The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the a subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to a-neurotoxins. We have previously reported that the resistance of snake AcChoR to a-bungarotoxin (a-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to a-neurotoxins. It was shown that mongoose AcChoR does not bind a-BTX in vivo or in vitro. cDNA fragments of the a subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coi. The expressed protein agments of the mongoose, as well as of snake receptors, do not bind a-BTX. The mongoose fragment is highly homologous (>90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand big site, close to cysteines 192 and 193. These changesare at positions 187 (Trp -Asn), 189 (Phe Thr), 191
The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the alpha-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193. We have been analyzing the binding site domain of AChRs from several animal species exhibiting various degrees of resistance to alpha-bungarotoxin (alpha-BTX). Our earlier work on the snake and mongoose AChR, both of which do not bind alpha-BTX, suggested that amino acid substitutions at positions 187, 189, and 194 of the AChR alpha-subunit are important in determining the resistance of these AChRs to alpha-BTX. In the present study, we have examined the correlation between alpha-BTX binding and the structure of the binding site domain of AChR from the hedgehog, shrew, cat, and human. Fragments of the AChR alpha-subunit corresponding to residues 122-205 from these species were cloned, sequenced, and expressed in Escherichia coli. The hedgehog fragment does not bind alpha-BTX, in common with the snake and mongoose AChR, and the human fragment is a partial binder. The shrew and cat fragments bind alpha-BTX to a similar extent as the mouse fragment. The hedgehog and human AChRs have nonaromatic amino acid residues at positions 187 and 189 of the alpha-subunit, as is seen with the "toxin resistant" snake and mongoose, and in contrast with the "toxin binders", which have aromatic residues at these two positions.(ABSTRACT TRUNCATED AT 250 WORDS)
The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is localized in the a-subunit within a domain containing the tandem analyzing the binding-site region of AcChoR from animal species that are resistant to a-neurotoxins, we have previously shown that four residues in this region, at positions 187, 189, 194, and 197, differ between animals sensitive (e.g., mouse) and resistant (e.g., mongoose and snake) to a-bungarotoxin (a-BTX). In the present study, we performed site-directed mutagenesis on a fragment of the mongoose AcChoR a-subunit (residues 122-205) and exchanged residues 187, 189, 194, and 197, either alone or in combination, with those present in the mouse a-subunit sequence. Only the mongoose fragment in which all four residues were mutated to the mouse ones exhibited a-BTX binding similar to that of the mouse fragment. The mongoose double mutation in which were replaced with proline residues, which are present at these positions in the mouse AcChoR and in all other toxin binders, bound a-BTX to '60%o of the level of binding exhibited by the mouse fragment. In addition, replacement of either in the mouse fragment with serine and histidine, respectively, markedly decreased a-BTX binding.All other mutations resulted in no or just a small increase in a-BTX binding. These results have led us to propose two subsites in the binding domain for a-BTX: the proline subsite, which includes and is critical for a-BTX binding, and the aromatic subsite, which includes amino acid residues 187 and 189 and determines the extent of a-BTX binding.The nicotinic acetylcholine (AcCho) receptor (AcChoR) is a complex multisubunit ligand-gated ion channel. The functional molecule is a pentameric complex of a2I3-Y subunits. Among the four subunits, the a-subunit has been shown to contain the ligand binding site (1-3). Previous reports from our laboratory demonstrated that a dodecapeptide containing residues 185-196 of the AcChoR a-subunit contains the major determinants of the binding site of the receptor (4, 5). Other studies using synthetic peptides (6), affinity-labeling experiments (7, 8), proteolytic fragmentation (9), and genetic constructs (10-12) also have localized the ligand binding site in close proximity or contiguous to the tandem . Recent studies with AcCho site-directed affinity reactions suggest that aromatic residues from other regions of the a-subunit (13) as well as negatively charged amino acid residues (aspartates and glutamates) at the junction with the -y and 8 subunits (14)(15)(16) participate also in the AcCho binding site. It is proposed from these studies that the two AcCho binding sites are formed at the interfaces between the a and y subunits and between the a and 6 subunits (15).In earlier studies from our laboratory, the binding site region of AcChoR from animal species that are resistant to a-neurotoxins had been analyzed. The domains of the a-subunit (corresponding to amino acid residues 122-205) from the snake (17) and the mongoose (18) and recently from the hedge...
Synthetic peptides and their respective antibodies have been used in order to map the a-bungarotoxin binding site within the a subunit of the acetylcholine receptor. By using antibodies to a synthetic peptide corresponding to residues 169-181 of the a subunit, we demonstrate that this sequence is included within the 18-kDa toxin binding fragment previously reported. Furthermore, the 18-kDa fragment was also found to bind a monoclonal antibody (5.5) directed against the cholinergic binding site. Sequential proteolysis of the acetylcholine receptor with trypsin, prior to Staphylococcus aureus V8 protease digestion, resulted in a 15-kl]a toxin binding fragment that is included within the 18-kDa fragment but is shorter than it only at its carboxyl terminus. This 15-kDa fragment therefore initiates beyond Asp-152 and terminates in the region of Arg-313/Lys-314. In addition, experiments are reported that indicate that in the intact acetylcholine receptor, Cys-128 and/or Cys-142 are not crosslinked by disulfide bridges with any ofthe cysteines (at positions 192, 193, and 222) that reside in the 15-kDa toxin binding fragment. Finally, the synthetic dodecapeptide Lys-His-Trp-Val-Tyr-Tyr-Thr-CysCys-Pro-Asp-Thr, which is present in the 15-kDa fragment (corresponding to residues 185-196 of the a subunit) was shown to bind a-bungarotoxin directly. This binding was completely inhibited by competition with d-tubocurarine.The nicotinic acetylcholine receptor (AcChoR) is probably the most well-characterized membrane receptor known today (for reviews, see refs. 1-3). The amino acid sequence of its four subunits has been the basis for models predicting the receptor's transmembrane orientation (4-7), the glycosylated amino acids, the main immunogenic regions (8), and the location of the ligand binding site (3,8).Based on the observation that a cysteine residue is situated 1 nm from the binding site (1), it was postulated (8) (18). The amino acid composition of the peptide was verified by amino acid analysis. The peptides were conjugated to bovine serum albumin (Sigma) by using 1-ethyl-3,3'-dimethylethylaminopropylcarbodiimide hydrochloride as the coupling reagent, as described (18,20).Immunological Procedures. Rabbits were immunized as described (18). In all experiments, total rabbit serum was adsorbed on a Sepharose 4B column of Staphylococcus aureus V8 protease prior to use.Proteolytic Digestion and Blot Analysis ofAcChoR. AcChoR was prepared by the method of Aharonov et al. (21). Tryptic digestion of the AcChoR was performed as described (22). S. aureus V8 protease digestion of intact a subunit or of the 27-kDa tryptic fragment was performed as described (23). Gel electrophoresis was performed with the Laemmli buffer system (24). Sample buffer (0.0625 M Tris/2% NaDodSO4/ 2% glycerol, pH 6.8) with or without 2-mercaptoethanol (3%) was subsequently added to the samples before separation on polyacrylamide gels. Blotting of the gels and the toxin or antibody overlays were done as described (16,18,25,26). RESULTS Previously...
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