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• W2155193072 abstract "Waglerin-1 (Wtx-1) is a 22-amino acid peptide that is a competitive antagonist of the muscle nicotinic receptor (nAChR). We find that Wtx-1 binds 2100-fold more tightly to the α-ε than to the α-δ binding site interface of the mouse nAChR. Moreover, Wtx-1 binds 100-fold more tightly to the α-ε interface from mouse nAChR than that from rat or human sources. Site-directed mutagenesis of residues differing in the extracellular domains of rat and mouse ε subunits indicates that residues 59 and 115 mediate the species difference in Wtx-1 affinity. Mutation of residues 59 (Asp in mouse, Glu in rat ε) and 115 (Tyr in mouse, Ser in rat ε) converts Wtx-1 affinity for the α-ε interface of one species to that of the other species. Studies of different mutations at position 59 indicate both steric and electrostatic contributions to Wtx-1 affinity, whereas at position 115, both aromatic and polar groups contribute to affinity. The human nAChR also has lower affinity for Wtx-1 than mouse nAChR, but unlike rat nAChR, residues in both α and ε subunits mediate the affinity difference. In human nAChR, polar residues (Ser-187 and Thr-189) confer low affinity, whereas in mouse nAChR aromatic residues (Trp-187 and Phe-189) confer high affinity. The overall results show that non-conserved residues at the nAChR binding site, although not crucial for activation by ACh, govern the potency of neuromuscular toxins. Waglerin-1 (Wtx-1) is a 22-amino acid peptide that is a competitive antagonist of the muscle nicotinic receptor (nAChR). We find that Wtx-1 binds 2100-fold more tightly to the α-ε than to the α-δ binding site interface of the mouse nAChR. Moreover, Wtx-1 binds 100-fold more tightly to the α-ε interface from mouse nAChR than that from rat or human sources. Site-directed mutagenesis of residues differing in the extracellular domains of rat and mouse ε subunits indicates that residues 59 and 115 mediate the species difference in Wtx-1 affinity. Mutation of residues 59 (Asp in mouse, Glu in rat ε) and 115 (Tyr in mouse, Ser in rat ε) converts Wtx-1 affinity for the α-ε interface of one species to that of the other species. Studies of different mutations at position 59 indicate both steric and electrostatic contributions to Wtx-1 affinity, whereas at position 115, both aromatic and polar groups contribute to affinity. The human nAChR also has lower affinity for Wtx-1 than mouse nAChR, but unlike rat nAChR, residues in both α and ε subunits mediate the affinity difference. In human nAChR, polar residues (Ser-187 and Thr-189) confer low affinity, whereas in mouse nAChR aromatic residues (Trp-187 and Phe-189) confer high affinity. The overall results show that non-conserved residues at the nAChR binding site, although not crucial for activation by ACh, govern the potency of neuromuscular toxins. The muscle nicotinic receptor (1Corringer P.J., Le Novère N. Changeux J.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (706) Google Scholar, 2Arias H.R. Neurochem. Int. 2000; 36: 595-645Crossref PubMed Scopus (186) Google Scholar, 3Karlin A. Akabas M.H. Neuron. 1995; 15: 1231-1244Abstract Full Text PDF PubMed Scopus (563) Google Scholar) contains five polypeptide subunits arranged with radial symmetry around a central pore. Two copies of α and one each of β, δ, and γ (in the embryonic receptor form) or ε (in the adult form) (4Reynolds J.A. Karlin A. Biochemistry. 1978; 17: 2035-2038Crossref PubMed Scopus (242) Google Scholar) are arranged around the central pore in the counterclockwise order: α-γ/ε-α-δ-β as established from the crystal structure determination of the acetylcholine binding protein from the freshwater snail Lymnaea stagnalis (5Brejc 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 (1574) Google Scholar, 6Smit 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 (463) Google Scholar). Each of the five subunits contains between 445 and 497 amino acids with residues 1 through ∼210 forming the extracellular ligand binding domain. Each receptor subunit has up to three N-linked glycosylation sites and four transmembrane spans, giving the pentamer a molecular mass of nearly 300 kDa with 20 membrane spans. The binding sites for agonists and competitive antagonists are found at interfaces of the α-δ and α-ε (or α-γ) subunits of the receptor. Full activation of the nAChR 1The abbreviations used are:nAChRnicotinic acetylcholine receptor125I-α-BgTx125I-α-bungarotoxinWtx-1Waglerin-1HEKhuman embryonic kidneyAChBPacetylcholine binding protein 1The abbreviations used are:nAChRnicotinic acetylcholine receptor125I-α-BgTx125I-α-bungarotoxinWtx-1Waglerin-1HEKhuman embryonic kidneyAChBPacetylcholine binding protein requires simultaneous binding of two agonist molecules, but antagonists block activation by occupying only one of the two sites. nicotinic acetylcholine receptor 125I-α-bungarotoxin Waglerin-1 human embryonic kidney acetylcholine binding protein nicotinic acetylcholine receptor 125I-α-bungarotoxin Waglerin-1 human embryonic kidney acetylcholine binding protein Snakes of the Elapidae and Hydrophidae families are notorious for producing toxins that target nicotinic receptors (7Endo T. Tamiya N. Harvey A.L. Snake Toxins. Pergamon Press, New York1991: 165-222Google Scholar). These small proteins of 57–80 amino acids are commonly called “3-fingered” snake toxins for their characteristic three loop topology, with each of the three fingers extending from a core “knuckle” region consisting of four conserved disulfide bonds. Three-fingered toxins such as α-bungarotoxin have been used to probe the nAChR for over 30 years (8Changeux J.P. Kasai M. Lee C.Y. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1241-1247Crossref PubMed Scopus (461) Google Scholar). On the other hand, the Viperidae family of snakes does not make 3-fingered toxins and were generally believed not to confer toxicity by targeting nicotinic receptors. Wagler's pit viper, Tropidolaemus wagleri, is unique among Viperids for morphological reasons as well as for the unique components of its venom. Four related peptides, whose sequence differences are boldfaced, have been isolated from the venom of this species, all of which cause paralysis by neuromuscular blockade (9Aiken S.P. Sellin L.C. Schmidt J.J. Weinstein S.A. McArdle J.J. Pharmacol. Toxicol. 1992; 70: 459-462Crossref PubMed Scopus (16) Google Scholar, 10Weinstein S.A. Schmidt J.J. Bernheimer A.W. Smith L.A. Toxicon. 1991; 29: 227-236Crossref PubMed Scopus (43) Google Scholar, 11Schmidt J.J. Weinstein S.A. Smith L.A. Toxicon. 1992; 30: 1027-1036Crossref PubMed Scopus (31) Google Scholar, 12Schmidt J.J. Weinstein S.A. Toxicon. 1995; 33: 1043-1049Crossref PubMed Scopus (14) Google Scholar) and are selective for the adult form of the receptor (13McArdle J.J. Lentz T.L. Witzemann V. Schwarz H. Weinstein S.A. Schmidt J.J. J. Pharmacol. Exp. Ther. 1999; 289: 543-550PubMed Google Scholar). The solved NMR structures for Waglerin-1 (14Sellin L.C. Mattila K. Annila A. Schmidt J.J. McArdle J.J. Hyvönen M. Rantala T.T. Kivistö T. Biophys. J. 1996; 70: 3-13Abstract Full Text PDF PubMed Scopus (14) Google Scholar, 15Chuang L.C., Yu, H.M. Chen C. Huang T.H., Wu, S.H. Wang K.T. Biochim. Biophys. Acta. 1996; 1292: 145-155Crossref PubMed Scopus (12) Google Scholar) show there is a single intramolecular disulfide between the two cysteines. The present work was inspired by observations of C. Y. Lee and colleagues who nearly forty years ago showed that α-bungarotoxin irreversibly blocks neuromuscular transmission (16Chang C.C. Lee C.Y. Arch. Int. Pharmacodyn. 1963; 144: 241-257PubMed Google Scholar). Their more recent studies indicate a remarkable difference in waglerin toxicity between mice and rats (17Lin W.W. Smith L.A. Lee C.Y. Toxicon. 1995; 33: 111-114Crossref PubMed Scopus (18) Google Scholar). Mice were paralyzed by a 0.5 μg/g intravenous injection in as little as 5 min; rats were completely resistant to 20 times the mouse lethal dose. A similar species selectivity was noted in isolated phrenic nerve-hemidiaphragm preparations, with over a 40-fold difference in sensitivity (17Lin W.W. Smith L.A. Lee C.Y. Toxicon. 1995; 33: 111-114Crossref PubMed Scopus (18) Google Scholar). Waglerin-1 also has no effect when applied to a chicken biventer cervicis muscle-nerve preparation (18Tan N.H. Tan C.S. Toxicon. 1989; 27: 349-357Crossref PubMed Scopus (21) Google Scholar). For obvious reasons, similar toxicity data are not available for human. Here we show that sequence differences between mouse, rat, and human nAChRs account for the specificity of Wtx-1. The identified residues are located in regions of the three-dimensional structure known to contribute to the nAChR binding site. Because the Wtx-1 binding profile for each species can be reconstructed by mutating only these key residues, the species selectivity is not likely due to global structural changes. Rather, the key residues likely interact directly with Wtx-1 or affect its bound orientation. The overall results show how non-conserved residues at the nAChR binding site interface govern species specificity of competitive antagonists. The crude peptides, synthesized by the American Peptide Company (San Jose, CA) or Synpep (Dublin, CA) were dissolved to 0.8 mg/ml in 30 mmTris-HCl, pH 8.2–8.5, sterile-filtered and left overnight at room temperature to form the single intramolecular disulfide. After disulfide bond cyclization, 0.1% trifluoroacetic acid was added to the solution to stop cyclization and prevent the formation of intermolecular disulfides. A 2-ml injection of the peptide solution was loaded onto a 5-ml high performance liquid chromatography sample loop and purified on a 10- × 250-mm semi-preparative C18 column (Vydac) and eluted using a 0.1% trifluoroacetic acid/70% acetonitrile solvent system changing at 1% for Solvent B every 3 min. Cyclized peptide elutes from the column 1–2 min earlier in the gradient than uncyclized peptides or dimerized peptides formed by intermolecular disulfide formation. Fractions containing the purified peptide were pooled, frozen, and lyophilized. Representative samples from different lots were checked for purity and correct mass by matrix-assisted laser desorption or ion-spray mass spectrometry. Cloned cDNAs for mouse α (19Isenberg K.E. Mudd J. Shah V. Merlie J.P. Nucleic Acids Res. 1986; 14: 5111Crossref PubMed Scopus (57) Google Scholar), β (20Buonanno A. Mudd J. Merlie J.P. J. Biol. Chem. 1989; 264: 7611-7616Abstract Full Text PDF PubMed Google Scholar), γ (21Yu L. LaPolla R.J. Davidson N. Nucleic Acids Res. 1986; 14: 3539-3555Crossref PubMed Scopus (47) Google Scholar), δ (22LaPolla R.J. Mayne K.M. Davidson N. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7970-7974Crossref PubMed Scopus (98) Google Scholar), and ε (23Gardner P.D. Nucleic Acids Res. 1990; 18: 6714Crossref PubMed Scopus (28) Google Scholar); rat α, β, δ, γ, and ε (24Witzemann V. Stein E. Barg B. Konno T. Koenen M. Kues W. Criado M. Hofmann M. Sakmann B. Eur. J. Biochem. 1990; 194: 437-448Crossref PubMed Scopus (95) Google Scholar); and human α (25Schoepfer R. Luther M. Lindstrom J. FEBS Lett. 1988; 226: 235-240Crossref PubMed Scopus (107) Google Scholar), β, δ, ε, and γ (gifts from A. Engel) were ligated into the mammalian expression vector pRBG4 at EcoRI sites for transient expression in HEK293 cells (26Sine S.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9436-9440Crossref PubMed Scopus (167) Google Scholar). Site-specific mutants of the wild-type mouse ε, rat ε, mouse α, and human α subunits were made by one of two methods. Complementary synthetic oligonucleotides (Sigma/Genosys, The Woodlands, TX) containing the desired mutation were ligated into the cDNA at unique restriction sites flanking the mutated region. If convenient restriction sites were not available, the Stratagene double primer method was used. In this procedure, complementary oligonucleotides containing the desired mutation served as primers in a PCR amplification reaction containing the wild-type plasmid andPfu polymerase. Following 16 to 18 amplification cycles in which the entire plasmid was made synthetically, the reaction was digested with DpnI restriction enzyme, which only digests the methylated, wild-type plasmid DNA, leaving behind the newly synthesized DNA. Mutations were subcloned into vectors not subjected to mutagenesis and verified initially by restriction digests and then by DNA sequencing. Large-scale plasmid preparations were made using DEAE columns (Invitrogen or Qiagen) or by cesium chloride ultracentrifugation. All individual subunit cDNAs for the nAChR are contained on unique plasmids using the cytomegalovirus promoter-based pRBG4 vector (27Lee B.S. Gunn R.B. Kopito R.R. J. Biol. Chem. 1991; 266: 11448-11454Abstract Full Text PDF PubMed Google Scholar). HEK293 cells were transfected by calcium phosphate, the media was changed 12–16 h later, and the experiments were performed 1–2 days after the media change. Transfected cells were removed from the culture plates by gentle agitation with 5 ml of phosphate-buffered saline with 5 mm EDTA. After incubating the dissociated cells with Wtx-1 for 45 min to 1 h, 5 nm125I-α-bungarotoxin (125I-α-BgTx) (PerkinElmer Life Sciences) was added and allowed to incubate for 20–40 min such that at 30–50% of the available binding sites become occupied (28Sine S. Taylor P. J. Biol. Chem. 1979; 254: 3315-3325Abstract Full Text PDF PubMed Google Scholar). Waglerin dissociation constants were determined from the fractional reduction of the initial rate of 125I-α-BgTx binding. The total number of sites was determined by incubating with 20 nm125I-α-BgTx for 1 h. Nonspecific binding was determined by incubating the cells with 10 mm carbachol before reacting with 125I-α-BgTx. We measured Wtx-1 binding by competition against the initial rate of 125I-α-BgTx binding to intact HEK cells transfected with cDNAs encoding adult nAChR from mouse or rat. Wtx-1 binds to mouse nAChR with two distinct dissociation constants differing by 2100-fold, whereas it binds to rat nAChR with dissociation constants differing by only 80-fold (Fig.1). Because full activation of the nAChR requires simultaneous binding of two agonist molecules, and antagonists block activation by occupying only one of the two sites, the very different dissociation constants at one site underlie the species specificity of Wtx-1 in vivo. To determine which nAChR subunits mediate Wtx-1 selectivity between mouse and rat nAChRs, we substituted the mouse ε subunit into the rat nAChR and the rat ε subunit into the mouse nAChR. Combining the mouse ε subunit with rat α, β, and δ subunits yields a binding profile approaching that of the wild-type mouse nAChR, with the dissociation constant for the high affinity site only 2-fold greater than the all mouse subunit reference. Conversely, combining the rat ε subunit with mouse α, β, and δ subunits yields a binding profile approaching that of the wild-type rat nAChR; the dissociation constant of the high affinity site in the hybrid nAChR is identical to that in the all rat reference, whereas the dissociation constant of the low affinity site coincides with that in the all mouse reference. These results show that the ε subunit mediates the mouse/rat species selectivity by affecting Wtx-1 affinity for the α-ε binding site interface. Experiments replacing the ε subunit with the fetal γ subunit confirm that the ε subunit confers high affinity binding of Wtx-1 (29Taylor P. Osaka H. Molles B.E. Sugiyama N. Marchot P. Ackermann E.J. Malany S. McArdle J.J. Sine S.M. Tsigelny I. J. Physiol. (Paris). 1998; 92: 79-83Crossref PubMed Scopus (17) Google Scholar). 2B. E. Molles, unpublished observations. Of the 218 amino acids forming the extracellular domain responsible for ligand recognition, only 10 differ between mouse and rat ε subunits. To determine which of the 10 residues mediates species specificity, we constructed chimeric ε subunits from rat and mouse using a common AflIII restriction site near the codon for amino acid 83. The isolated restriction fragments were ligated to make chimeric subunits, designated εrat83mouse and εmouse83rat, roughly bisecting the extracellular domain. When combined with mouse α, β, and δ subunits, each chimera produced Wtx-1 affinities intermediate between those for wild-type mouse and rat (Fig.2). The intermediate affinities indicate at least two determinants of Wtx-1 selectivity, one N-terminal and one C-terminal to the junction at position 83. Of the four N-terminal residue differences, only positions 59 and 61 are near the ligand recognition site (1Corringer P.J., Le Novère N. Changeux J.P. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 431-458Crossref PubMed Scopus (706) Google Scholar), whereas positions 67 and 76 are in or near the main immunogenic region removed from the binding site (30Tzartos S.J. Cung M.T. Demange P. Loutrari H. Mamalaki A. Marraud M. Papadouli I. Sakarellos C. Tsikaris V. Mol. Neurobiol. 1991; 5: 1-29Crossref PubMed Scopus (48) Google Scholar). Of the six C-terminal residues, positions 168 and 115 are near the ligand recognition site. Candidate residues C-terminal to 115 at the interface can be ruled out from a comparison of affinities of two mouse γ-ε chimeras that we generated. γ165ε and γ171ε yielded near identical K D values, 11.4 and 9.3 μm. Hence, residue 168, which differs between mouse and rat ε as well as between mouse ε and mouse γ by having a T to A substitution, is an unlikely determinant of Waglerin affinity. To determine which residues mediate species selectivity, we worked with the mouse ε subunit and mutated all four N-terminal residue differences and one C-terminal difference. Of the four N-terminal mutations, only εD59E reduced affinity to that observed for the εrat83mouse chimera (Fig. 3 and TableI). In the C-terminal region, εY115S reduced affinity to that observed for the εmouse83rat chimera (Fig.3). These intermediate Wtx-1 affinities suggest that residues at positions 59 and 115 account for selectivity between mouse and rat nAChRs.Table IMouse and rat ɛ subunit species mutations and dissociation constantsMutant subunitK D, α-ɛK D, α-δK D, α-δ/K D, α-ɛΔK D, α-ɛΔK D, α-δnnmμmMouse αβδɛ9.820.42100(1.0)(1.0)26Rat αβδɛ112081681224.03αβδ rat + ɛ mouse16.790.454001.74.42Rat ɛ124021.9181271.15ɛmouse83rat13424.0180141.22ɛrat83mouse79.714.518080.72Mouse ɛD59E28027.998291.44Mouse ɛD59N29.017.159030.83Mouse ɛD59Q47.033.972051.73Mouse ɛD59A10.818.117001.10.92Mouse ɛH61Q5.116.323000.50.81Mouse ɛY67F4.713.729000.50.71Mouse ɛG76E4.610.222000.50.51Mouse ɛY115S89.221.224091.03Mouse ɛY115F17.418.511001.80.93Mouse ɛD59E+Y115S47526.456481.33Rat ɛE59D8417.621090.93Rat ɛE59N18934.4180191.74Rat ɛE59Q43035.482441.73Rat ɛS115Y21228.5130221.43Rat ɛS115F40825.462421.22Rat ɛE59D+S115Y5.222.346000.51.32The ɛ subunit of the given species was cotransfected with mouse α, β, and δ subunits unless indicated otherwise.K D, α-ɛ and K D, α-δwere determined from full concentration curves as shown in Figs. 1 and2. ΔK D, α-ɛ and ΔK D, α-δ is the ratio of the dissociation constant to the corresponding subunit interface in the all mouse wild-type receptor. Open table in a new tab The ɛ subunit of the given species was cotransfected with mouse α, β, and δ subunits unless indicated otherwise.K D, α-ɛ and K D, α-δwere determined from full concentration curves as shown in Figs. 1 and2. ΔK D, α-ɛ and ΔK D, α-δ is the ratio of the dissociation constant to the corresponding subunit interface in the all mouse wild-type receptor. To determine whether residues at positions 59 and 115 together confer Wtx-1 selectivity, we constructed the corresponding double mutations in mouse and rat ε subunits. The mouse double mutant ε(D59E/Y115S) reduces Wtx-1 affinity to within 2-fold of the wild-type rat ε subunit. Conversely, the rat double mutant ε(E59D/S115Y) increases Wtx-1 affinity to within 2-fold of the wild-type mouse ε subunit (Fig. 3 and Table I). Thus species selectivity of Wtx-1 can be closely mimicked by substituting residues at positions 59 and 115 of the ε subunit. Once the primary determinants of Waglerin affinity were identified, we characterized how each receptor determinant interacts with Wtx-1. Working with the mouse ε subunit, we mutated Asp-59 to Ala (the corresponding residue in mouse δ), Asn (to remove the negative charge), and Gln (the corresponding residue of mouse γ). The resulting rank order of affinity for mouse ε 59 residues is Asp = Ala > Asn ≥ Gln > Glu (Table I). Removing the charge does not in itself significantly decrease affinity (no change, 3-fold, or 5-fold for the Ala, Asn, and Gln substitutions, respectively). Changing the Asp to a Glu, which increases the length of the side chain by one methylene group, causes the largest decrease. These observations indicate that the interaction at residue 59 is not purely electrostatic, but instead is mediated by a combination of electrostatic and other stabilizing forces within a confined space. Steric occlusion incurred by addition of a single methylene in the side chain may alter the position of the charge or the orientation of bound Waglerin. In the rat ε mutants tested, the affinities followed a similar rank order, Asp > Asn > Gln > Glu (Table I), further showing that both electrostatic forces and steric constraints mediate the interaction at residue 59. Position 115 of the ε subunit of both rat and mouse nAChR was mutated to Phe and Ser to determine the role of the aromatic ring in governing affinity for Wtx-1. For both rat and mouse ε subunits, the rank order of Wtx-1 affinity was Tyr > Phe > Ser at position 115, indicating that both the aromatic ring and the hydroxyl group contribute to high affinity binding of Wtx-1 at the α-ε interface. We next compared Wtx-1 binding to human nAChR with that for mouse nAChR. Wtx-1 binds to human nAChR with two distinct affinities, but compared with mouse nAChR, it shows 70-fold lower affinity at the α-ε site and 10-fold lower affinity at the α-δ site (Fig. 4). To determine whether the ε subunit contributes to the low affinity of human nAChR, we coexpressed mouse α, β, and δ subunits with human ε. The resulting hybrid nAChRs bind Wtx-1 with 14-fold lower affinity at the α-ε site compared with the all mouse reference, indicating that some of the lower affinity for human nAChR originates in the ε subunit. Although no human ε mutants were tested, the human ε, like rat, has a Ser at residue 115, a residue shown to mediate reduced affinity of rat ε. On the other hand, human ε contains Asp at position 59, like mouse, which promotes high affinity for the nAChR. Thus, Ser-115 in human ε should be a primary determinant for the human-mouse difference in affinity. To determine whether the α subunit contributes to low affinity of human nAChR, we cotransfected human α with mouse β, ε, and δ subunits. The resulting hybrid nAChRs bind Wtx-1 with 10-fold lower affinity at both binding sites compared with the all mouse reference, indicating that the α subunit is also responsible for the low affinity of human nAChR. We therefore constructed chimeric α subunits composed of mouse and human sequences to identify residues responsible for the mouse/human affinity difference. Residues 1–124 of the mouse α subunit were joined with residues 125–496 of human α, and residues 1–172 of human were joined to 173–496 of the mouse α subunit. When mouse sequence occupies positions 1–124 of the α subunit, Wtx-1 binding coincides with that of the wild-type human α subunit (Fig. 4 B), indicating that residues C-terminal to residue 124 affect affinity. On the other hand, when human sequence occupies positions 1–172, Wtx-1 binding coincides with that of the all mouse reference, indicating that residues C-terminal to position 172 affect affinity. The mouse-human α subunit chimeras, therefore, indicate that the determinants of the affinity difference are C-terminal to position 172 in the α subunit. Between position 172 and the first transmembrane span at position 210, four residues differ between the mouse and human wild-type α subunits: positions 181, 187, 189, and 195. We therefore generated point mutations at each of these positions in both mouse and human subunits, mutating each residue to that in the other species. Each mutant α subunit was cotransfected with mouse wild-type β, δ, and ε subunits, and Wtx-1 binding was measured. For mutations at positions 181 and 195, virtually no change in affinity is observed in either mouse or human α subunits (TableII). For positions 187 and 189, a more complex interplay of the two residues is evident. The mouse αW187S mutation decreases Wtx-1 affinity only 2-fold at both sites, and the corresponding human mutant (human αS187W) increases affinity 2- and 4-fold (Fig. 5, A andB). At position 189, the mouse mutation (αF189T) reduces affinity to that for human α, but the human mutant (αT189F) increases affinity only 2-fold at each site (Fig. 5, A andB). Changing both residues simultaneously in the human αS187W/T189F double mutant gives nearly identical affinities to the wild-type mouse α at both sites. However, the mouse αW187S/F189T double mutant decreases affinity 3-fold at each site, less than the αF189T single mutant alone (Table II).Table IIMouse and human α subunit mutations to mimic species differences between nAchRSubunit transfectedK D, α-ɛK D, α-δK D, α-δ/K D, α-ɛΔK D, α-ɛΔK D, α-δnnmμmMouse α9.820.42100(1.0)(1.0)26Human αβδɛ69220029071106Human α8420024009107Human ɛ (+mouse αβδ)14219.3140140.92α Mouse 124human78.11612100882α Human 172mouse8.616.920000.90.82Human αS181A11214913001176Human αS187W27.21294800363Human αT189F48.893.71900555Human αD195T11515714001283Human αS187W + T189F10.137.437001.01.85Mouse αA181S17.420.312001.81.04Mouse α W187S23.132.9140021.64Mouse αF189T135101750145.05Mouse αT195D16.613.07801.70.63Mouse αW187S + F189T26.466.52500334All experiments performed on HEK cells transfected with mouse wild-type β, δ, and ɛ subunits and the given α subunit unless otherwise specified. Column headings are identical to those in Table I. Open table in a new tab All experiments performed on HEK cells transfected with mouse wild-type β, δ, and ɛ subunits and the given α subunit unless otherwise specified. Column headings are identical to those in Table I. Careful inspection of the data in Fig. 5 reveals that a Wtx-1 determinant in the α subunit also affects the rate of α-BgTx association. As is evident from the plateau in the curves in Fig. 5, nAChRs containing the human α subunit typically have a lower apparent proportion of α-ε binding sites. This most likely results from a disparity between 125I-α-BgTx association rates at the α-ε site compared with the α-δ site, where association is faster at the α-δ site. Our results clearly show that the 187 position is critical to determining this difference in association rates. The presence of Ser at position 187, whether in the wild-type human α, in the mouse αW187S mutant or the mouse αW187S/F189T double mutant, leads to a lower apparent ratio of α-ε to α-δ sites. Conversely, the presence of Trp in the wild-type mouse, the human αS187W mutant, or the human αS187W/T189F double mutant, leads to a 1:1 ratio of α-ε to α-δ sites (Fig. 5 B). Thus the residue at 187 position governs the relative rate of α-BgTx association at the α-ε and α-δ sites. A wide variety of toxins from both animal and plant sources are known to aid the predatory species or protect other species from predation by blocking motor activity. Because acetylcholine is the motor transmitter in fish and higher forms of terrestrial animals, the nicotinic receptor is a prevalent target for lophotoxins from coral, α-conotoxins from fish-hunting cone snails, alkaloids from certain plants and amphibians, and α-neurotoxins from Elapid snakes (29Taylor P. Osaka H. Molles B.E. Sugiyama N. Marchot P. Ackermann E.J. Malany S. McArdle J.J. Sine S.M. Tsigelny I. J. Physiol. (Paris). 1998; 92: 79-83Crossref PubMed Scopus (17) Google Scholar). The Waglerins add to the structural diversity of nicotinic receptor blocking agents in that they contain a novel structural motif and reveal a structural specificity distinct from other peptide toxins. For example, α-conotoxin MI shows the binding site interface selectivity α-δ > α-ε > α-γ in the mouse nAChR (31Sine S.M. Kreienkamp H.J. Bren N. Maeda R. Taylor P. Neuron. 1995; 15: 205-211Abstract Full Text PDF PubMed Scopus (175) Google Scholar), whereas the short three-fingered α-toxin from Naja mossambica mossambica has interface selectivity α-γ = α-δ > α-ε (32Osaka H. Malany S. Kanter J.R. Sine S.M. Taylor P. J. Biol. Chem. 1999; 274: 9581-9586Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The Waglerin preference is α-ε > α-δ = α-γ in mouse, the species in which the highest affinity has been detected (29Taylor P. Osaka H. Molles B.E. Sugiyama N. Marchot P. Ackermann E.J. Malany S. McArdle J.J. Sine S.M. Tsigelny I. J. Physiol. (Paris). 1998; 92: 79-83Crossref PubMed Scopus (17) Google Scholar). Hence, characterization of the specificity of this relatively simple peptide should yield valuable information on receptor structure. The present work illustrates an emerging principle relating nAChR binding site structure to agonist and competitive antagonist binding. The subunit interface is designed to bind ACh with low affinity in the resting state and to bind ACh much more tightly when in the open channel and desensitized states. This state dependence of the nAChR binding site implies a very delicate design, which is mediated by conserved residues that do not tol" @default.
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