrod S. Kem W.R. Taylor P. Marchot P. Bourne Y. EMBO J. 2009; 28: 3040-3051Crossref PubMed Scopus (136) Google Scholar), molecular dynamics simulation (38Gao F. Bren N. Burghardt T.P. Hansen S. Henchman R.H. Taylor P. McCammon J.A. Sine S.M. J. Biol. Chem. 2005; 280: 8443-8451Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 39Cheng X. Wang H. Grant B. Sine S.M. McCammon J.A. PLoS Comput. Biol. 2006; 2: e134Crossref PubMed Scopus (108) Google Scholar, 40Yi M. Tjong H. Zhou H.X. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 8280-8285Crossref PubMed Scopus (44) Google Scholar), site-directed mutagenesis (41Horenstein N.A. McCormack T.J. Stokes C. Ren K. Papke R.L. J. Biol. Chem. 2007; 282: 5899-5909Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 42Hibbs R.E. Radic Z. Taylor P. Johnson D.A. J. Biol. Chem. 2006; 281: 39708-39718Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 43Suryanarayanan A. Joshi P.R. Bikádi Z. Mani M. Kulkarni T.R. Gaines C. Schulte M.K. Biochemistry. 2005; 44: 9140-9149Crossref PubMed Scopus (20) Google Scholar, 44Venkatachalan S.P. Czajkowski C. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 13604-13609Crossref PubMed Scopus (54) Google Scholar), and electrophysiology (45Pless S.A. Lynch J.W. J. Biol. Chem. 2009; 284: 27370-27376Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 46Toshima K. Kanaoka S. Yamada A. Tarumoto K. Akamatsu M. Sattelle D.B. Matsuda K. Neuropharmacology. 2009; 56: 264-272Crossref PubMed Scopus (24) Google Scholar). The data from these studies demonstrates that loop C is flexible in the non-liganded form (22Shi J. Koeppe J.R. Komives E.A. Taylor P. J. Biol. Chem. 2006; 281: 12170-12177Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 37Hibbs R.E. Sulzenbacher G. Shi J. Talley T.T. Conrod S. Kem W.R. Taylor P. Marchot P. Bourne Y. EMBO J. 2009; 28: 3040-3051Crossref PubMed Scopus (136) Google Scholar) and adopts distinct conformations upon agonist or antagonist binding. Loop C assumes a contracted configuration with agonists bound (corresponding to either the open or desensitized state of the receptor) and takes on an extended configuration with antagonists bound (corresponding to the closed state of the channel (11Ulens C. Hogg R.C. Celie P.H. Bertrand D. Tsetlin V. Smit A.B. Sixma T.K. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 3615-3620Crossref PubMed Scopus (180) Google Scholar, 35Hansen S.B. Sulzenbacher G. Huxford T. Marchot P. Taylor P. Bourne Y. EMBO J. 2005; 24: 3635-3646Crossref PubMed Scopus (569) Google Scholar, 36Dutertre S. Lewis R.J. Biochem. Pharmacol. 2006; 72: 661-670Crossref PubMed Scopus (56) Google Scholar). More recent work suggests that the degree of loop C movement may correspond to agonist efficacy (37Hibbs R.E. Sulzenbacher G. Shi J. Talley T.T. Conrod S. Kem W.R. Taylor P. Marchot P. Bourne Y. EMBO J. 2009; 28: 3040-3051Crossref PubMed Scopus (136) Google Scholar). Movement in the ligand binding domain is thought to propagate from the extracellular domain to the pore region to allow activation or inhibition of ion flux. This transduction of signal can be understood both as a sequence of chemical events (47Sine S.M. Engel A.G. Nature. 2006; 440: 448-455Crossref PubMed Scopus (421) Google Scholar) or as coordinated movements or rotations of the whole extracellular domain (40Yi M. Tjong H. Zhou H.X. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 8280-8285Crossref PubMed Scopus (44) Google Scholar, 48Law R.J. Henchman R.H. McCammon J.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 6813-6818Crossref PubMed Scopus (130) Google Scholar, 49Purohit P. Mitra A. Auerbach A. Nature. 2007; 446: 930-933Crossref PubMed Scopus (128) Google Scholar).Unexpectedly, we observed electron density that could be interpreted as phosphate ions in the vestibule of AChBP at a location that is very near to the interface with the transmembrane domain in integral Cys-loop receptors. The contribution of rings of charged amino acids to channel conductance has previously been demonstrated in the Torpedo nAChR (50Imoto K. Busch C. Sakmann B. Mishina M. Konno T. Nakai J. Bujo H. Mori Y. Fukuda K. Numa S. Nature. 1988; 335: 645-648Crossref PubMed Scopus (603) Google Scholar), but detailed insight into the mechanism of ion conduction was lacking. Structural insight into selection of ions in the extracellular domain of Cys-loop receptors was obtained from a crystal structure containing 5 sulfate ions near residue Arg-95 in the vestibule of AChBP (23Hansen S.B. Wang H.L. Taylor P. Sine S.M. J. Biol. Chem. 2008; 283: 36066-36070Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Hansen et al. demonstrated that this residue, which corresponds to a highly conserved Asp in cation-selective Cys-loop receptors, affects single channel conductance upon charge reversal mutations in the muscle nAChR. In our structure, we observe 10 phosphate ions bound to a cluster of charged residues that involve Lys-40, Glu-47, Asp-49, and Arg-95. The phosphate ions are arranged in two pentagonal layers separated by a distance of less than 4 Å. The 5 phosphate ions in the upper layer (ring 1) occupy the same positions as the sulfate ions in the study from Hansen et al. and are arranged at a distance of 9 Å apart. Phosphate ions in the lower layer (ring 2) are spaced at a distance 7 Å apart and interact with Lys-40, Glu-47, and Asp-49. The observation that sulfates as well phosphates bind in a defined location of the vestibule of AChBP suggests that this region functions as a general anion-binding site. One of the binding site residues, Glu-47, is strictly conserved as a negatively charged amino acid (Asp or Glu) in anion-selective Cys-loop receptors, and hydrophobic (M, I, or V) in cation-selective Cys-loop receptors. Therefore, we hypothesized that chloride anions may interact in a similar manner with conserved negatively charged residues in anion-selective Cys-loop receptors, such as GABAA-R and GlyR. We demonstrated that mutation of homologous residues in ring 1 and ring 2 of the α1 GlyR causes a pronounced reduction in the single channel conductance. This result suggests a functional role of the extracellular domain of α1 GlyR in selection and permeation of anions. Our observation fits with molecular dynamics simulations of the nAChR (51Wang H.L. Cheng X. Taylor P. McCammon J.A. Sine S.M. PLoS Comput. Biol. 2008; 4: e41Crossref PubMed Scopus (49) Google Scholar) and the bacterial homolog GLIC (52Nury H. Poitevin F. Van Renterghem C. Changeux J.P. Corringer P.J. Delarue M. Baaden M. Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 6275-6280Crossref PubMed Scopus (129) Google Scholar), which demonstrated the existence of one or more cation reservoirs in the extracellular domain of these cation-selective Cys-loop receptors.In conclusion, our study shows a good correlation between structure and function, and offers possible explanations for the opposite effects of MTSET+ and MMTS on α7 W55C nAChRs. We show that targeted modification of loop D plays a key role in defining the conformational state of AChBP. This hints at a contribution of a conserved aromatic residue in loop D that goes beyond its established role of shaping the ligand-binding site. This residue may contribute to a structural switch that discriminates between the activated and non-activated state of Cys-loop receptors. In addition, the unexpected observation of phosphate anions bound in the vestibule of AChBP parallels the functional role of two rings of charged residues in the extracellular domain of the α1 GlyR that contribute to ion selection and permeation. IntroductionCys-loop receptors (CLRs) 2The abbreviations used are: CLR, Cys-loop receptor; AChBP, acetylcholine-binding protein; nAChR, nicotinic acetylcholine receptor; MMTS, methyl methanethiolsulfonate; MTSET+, [2-(trimethylammonium)ethyl] methanethiosulfonate; RMSD, root mean square deviation from ideal geometry. belong to a class of ligand-gated ion channels that are involved in fast synaptic transmission in the central nervous system and the neuromuscular junction. This transmission can either be excitatory or inhibitory depending on the charge of ions that pass through the ion conduction pathway of the channel. Excitatory transmission is mediated by nicotinic acetylcholine receptors (nAChR) and 5-HT3 serotonin receptors, which selectively pass cations. On the other hand, inhibitory transmission is mediated by glycine receptors (GlyR) and γ-aminobutyric acid (GABAA and GABAC) receptors, which selectively conduct anions. In both types of receptors, the ion conduction pathway lies along the central axis formed by five channel subunits, which can either be identical for homomeric CLRs or non-identical for heteromeric CLRs. Opening and closing of the ion conduction pathway is controlled by a gate that is allosterically coupled to the extracellular ligand-binding domain (for a recent review see Ref. 1Taly A. Corringer P.J. Guedin D. Lestage P. Changeux J.P. Nat. Rev. Drug Discov. 2009; 8: 733-750Crossref PubMed Scopus (540) Google Scholar).Detailed structural data are still lacking for an intact eukaryotic CLR, but structural information has been obtained from 4 Å resolution electron microscopic images of the Torpedo nAChR (2Unwin N. J. Mol. Biol. 2005; 346: 967-989Crossref PubMed Scopus (1404) Google Scholar) and higher resolution x-ray crystal structures of AChBP, a molluscan homolog of the extracellular domain of nAChRs (3Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1571) Google Scholar, 4Smit A.B. Syed N.I. Schaap D. van Minnen J. Klumperman J. Kits K.S. Lodder H. van der Schors R.C. van Elk R. Sorgedrager B. Brejc K. Sixma T.K. Geraerts W.P. Nature. 2001; 411: 261-268Crossref PubMed Scopus (460) Google Scholar), the monomeric α1 nAChR subunit (5Dellisanti C.D. Yao Y. Stroud J.C. Wang Z.Z. Chen L. Nat. Neurosci. 2007; 10: 953-962Crossref PubMed Scopus (354) Google Scholar), and two prokaryotic homologs ELIC (6Hilf R.J. Dutzler R. Nature. 2008; 452: 375-379Crossref PubMed Scopus (574) Google Scholar) and GLIC (7Hilf R.J. Dutzler R. Nature. 2009; 457: 115-118Crossref PubMed Scopus (467) Google Scholar, 8Bocquet N. Nury H. Baaden M. Le Poupon C. Changeux J.P. Delarue M. Corringer P.J. Nature. 2009; 457: 111-114Crossref PubMed Scopus (586) Google Scholar), which presumably represent the closed and open state of a CLR. Despite their different pharmacological properties, these CLRs share a common architectural arrangement of aromatic residues in their ligand-binding site. The ligand-binding site for CLRs is found at the interface between two subunits and ligands interact with amino acids from both the principal face (containing loops A, B, and C) and complementary face (containing loops D, E, and F). We have focused our attention on an aromatic residue that lies on the complementary face of the binding pocket and is highly conserved among eukaryotic and prokaryotic CLRs.Recently, we found that the tryptophan residue at position 55 of the rat α7 nAChR (Trp-55) was the site where synthetic peptides derived from apolipoprotein E non-competitively inhibited α7 receptors through hydrophobic interactions (9Gay E.A. Bienstock R.J. Lamb P.W. Yakel J.L. Mol. Pharmacol. 2007; 72: 838-849Crossref PubMed Scopus (19) Google Scholar). In addition, when Trp-55 was mutated to alanine, the α7 W55A nAChR desensitized more slowly and recovered from desensitization more rapidly than wild-type receptor (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar). Mutating Trp-55 to other aromatic residues (Phe or Tyr) had no significant effect on the kinetics of desensitization, whereas mutation to various hydrophobic residues (Ala, Cys, or Val) significantly decreased the rate of onset and increased the rate of recovery from desensitization (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar). To gain insight into possible structural rearrangements during desensitization, we probed the accessibility of Trp-55 by mutating Trp-55 to cysteine (α7 W55C) and tested the ability of various sulfhydryl reagents to react with this cysteine (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar). Modification with several positively charged sulfhydryl reagents, including [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+), produced α7 W55C nAChRs that became unresponsive to acetylcholine, whereas a neutral sulfhydryl reagent methyl methanethiolsulfonate (MMTS) enhanced acetylcholine-activated currents by nearly 60% (10Gay E.A. Giniatullin R. Skorinkin A. Yakel J.L. J. Physiol. 2008; 586: 1105-1115Crossref PubMed Scopus (45) Google Scholar). These data suggested that Trp-55 plays an important role in both the onset and recovery from desensitization in the rat α7 nAChR, and suggested that Trp-55 may be a potential target for modulatory agents operating via hydrophobic interactions.However, these data left unresolved how modification of loop D with MTSET+ or MMTS leads to two clearly distinct functional effects. MTSET+ modification renders α7 W55C receptors unresponsive to acetylcholine, whereas MMTS modification produces receptors with enhanced responses to acetylcholine. In this study, we used AChBP to determine x-ray crystal structures of the homologous Y53C mutant modified either with MTSET+ alone or MMTS in the presence of acetylcholine. We investigated whether structural changes of methanethiosulfonate (MTS)-modified AChBP correlate with the opposing functional effects of MTSET+ and MMTS observed on α7 W55C nAChRs.
Referência(s)