Mapping the Agonist Binding Site of the Nicotinic Acetylcholine Receptor

Abstract

To characterize the structural requirements for ligand orientation compatible with activation of theTorpedo nicotinic acetylcholine receptor (nAChR), we used Cys mutagenesis in conjunction with sulfhydryl-reactive reagents to tether primary or quaternary amines at defined positions within the agonist binding site of nAChRs containing mutant α- or γ-subunits expressed in Xenopus oocytes. 4-(N-Maleimido)benzyltrimethylammonium and 2-aminoethylmethanethiosulfonate acted as irreversible antagonists when tethered at αY93C, αY198C, or γE57C, as well as at αN94C (2-aminoethylmethanethiosulfonate only). [2-(Trimethylammonium)-ethyl]-methanethiosulfonate (MTSET), which attaches thiocholine to binding site Cys, also acted as an irreversible antagonist when tethered at αY93C, αN94C, or γE57C. However, MTSET modification of αY198C resulted in prolonged activation of the nAChR not reversible by washing but inhibitable by subsequent exposure to non-competitive antagonists. Modification of αY198C (or any of the other positions tested) by [(trimethylammonium)methyl]methanethiosulfonate resulted only in irreversible inhibition, while modification of αY198C by [3-(trimethylammonium)propyl]methanethiosulfonate resulted in irreversible activation of nAChR, but at lower efficacy than by MTSET. Thus changing the length of the tethering arm by less than 1 Å in either direction markedly effects the ability of the covalent trimethylammonium to activate the nAChR, and agonist activation depends on a very selective orientation of the quaternary ammonium within the agonist binding site. To characterize the structural requirements for ligand orientation compatible with activation of theTorpedo nicotinic acetylcholine receptor (nAChR), we used Cys mutagenesis in conjunction with sulfhydryl-reactive reagents to tether primary or quaternary amines at defined positions within the agonist binding site of nAChRs containing mutant α- or γ-subunits expressed in Xenopus oocytes. 4-(N-Maleimido)benzyltrimethylammonium and 2-aminoethylmethanethiosulfonate acted as irreversible antagonists when tethered at αY93C, αY198C, or γE57C, as well as at αN94C (2-aminoethylmethanethiosulfonate only). [2-(Trimethylammonium)-ethyl]-methanethiosulfonate (MTSET), which attaches thiocholine to binding site Cys, also acted as an irreversible antagonist when tethered at αY93C, αN94C, or γE57C. However, MTSET modification of αY198C resulted in prolonged activation of the nAChR not reversible by washing but inhibitable by subsequent exposure to non-competitive antagonists. Modification of αY198C (or any of the other positions tested) by [(trimethylammonium)methyl]methanethiosulfonate resulted only in irreversible inhibition, while modification of αY198C by [3-(trimethylammonium)propyl]methanethiosulfonate resulted in irreversible activation of nAChR, but at lower efficacy than by MTSET. Thus changing the length of the tethering arm by less than 1 Å in either direction markedly effects the ability of the covalent trimethylammonium to activate the nAChR, and agonist activation depends on a very selective orientation of the quaternary ammonium within the agonist binding site. nicotinic acetylcholine receptor acetylcholine 4-(N-maleimido)benzyltrimethylammonium 2-aminoethylmethanethiosulfonate ([2-(trimethylammonium)-ethyl]-methanethiosulfonate d-tubocurarine bromoacetylcholine α-bungarotoxin [(trimethylammonium)methyl]methanethiosulfonate [3-(trimethylammonium)propyl]methanethiosulfonate methanethiosulfonate polymerase chain reaction The Torpedo nicotinic acetylcholine receptor (nAChR)1 is made up of four homologous, transmembrane subunits (α2βγδ) that associate pseudosymmetrically about a central axis that forms the cation-selective ion channel. Efficient channel gating depends upon the binding of two acetylcholine (ACh) molecules. The binding sites for agonist and competitive antagonists are within the extracellular domain of the nAChR at the α-γ and α-δ subunit interfaces (1.Blount P. Merlie J.P. Neuron. 1989; 3: 349-357Abstract Full Text PDF PubMed Scopus (225) Google Scholar, 2.Pedersen S.E. Cohen J.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2785-2789Crossref PubMed Scopus (223) Google Scholar, 3.Sine S.M. Claudio T. J. Biol. Chem. 1991; 266: 19369-19377Abstract Full Text PDF PubMed Google Scholar). Affinity labeling and mutagenesis studies support a model for agonist site structure with contributing amino acids from three distinct regions of the α-subunits and from multiple regions of the γ (or δ)-subunit (reviewed in Refs. 4.Hucho F. Tsetlin V.I. Machold J. Eur. J. Biochem. 1996; 239: 539-557Crossref PubMed Scopus (204) Google Scholar, 5.Prince R.J. Sine S.M. Barrantes F.J. The Nicotinic Acetylcholine Receptor: Current Views and Future Trends. Springer Verlag/R. G. Landes Co., Austin, TX1998: 31-59Crossref Google Scholar, 6.Corringer, P. J., Le Novere, N., and Changeux, J.-P. (2000) Annu. Rev. Pharmacol. Toxicol., in pressGoogle Scholar). For Torpedo nAChR, photoaffinity labeling with the competitive antagonistp-[3H]dimethylaminobenzenediazonium fluoroborate identified residues in three distinct regions of the α-subunit primary sequence: αTyr93 (loop A), αTrp149 (loop B), and αTyr190 (loop C) (7.Dennis M. Giraudat J. Kotzyba-Hibert F. Goeldner M. Hirth C. Chang J.-Y. Lazure C. Chrétien M. Changeux J.-P. Biochemistry. 1988; 27: 2346-2357Crossref PubMed Scopus (248) Google Scholar,8.Galzi J.L. Revah F. Black D. Goeldner M. Hirth C. Changeux J.-P. J. Biol. Chem. 1990; 265: 10430-10437Abstract Full Text PDF PubMed Google Scholar). Photoaffinity labeling with [3H]d-tubocurarine (dTC), a competitive antagonist, and with [3H]nicotine, an agonist, identified α-subunit residues in loop C as well as γTrp55 (loop D), and [3H]dTC also reacted with γTyr111/Tyr117 (loop E) (9.Chiara D.C. Cohen J.B. J. Biol. Chem. 1997; 272: 32940-32950Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 10.Chiara D.C. Middleton R.E. Cohen J.B. FEBS Lett. 1998; 423: 223-226Crossref PubMed Scopus (46) Google Scholar, 11.Chiara D.C. Xie Y. Cohen J.B. Biochemistry. 1999; 38: 6689-6698Crossref PubMed Scopus (59) Google Scholar). Using a heterobifunctional cross-linker ∼9 Å in length, αCys192/193 was cross-linked to a negatively charged residue in the δ-subunit identified as δAsp180 (loop F) (12.Czajkowski C. Karlin A. J. Biol. Chem. 1995; 270: 3160-3164Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). In embryonic mouse muscle nAChR (α2βγδ), the site selectivity of dimethyl-d-tubocurarine has been attributed to amino acid differences in loop E (γIle116Tyr117/δVal118Thr119) and loop F (γSer161/δLys163) (13.Sine S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9436-9440Crossref PubMed Scopus (164) Google Scholar, 14.Fu D.X. Sine S.M. J. Biol. Chem. 1994; 269: 26152-26157Abstract Full Text PDF PubMed Google Scholar), while for adult mouse nAChR (α2βεδ) antagonist affinity determinants have been identified in loop D (γGlu57) and other regions of non-α subunit (15.Bren N. Sine S.M. J. Biol. Chem. 1997; 272: 30793-30798Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Despite the availability of this large body of information about the identity of amino acids that contribute to the agonist binding site, little is known about their overall three-dimensional distribution or about the orientation of agonists or antagonists bound within the site. ACh has two functional groups, the acetyl group and a quaternary ammonium group located within 6.4 Å of the carbonyl carbon. Simpler quaternary ammonium compounds including choline and tetramethylammonium also act as agonists, although they are less potent than ACh, and when bound the probability of channel opening is substantially lower than for ACh (16.Akk G. Auerbach A. Biophys. J. 1996; 70: 2652-2658Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 17.Zhou M. Engel A.G. Auerbach A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10466-10471Crossref PubMed Scopus (72) Google Scholar). While 4-(N-maleimido)benzyltrimethylammonium (MBTA) acts as an irreversible antagonist after reaction with αCys192/193in reduced, wild type nAChR (18.Kao P. Dwork A.J. Kaldany R.J. Silver M.L. Wideman J. Stein S. Karlin A. J. Biol. Chem. 1984; 259: 11662-11665Abstract Full Text PDF PubMed Google Scholar), bromoacetylcholine (BrACh) acts as a covalent agonist after reaction with αCys192/193 (19.Damle V.N. McLaughlin M. Karlin A. Biochem. Biophys. PubMed Scopus Google J. PubMed Scopus Google Scholar). in the nAChR, of the group within Å of αCys192/193 are for channel Affinity labeling with containing the within a that the of ACh with αTyr93 in the of the nAChR J.B. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). of αTrp149 by an amino in a receptor J.P. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), that an the quaternary ammonium ion and the chain for nAChR To characterize the agonist binding site we have Cys in α-subunit in ACh site and C and in loop D and the functional of mutant nAChRs after reaction with Cys mutagenesis has been used to structure in a of ion and residue and transmembrane of S. M. J. J. 1998; PubMed Scopus Google A. 1998; PubMed Scopus Google Scholar). In studies of nAChR, has been used to the structure of the ion channel C. P. Karlin A. Neuron. 1994; Full Text PDF PubMed Scopus Google Scholar, Karlin A. Biochemistry. 1997; PubMed Scopus Google Scholar, Karlin A. Biochemistry. 1998; PubMed Scopus Google and to residues in binding site C and E for binding G. Biochem. J. 1995; PubMed Scopus Google Scholar, S.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. McLaughlin Biochemistry. 1999; 38: PubMed Scopus Google Scholar). studies of Torpedo nAChR wild type and Cys mutant subunits expressed in Xenopus and by in the and by receptor The effects of covalent modification within the binding site including and methanethiosulfonate which to a of charged and Karlin A. Biochemistry. 1994; PubMed Scopus Google Scholar). a charged primary to attaches thiocholine to which is than a group to Cys, and which is than The to the orientation of the quaternary ammonium ion within the agonist binding site that is for nAChR containing the wild type and from of and and the in was from of which of two Scholar). used for and from from and used to polymerase and the was by at on The for the and was αTyr93 made by an that was sites within the α-subunit and at αTrp149 mutagenesis by the the which at the site, and the which the site. in the α-subunit at αTyr190 and that which the site and site the of the γTrp55 made by an that was the site in the and the site in the of and in the reaction with the For αTyr93 the for with a at 1 at 1 at the for at at and at The was also used for for at αTyr190 and and also for The from or by The containing the was the subunit of the and the of the by the with either and or and in with polymerase and at a of to a of for by and in 1 by ACh from after a at a of with and of The was by with 1 containing 1 of ACh, and other the for ACh, and from was from of competitive and antagonists in to of reagents at in and on with for which was for only on For nAChR ACh to the and are the at a of ACh and the is the activation for ACh, and is the and used for For the Cys reagents irreversible of ACh the of the reaction with a Cys mutant in the of ACh was by the to ACh and the to ACh after of for of was by a three ACh and a ACh was at a after as a of modification and by a is the of the and the Karlin A. J. 1998; PubMed Scopus Google Scholar). two of on two For reagents in irreversible activation of after and ACh to the To binding to nAChRs expressed on the with for at in a of of and three with of to and for in a γ Cys at positions by or mutagenesis as contributing to the agonist binding and To the of Cys at any of positions nAChR we functional by and the binding of to nAChRs on the of the Cys well when expressed with other wild type subunits for wild type nAChR, for ACh was with a of and of of subunit resulted in the on the of binding after The Cys in at and little any or as with wild and the and binding also to wild Cys at αTyr93 and of with at positions also resulted in in ACh S.M. P. F. P. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google M.L. FEBS Lett. 1994; PubMed Scopus Google Scholar). For mutant binding was of wild but the only of wild nAChRs and receptor of wild as for the in the mouse muscle nAChR G. Biochem. J. 1995; PubMed Scopus Google Scholar). nAChRs also although their receptor to of and αY198C The and receptor that Cys at and resulted in functional expressed at for wild type nAChR or mutant nAChRs containing agonist site reacted with or in the of agonist and for to a sulfhydryl-reactive is known to act as an irreversible antagonist of wild type nAChR after of the (18.Kao P. Dwork A.J. Kaldany R.J. Silver M.L. Wideman J. Stein S. Karlin A. J. Biol. Chem. 1984; 259: 11662-11665Abstract Full Text PDF PubMed Google Scholar), and we that reacted with at other positions in the site also act as an irreversible with a charged primary within the binding site. The was to the to ACh at a to the to or for and to the ACh after a to bound are for wild type nAChRs and for and αY198C mutant nAChRs with and B), as well as for as well as the and For wild type nAChR, after with and was a in the to ACh, substantially less than the and for and αY198C For wild type nAChR, exposure to resulted in irreversible inhibition, while was of ACh for and αY198C mutant For Cys within loop D of of nAChRs with either or resulted in irreversible inhibition, while of nAChRs resulted in than that for wild type after of nAChRs with for after washing the ACh was by not that γE57C, αY93C, and αY198C for modification and that reaction at positions with either or resulted in the of covalent antagonists. The of at that Cys at that is not for The ACh of γE57C, αY93C, and αY198C nAChRs by To characterize this inhibition, we the ACh and after a of with irreversible of a in the for of the mutant nAChRs with little any in the The effects of the of thiocholine to agonist site by mutant nAChRs with MTSET MTSET at acted as an agonist for wild type nAChR irreversible of ACh The MTSET resulted in a irreversible of nAChRs and a of nAChR The resulted in for ACh for and for exposure to MTSET for as as to of the ACh an of to that for wild type nAChR not MTSET of αY198C nAChRs a very of αY198C nAChRs with MTSET resulted in the of a which was upon MTSET In with the to ACh with the after with MTSET was as MTSET of with or nAChRs not any not While thiocholine tethered at positions or acted as a covalent antagonist, to act as a covalent agonist when tethered at To MTSET was the agonist binding site, we binding of the competitive antagonist, reaction of MTSET with the αY198C nAChR when at was with ACh, was a reversible of ACh with 1 the reaction of MTSET with αY198C, as by the of was but subsequent to 1 MTSET an that of ACh was after of the at a of 1 ACh acts as an channel D.C. D. Proc. Biol. Sci. PubMed Scopus Google Scholar). The after with MTSET by in 1 and of the with 1 a to after which ACh not studies to the of the of the mutant nAChR channel gating at resulted in a of within the The after reaction of αY198C nAChRs with MTSET was inhibitable by nAChR antagonists The nAChR channel P. C. R.J. A. N. Neuron. 1990; Full Text PDF Scopus Google a reversible of the A), as B), which is known to to a affinity site within the of the nAChR ion channel Cohen J.B. Pharmacol. 1999; PubMed Scopus Google Scholar). which to the of the nAChR Cohen J.B. Biochemistry. 1984; PubMed Scopus Google Scholar), was a potent of the but in to was for and by was not reversible with quaternary ammonium reagents used to characterize the structural requirements for nAChR activation which is than a group to which is than The of modification was to that of and to for resulted in irreversible of αY93C, αY198C, and mutant and was that reacted with The of modification was to that for MTSET. with resulted in irreversible of and mutant while for αY198C, modification resulted in a in than the by 1 To the from modification of αY198C nAChRs by MTSET with mutant nAChR to of either For MTSET and exposure to and for after exposure to of For αY198C the by MTSET was the of and the by 1 either MTSET reacted with nAChRs or after covalent modification the αY198C mutant nAChR in a than ACh or C that after an been by with MTSET in that the two reagents with the of For the Cys by or the of reaction with mutant nAChRs by the to ACh after reaction and for For the αY198C mutant nAChR, the from exposure to MTSET as a of reaction The reaction from the of which with amino acid the three reagents reacted at However, upon the amino the reaction as as with the αY198C γE57C. αY198C the reaction the for which acted as an irreversible antagonist, as for MTSET and which acted as irreversible For the three αY198C was and γE57C. For was the as for the but reacted and than the reagents at and αY198C, of nAChR not for the to in for modification of αY198C, αY93C, and mutant nAChRs by and 1 1 as and in The in are the of in a as and in The in are the of also of and αY198C mutant nAChRs with a agonist that acts as an irreversible agonist of wild type nAChR after of the αCys192/193 (19.Damle V.N. McLaughlin M. Karlin A. Biochem. Biophys. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). acted as a reversible agonist of wild type nAChR and about that of a reversible agonist, prolonged exposure to resulted in nAChR exposure to 1 for after a of subsequent to ACh by with not a with was a in to that upon that to but after a the to an ACh was by with with little after For mutant nAChR and acted as a reversible agonist a about that of exposure to 1 for subsequent to ACh within of the not of αY198C nAChRs to 1 for resulted in a In to was for wild type nAChR, this after and was by a 1 ACh of MTSET resulted in only a of the For the αY198C mutant nAChR with to acted as a reversible agonist in to ACh, with the for that for ACh on the not However, upon washing after a exposure to 1 the not to was a of the of for resulted in a of the with an of the to a to that for the a exposure to 1 for subsequent reaction with MTSET resulted in a of the but only to a about of the ACh The of this to the of αY198C nAChRs reacted with MTSET exposure to in which the than the ACh that with αY198C, and that when was a covalent However, the reaction for was lower than that for and the tethered acetylcholine was less in gating the channel than tethered thiocholine at this The amino acids from and mutagenesis to contribute to the agonist site. mutant subunits containing Cys at positions αTyr93 or γTrp55 to other which irreversible or activation after reaction with or the The of Cys in loop are in Cys in this in In to the that was than that of wild type Cys and and in the ACh Cys at the other positions resulted in less than of Cys at not but the less than of wild type and in to for However, the for the αN94C mutant with lower of nAChR binding of wild not The irreversible effects of and MTSET on Cys as in and than αY93C, the only residue in this to of modification was reaction with for ACh of the αN94C mutant nAChR by and reaction with for resulted in by However, reaction with 1 resulted in than that for wild type Cys also at positions γTrp55 in loop D For of the for ACh was within a of of wild type not and the also to that for wild type reaction of or MTSET with resulted in irreversible of ACh However, for of the other ACh either the other positions to modification or the modification on ACh of mutant nAChRs containing Cys in loop of ACh binding ACh and as in with and the of from at three oocytes. of by 1 and MTSET as in in a ACh and as in with and the of from at three oocytes. of by 1 and MTSET as in The of Cys mutagenesis in conjunction with agonists and antagonists a for the agonist binding site that has structural information the orientation of bound within the site. Cys in and C in the α-subunit and loop D of the resulted in functional nAChRs and amino acid at positions within the agonist binding site. ACh for two of the and For the binding on the was to that for the and αY198C the of ACh not from a of For was binding of on the as was also for mutant mouse nAChR G. Biochem. J. 1995; PubMed Scopus Google Scholar), although the mouse mutant is expressed on the of S.M. P. F. P. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). Using and the we modification that resulted in a functional at four and the of the of and the agonist binding site and for the that also to the agonist binding site. The of of on nAChR after reaction was and was in distinct to the of γTrp55 is the primary site of in of [3H]dTC (9.Chiara D.C. Cohen J.B. J. Biol. Chem. 1997; 272: 32940-32950Abstract Full Text Full Text PDF PubMed Scopus (86) Google and D.C. Middleton R.E. Cohen J.B. FEBS Lett. 1998; 423: 223-226Crossref PubMed Scopus (46) Google Scholar), and at that in Torpedo J. 1994; 266: PubMed Google or J.L. J.L. S. Changeux J.P. D. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google nAChR agonist and antagonist the of functional of and the reagents on nAChR a of and within the binding site in a than γE57C. In Cys at which to nAChR as well as at and to modification by and the modification resulted in of A.J. C. J. 1999; PubMed Google Scholar). studies of the effects of on as well as on the other Cys in ACh binding site and D this modification ACh in the of functional effects of MTSET or a as as tetramethylammonium act as an agonist, that are very and structural requirements for a tethered to act as an In and nAChR Cys we only αY198C, at which MTSET modification resulted in irreversible nAChR acts as a tethered agonist when at αY198C, while acts as an antagonist when at three other and αY198C, the structural requirements of the group are also the of of the compounds used in this which thiocholine and the quaternary ammonium group at a of Å from the site, the mutant nAChR in substantially than for the length of the tethering arm by less than 1 Å in either direction markedly effects the ability to activate the which positions the quaternary ammonium group at a of not activate the αY198C which the quaternary ammonium group at a of a the nAChR, but not as well as tethered modification at αY198C in for that is for tethered While MTSET the of tethered reaction with to the tethering of acetylcholine with the group Å the acetyl and at a of Å from the also with αY198C mutant nAChR, with a lower than MTSET. Acetylcholine tethered at αY198C only that for MTSET. of the of acetylcholine and thiocholine tethered at αCys192/193 in reduced, wild type nAChR as well as at other Cys in loop C of the requirements for tethered trimethylammonium to act as However, studies with are by reaction which the of and reaction that nAChR by this reversible αY198C nAChRs by tethered thiocholine that for tethered a that the two tethered agonists the channel to not to this an in opening or an in channel is that thiocholine modification of αY198C in a channel with in that after with αCys192/193in reduced, wild type is a of the channel and not an or of channel opening J. PubMed Scopus Google Scholar). that αY198C nAChRs have the as wild type nAChRs not The activation of the αY198C nAChR by tethered thiocholine for a nAChR other and with an structure of the ion channel. In the receptor which from of αTrp149 by an amino only when the mutant α-subunit is with a mutant containing a in the ion channel domain that the and channel opening in the of agonist J.P. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The by MTSET of αY198C nAChRs was at in than from the oocytes. is that a Cys at residue the structure of the agonist binding site in a as to activation by agonists in structure to thiocholine than to To this we the ability of agonists to activate the nAChR not For wild type nAChR, not for choline of ACh while tetramethylammonium was a agonist about that for For the αY198C for and only for of tetramethylammonium that the of ACh for the αY198C the for was that for that the αY198C mutant is by than by The reaction for the reagents by than upon the of the Cys For the three reagents as well as the reaction was for αY198C, with the for the reagents αY198C than for The for reaction of reagents at this than at and than at γE57C. For the by only used to the reaction with for and while for reaction with αCys192/193 in reduced, wild nAChR, for as well as and MTSET Karlin A. Biochemistry. 1994; PubMed Scopus Google Scholar). studies are to the of in the of the in an of the reaction of the reagents at However, not their lower for and γE57C. While αY198C was identified as the of the positions within the ACh binding site, and also for is that the reaction for the ligand to with a orientation within the ACh site, the of the at the three positions that this The the selective activation of the nAChR after modification of αY198C, in conjunction with activation of nAChRs after modification of αCys192/193 with that activation depends upon of the acetyl of ACh with amino acids in loop C. studies of the regions of the agonist binding site with the charged group channel have identified in and C S.M. P. F. P. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, M.L. FEBS Lett. 1994; PubMed Scopus Google as well as in and F N. P. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google as G. M. Auerbach A. Biophys. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). of an amino acid at αTrp149 that of the group with the chain of αTrp149 for gating J.P. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). a charged quaternary group and that at in the of the nAChR this to the domain of the site J.B. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). the ion with of the of ACh with mutant nAChRs containing ACh site Cys an to binding site with the of