Alpha-toxin binding to acetylcholine receptor alpha 179-191 peptides: intrinsic fluorescence studies

Interactions between two alpha-toxins and the synthetic peptides alpha 179-191 from both calf and human acetylcholine receptor alpha-subunit sequences have been studied by measurements of quenching of intrinsic fluorescence after toxin addition. Dissociation constants of approx. 5 x 10(-8) M for binding of calf peptide by both alpha-cobratoxin and erabutoxin a have been estimated. The binding of alpha-cobratoxin to calf peptide, which leads to marked quenching of fluorescence intensity, is inhibited by a 10(4) molar excess of acetylcholine. The human alpha 179-191 peptide binds to alpha-cobratoxin, but not, under comparable conditions, to erabutoxin a.     

NMR structural analysis of alpha-bungarotoxin and its complex with the principal alpha-neurotoxin-binding sequence on the alpha 7 subunit of a neuronal nicotinic acetylcholine receptor.

We report a new, higher resolution NMR structure of alpha-bungarotoxin that defines the structure-determining disulfide core and beta-sheet regions. We further report the NMR structure of the stoichiometric complex formed between alpha-bungarotoxin and a recombinantly expressed 19-mer peptide ((178)IPGKRTESFYECCKEPYPD(196)) derived from the alpha7 subunit of the chick neuronal nicotinic acetylcholine receptor. A comparison of these two structures reveals binding-induced stabilization of the flexible tip of finger II in alpha-bungarotoxin. The conformational rearrangements in the toxin create an extensive binding surface involving both sides of the alpha7 19-mer hairpin-like structure. At the contact zone, Ala(7), Ser(9), and Ile(11) in finger I and Arg(36), Lys(38), Val(39), and Val(40) in finger II of alpha-bungarotoxin interface with Phe(186), Tyr(187), Glu(188), and Tyr(194) in the alpha7 19-mer underscoring the importance of receptor aromatic residues as critical neurotoxin-binding determinants. Superimposing the structure of the complex onto that of the acetylcholine-binding protein (1I9B), a soluble homologue of the extracellular domain of the alpha7 receptor, places alpha-bungarotoxin at the peripheral surface of the inter-subunit interface occluding the agonist-binding site. The disulfide-rich core of alpha-bungarotoxin is suggested to be tilted in the direction of the membrane surface with finger II extending into the proposed ligand-binding cavity.     

Phosphorescence and ODMR study of the binding interactions of acetylcholine receptor alpha-subunit peptides with alpha-cobratoxin.

Optical detection of magnetic resonance (ODMR) and phosphorescence spectroscopy have been applied to synthetic peptides derived from the alpha-subunit of the nicotinic acetylcholine receptor of Torpedo californica and their complexes with alpha-cobratoxin (CBTX). The CBTX Trp phosphorescence is strongly quenched by the proximal disulfide linkage, while the emission wavelengths and ODMR frequencies of the 18-mer alpha 181-198 indicate a more hydrophobic Trp environment than in the 12-mer alpha 185-196. Binding to CBTX produces a subtle increase in the hydrophobicity of the Trp environment for the peptides, in qualitative agreement with a recently proposed binding model, in which a receptor Trp residue interacts strongly with a hydrophobic cleft of the toxin.     

Alpha-bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite

In the nicotinic acetylcholine receptors (AChRs), the sequence segment surrounding two invariant vicinal cysteinyl residues at positions 192 and 193 of the alpha subunit contains important structural component(s) of the binding site for acetylcholine and high molecular weight cholinergic antagonists, like snake alpha-neurotoxins. At least a second sequence region contributes to the formation of the cholinergic site. Studying the binding of alpha-bungarotoxin and three different monoclonal antibodies, able to compete with alpha-neurotoxins and cholinergic ligands, to a panel of synthetic peptides as representative structural elements of the AChR from Torpedo, we recently identified the sequence segments alpha 181-200 and alpha 55-74 as contributing to form the cholinergic site (Conti-Tronconi et al., 1990). As a first attempt to elucidate the structural requirements for ligand binding to the subsite formed by the sequence alpha 181-200, we have now studied the binding of alpha-bungarotoxin and of antibody WF6 to the synthetic peptide alpha 181-200, and to a panel of peptide analogues differing from the parental sequence alpha 181-200 by substitution of a single amino acid residue. CD spectral analysis of the synthetic peptide analogues indicated that they all have comparable structures in solution, and they can therefore be used to analyze the influence of single amino acid residues on ligand binding. Distinct clusters of amino acid residues, discontinuously positioned along the sequence 181-200, seem to serve as attachment points for the two ligands studied, and the residues necessary for binding of alpha-bungarotoxin are different from those crucial for binding of antibody WF6. In particular, residues at positions 188-190 (VYY) and 192-194 (CCP) were necessary for binding of alpha-bungarotoxin, while residues W187, T191, and Y198 and the three residues at positions 193-195 (CPD) were necessary for binding of WF6. Comparison of the CD spectra of the toxin/peptide complexes, and those obtained for the same peptides and alpha-bungarotoxin in solution, indicates that structural changes of the ligand(s) occur upon binding, with a net increase of the beta-structure component. The cholinergic binding site is therefore a complex surface area, formed by discontinuous clusters of amino acid residues from different sequence regions. Such complex structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody/antigen complexes [reviewed in Davies et al. (1988)]. Within this relatively large structure, cholinergic ligands bind with multiple points of attachment, and ligand-specific patterns of the attachment points exist.(ABSTRACT TRUNCATED AT 400 WORDS)     

The mechanism for acetylcholine receptor inhibition by alpha-neurotoxins and species-specific resistance to alpha-bungarotoxin revealed by NMR

The structure of a peptide corresponding to residues 182-202 of the acetylcholine receptor alpha1 subunit in complex with alpha-bungarotoxin was solved using NMR spectroscopy. The peptide contains the complete sequence of the major determinant of AChR involved in alpha-bungarotoxin binding. One face of the long beta hairpin formed by the AChR peptide consists of exposed nonconserved residues, which interact extensively with the toxin. Mutations of these receptor residues confer resistance to the toxin. Conserved AChR residues form the opposite face of the beta hairpin, which creates the inner and partially hidden pocket for acetylcholine. An NMR-derived model for the receptor complex with two alpha-bungarotoxin molecules shows that this pocket is occupied by the conserved alpha-neurotoxin residue R36, which forms cation-pi interactions with both alphaW149 and gammaW55/deltaW57 of the receptor and mimics acetylcholine.     

Role of C-terminal tail of long neurotoxins from snake venoms in molecular conformation and acetylcholine receptor binding: proton nuclear magnetic resonance and competition binding studies

The role of the "C-terminal tail" segment of long neurotoxins has been investigated. The C-terminal four to five residues of alpha-bungarotoxin and Laticauda colubrina b have been cleaved off by carboxypeptidase P. The effect of such deletion on the toxin conformation has been monitored in proton nuclear magnetic resonance spectra and circular dichroism spectra. The removal of the C-terminal residues primarily affects the chemical shifts of proton resonances of the residues close to the cleavage site and does not induce a major conformational change. Therefore, the C-terminal tail of long neurotoxins does not appear to be important in maintaining the specific polypeptide chain folding. On the other hand, competition binding with tritium-labeled toxin alpha to Narke japonica acetylcholine receptor has revealed that cleavage of the C-terminal residues reduces the binding activity of alpha-bungarotoxin or Laticauda colubrina b to acetylcholine receptor. Thus it is likely that (the basic amino acid residues in) the C-terminal tail is directly involved in the binding of long neurotoxins to electric organ (and muscle) acetylcholine receptor.     

Rabies virus binding to an acetylcholine receptor alpha-subunit peptide

The binding of 125I-labeled rabies virus to a synthetic peptide comprising residues 173-204 of the alpha 1-subunit of the nicotinic acetylcholine receptor was investigated. Binding of rabies virus to the receptor peptide was dependent on pH, could be competed with by unlabeled homologous virus particles, and was saturable. Synthetic peptides of snake venom, curaremimetic neurotoxins and of the structurally similar segment of the rabies virus glycoprotein, were effective in competing with labeled virus binding to the receptor peptide at micromolar concentrations. Similarly, synthetic peptides of the binding domain on the acetylcholine receptor competed for binding. These findings suggest that both rabies virus and neurotoxins bind to residues 173-204 of the alpha 1-subunit of the acetylcholine receptor. Competition studies with shorter alpha-subunit peptides within this region indicate that the highest affinity virus binding determinants are located within residues 179-192. A rat nerve alpha 3-subunit peptide, that does not bind alpha-bungarotoxin, inhibited binding of virus to the alpha 1 peptide, suggesting that rabies binds to neuronal nicotinic acetylcholine receptors. These studies indicate that synthetic peptides of the glycoprotein binding domain and of the receptor binding domain may represent useful antiviral agents by targeting the recognition event between the viral attachment protein and the host cell receptor, and inhibiting attachment of virus to the receptor.     

Naturally occurring disulfide-bound dimers of three-fingered toxins: a paradigm for biological activity diversification

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of alpha-cobratoxin (a long-chain alpha-neurotoxin) and heterodimers formed by alpha-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26-30 in polypeptide loop II of alpha-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the alpha-cobratoxin capacity to compete with alpha-bungarotoxin for binding to Torpedo and alpha7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that alpha-cobratoxin dimer not only interacts with alpha7 nAChR but, in contrast to alpha-cobratoxin monomer, also blocks alpha3beta2 nAChR. In the latter activity it resembles kappa-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric alpha3beta2 nAChRs.     

Binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of the alpha subunit of Torpedo, calf, and human acetylcholine receptor and restoration of high-affinity binding by sodium dodecyl sulfate

In order to investigate structure-function relationships of a segment of the acetylcholine receptor alpha subunit, binding of alpha-bungarotoxin to synthetic peptides corresponding to residues 173-204 of Torpedo, calf, and human alpha subunits was compared using a solid-phase radioassay. The affinities of 125I-alpha-bungarotoxin for the calf and human peptides were 15- and 150-fold less, respectively, than for the Torpedo peptide. On the basis of nonconservative substitutions in the calf and human sequences, aromatic residues (Tyr-181, Trp-187, and Tyr-189) are important for the higher affinity binding of the Torpedo peptide. Substitution of negatively charged Glu-180 with uncharged Gln in the calf peptide did not significantly affect toxin binding, indicating Glu-180 alone does not comprise the anionic subsite on the receptor to which the cationic quaternary ammonium groups of cholinergic agents bind. d-Tubocurarine competed toxin binding to the modified calf 32-mer which lacks Glu-180 and Asp-195 present in Torpedo. Thus, the negative subsite could be formed by another negatively charged residue or by more than one amino acid side chain. It is possible that the positive charges on cholinergic ligands are countered by a negative electrostatic potential provided by polar groups, such as the hydroxyl group of tyrosine, present on several residues in this region, and the negative charges present on any of residues 175, 180, 195, or 200. Equilibrium saturation binding of alpha-bungarotoxin to Torpedo peptide 173-204 revealed a minor binding component with an apparent KD of 4.2 nM and a major component with a KD of 63 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrinsic fluorescence of binding-site fragments of the nicotinic acetylcholine receptor: perturbations produced upon binding alpha-bungarotoxin

Synthetic peptides corresponding to sequences contained within residues 173-204 of the alpha-subunit in the nicotinic acetylcholine receptor (nAChR) of Torpedo californica bind the competitive antagonist alpha-bungarotoxin (BGTX) with relative high affinity. Since the synthetic peptide fragments of the receptor and BGTX each contain a small number of aromatic residues, intrinsic fluorescence studies were used to investigate their interaction. We examined a number of receptor-derived peptide fragments of increasing length (4-32 amino acids). Changes in the lambda max and quantum yield with increasing polypeptide chain length suggest an increase in the hydrophobicity of the tryptophan environment. When selective excitation and subtraction were used to reveal the tyrosine fluorescence of the peptides, a significant red shift in emission was observed and was found to be due to an excited-state tyrosinate. The binding of BGTX to the receptor-derived peptide fragments resulted in a large increase in fluorescence. In addition, at equilibrium, the lambda max of tryptophan fluorescence was shifted to shorter wavelengths. The. fluorescence enhancement, which was saturable with either peptide or BGTX, was used to determine the dissociation constants for the complexes. At pH 7.4, the apparent Kd for a dodecameric peptide (alpha 185-196), consisting of residues 185-196 in the alpha-subunit of the nAChR from Torpedo californica, was 1.4 microM. The Kd for an 18-mer (alpha 181-198), consisting of residues 181-198 of the Torpedo alpha-subunit, was 0.3 microM. No binding or enhanced fluorescence was observed with an irrelevant synthetic peptide of comparable composition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing local secondary structure by fluorescence: time-resolved and circular dichroism studies of highly purified neurotoxins.

The relationship between beta-sheet secondary structure and intrinsic tryptophan fluorescence parameters of erabutoxin b, alpha-cobratoxin, and alpha-bungarotoxin were examined. Nuclear magnetic resonance and x-ray crystallography have shown that these neurotoxins have comparable beta-sheet, beta-turn, and random coil secondary structures. Each toxin contains a single tryptophan (Trp) residue within its beta-sheet. The time-resolved fluorescence properties of native erabutoxin b and alpha-cobratoxin are best described by triple exponential decay kinetics, whereas native alpha-bungarotoxin exhibits more than four lifetimes. The disulphide bonds of each toxin were reduced to facilitate carboxymethylation and amidocarboxymethylation. The two different toxin derivatives of all three neurotoxins displayed triple exponential decay kinetics and were completely denatured as evidenced by circular dichroism (random coil). The concentration (c) values of the three fluorescence decay times (time-resolved fluorescence spectroscopy (TRFS)) were dramatically different from those of the native toxins. Each neurotoxin, treated with different concentrations of guanidinium hydrochloride (GuHCl), was studied both by circular dichroism and TRFS. Disappearance of the beta-sheet secondary structural features with increasing concentrations of GuHCl was accompanied by a shift in the relative contribution (c value) of each fluorescence decay time (TRFS). It was found that certain disulphide residues confer added stability to the beta-sheet secondary structure of these neurotoxins and that the center of the beta-sheet is last to unfold. These titrations show that Trp can be used as a very localized probe of secondary structure.     

A novel pharmatope tag inserted into the beta4 subunit confers allosteric modulation to neuronal nicotinic receptors

alpha-Bungarotoxin, the classic nicotinic antagonist, has high specificity for muscle type alpha1 subunits in nicotinic acetylcholine receptors. In this study, we show that an 11-amino-acid pharmatope sequence, containing residues important for alpha-bungarotoxin binding to alpha1, confers functional alpha-bungarotoxin sensitivity when strategically placed into a neuronal non-alpha subunit, normally insensitive to this toxin. Remarkably, the mechanism of toxin inhibition is allosteric, not competitive as with neuromuscular nicotinic receptors. Our findings argue that alpha-bungarotoxin binding to the pharmatope, inserted at a subunit-subunit interface diametrically distinct from the agonist binding site, interferes with subunit interface movements critical for receptor activation. Our results, taken together with the structural similarities between nicotinic and GABAA receptors, suggest that this allosteric mechanism is conserved in the Cys-loop ion channel family. Furthermore, as a general strategy, the engineering of allosteric inhibitory sites through pharmatope tagging offers a powerful new tool for the study of membrane proteins.     

The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.     

Characterization of the binding of alpha-bungarotoxin to bacterially expressed cholinergic binding sites

Bacterially expressed cDNA fragments of the alpha-subunit of the nicotinic acetylcholine receptor previously have been shown to bind alpha-bungarotoxin (Gershoni, J. M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 4318-4321). Here, a novel system has been developed in which totally synthetic alpha-bungarotoxin binding sites are expressed in Escherichia coli transformants. The amino acid sequences, alpha 184-200 and alpha 184-196 of the Torpedo californica alpha-subunit of the nicotinic acetylcholine receptor were expressed as trpE fusion proteins via the expression vector pATH2 and a method for the enrichment of these fusion proteins is described. Quantitative analysis of toxin binding to the recombinant binding sites demonstrates that they bind toxin with affinities of KD = 2.5 X 10(-7) and 4.7 X 10(-6) M, respectively. Furthermore, the pharmacological profile of alpha 184-200 qualitatively reflects that of the intact receptor. These data not only indicate that the area of alpha 184-200 is an essential element of the cholinergic binding site but that residues alpha 197-200 contribute a point of contact between the receptor and alpha-bungarotoxin.     

Histidine 186 of the nicotinic acetylcholine receptor alpha subunit requires the presence of the 192-193 disulfide bridge to interact with alpha-bungarotoxin

We have previously shown that two histidine residues of the nicotinic acetylcholine receptor are relevant for alpha-bungarotoxin binding. This paper studies: (1) the interaction between alpha-bungarotoxin and the peptide alpha173-202--synthesized according to the sequence of the Torpedo californica receptor alpha subunit--and between the toxin and the same peptide containing His186 modified with ethoxyformic anhydride or substituted by Ala; (2) the influence of the presence of Cys192-Cys193 disulfide bridge on such interactions. Solid-phase and in-solution competition assays were performed: ethoxyformylation of His186 or its substitution by Ala led to a significant drop in the toxin binding capacity only for peptides containing the bridge. Circular dichroism and fourth derivate spectra of all peptides were also analyzed. Results strongly indicate the involvement of His186 in the toxin binding to those peptides with the bridge--also present in the native receptor molecules--but not to their reduced forms; on the other hand, they give further support to the already established premise that, though the bridge does not participate directly in receptor-toxin binding, its presence is relevant to define the appropriate conformation of the interaction area.     

An H-bond between two residues from different loops of the acetylcholine binding site contributes to the activation mechanism of nicotinic receptors

The molecular mechanisms of nicotinic receptor activation are still largely unknown. The crystallographic structure of the acetylcholine binding protein (AChBP) reveals a single H-bond between two different acetylcholine binding loops. Within these homologous loops we systematically introduced alpha4 residues into the alpha7/5HT(3) chimeric receptor and found that the single point mutations G152K (loop B) and P193I (loop C) displayed a non-additive increase of equilibrium binding affinity for several agonists compared with the double mutant G152K/P193I. In whole-cell patch-clamp recordings, G152K, P193I and G152K/P193I mutants displayed an increase up to 5-fold in acetylcholine potency with a large decrease of the apparent Hill coefficients (significantly smaller than one). Concomitantly, the G152K/P193I mutant showed a dramatic loss of high-affinity alpha-bungarotoxin binding (100-fold decrease), thus pinpointing a new contact area for the toxin. Fitting the data with an allosteric-kinetic model, together with molecular dynamic simulations, suggests that the presence of the inter-backbone H-bond between positions 152 and 193, revealed in alpha4 and in alpha7 double mutant but not in alpha7, coincides with a large stabilization of both open and desensitized states of nicotinic receptors.     

High affinity binding of alpha-bungarotoxin to the purified alpha-subunit and to its 27,000-dalton proteolytic peptide from Torpedo marmorata acetylcholine receptor. Requirement for sodium dodecyl sulfate

Intact nicotinic acetylcholine receptor (AChR) tightly binds alpha-bungarotoxin. The two toxin-binding sites are presumed to be on the two alpha-subunits, either on or near the ACh-binding sites. Isolated alpha-subunits have been found to maintain weak binding to alpha-bungarotoxin (KD approximately 0.2 microM). We describe here conditions under which the alpha-subunit and a 27,000-dalton proteolytic peptide bound alpha-bungarotoxin with high affinity. The four subunits of Torpedo marmorata AChR, as well as several proteolytic peptides of the alpha-subunit, were first purified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. We found that the purified alpha-subunit (but not the beta-, gamma- or delta-subunits) and its 27,000-dalton peptide specifically bound 125I-labeled alpha-bungarotoxin with KD approximately 3 and 6 nM, i.e., about two orders of magnitude lower than the intact AChR. Nearly 100% of the sites were recovered. The recovery of this high affinity binding required the presence of SDS (approximately 0.02%) but non-denaturing detergents had a strongly inhibitory effect. Unlabeled alpha-toxins competed with labeled alpha-bungarotoxin, alpha-bungarotoxin being more effective than all the other toxins tested. Decamethonium and hexamethonium competed efficiently with alpha-bungarotoxin binding but carbamylcholine had only a weak effect. The main immunogenic region of the AChR was only partially preserved since conformation-dependent monoclonal antibodies to this region bound the alpha subunit-toxin complexes, but much less efficiently than the intact AChR. We conclude that SDS can be advantageous to the recovery of high toxin binding to the alpha subunit which still has not completely recovered its native conformation.     

Substitution of Torpedo acetylcholine receptor alpha 1-subunit residues with snake alpha 1- and rat nerve alpha 3-subunit residues in recombinant fusion proteins: effect on alpha-bungarotoxin binding.

A fusion protein consisting of the TrpE protein and residues 166-211 of the Torpedo acetylcholine receptor alpha 1 subunit was produced in Escherichia coli using a pATH10 expression vector. Residues in the Torpedo sequence were changed by means of oligonucleotide-directed mutagenesis to residues present in snake alpha 1 subunit and rat nerve alpha 3 subunit which do not bind alpha-bungarotoxin. The fusion protein of the Torpedo sequence bound 125I-alpha-bungarotoxin with high affinity (IC50 = 2.5 x 10(-8) M from competition with unlabeled toxin, KD = 2.3 x 10(-8) M from equilibrium saturation binding data). Mutation of three Torpedo residues to snake residues, W184F, K185W, and W187S, had no effect on binding. Conversion of two additional Torpedo residues to snake, T191S and P194L, reduced alpha-bungarotoxin binding to undetectable levels. The P194L mutation alone abolished toxin binding. Mutation of three Torpedo alpha 1 residues to neuronal alpha 3-subunit residues, W187E, Y189K, and T191N, also abolished detectable alpha-bungarotoxin binding. Conversion of Try-189 to Asn which is present in the snake sequence (Y189N) abolished toxin binding. It is concluded that in the sequence of the alpha subunit of Torpedo encompassing Cys-192 and Cys-193, Try-189 and Pro-194 are important determinants of alpha-bungarotoxin binding. Tyr-189 may interact directly with cationic groups or participate in aromatic-aromatic interactions while Pro-194 may be necessary to maintain a conformation conductive to neurotoxin binding.