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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)

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].

Using 2,8,5'-[H-3]ATP as a direct photoaffinity label for membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata, we have identified a binding site for ATP in the extracellular region of the beta-subunit of the receptor. Photolabeling was completely inhibited in the presence of saturating concentrations of nonradioactive ATP, whereas neither the purinoreceptor antagonists suramin, theophyllin, and caffeine nor the nAChR antagonists alpha-bungarotoxin and d-tubocurarine affected the labeling reaction. Competitive and noncompetitive nicotinic agonists and Ca2+ increased the yield of the photoreaction by up to 50%, suggesting that the respective binding sites are allosterically linked with the ATP site. The dissociation constant K-D of binding of ATP to the identified site on the nAChR was of the order of 10(-4) M. Sites of labeling were found in the sequence regions Leu 11-Pro17 and Asp 152-His163 of the nAChR beta-subunit. These regions may represent parts of a single binding site for ATP, which is discontinuously distributed within the primary structure of the N-terminal extracellular domain. The existence of an extracellular binding site for ATP confirms, on the molecular level, that this nucleotide can directly act on nicotinic receptors, as has been suggested from previous electrophysiological and biochemical studies.

We have studied by means of equilibrium binding and kinetic experiments the interaction of the membrane-bound nicotinic acetylcholine receptor (nACHR) from Torpedo marmorata with [3H]acetylcholine and the fluorescent agonist NBD-5-acylcholine. In agreement with previous studies by others, we observed the preexistence, in the absence of ligand, of an equilibrium between two states of the nAChR, one with high affinity and the other with low affinity for agonist. As additional requirements for a minimal reaction scheme, we recognized (i) the existence of two ligand-binding sites, each of which may exist in two conformational states when occupied, and (ii) ligand-induced transitions between these conformations. Employing a special form of the allosteric model which considers these requirements, we then developed a suitable algorithm in order to simultaneously fit the whole set of equilibrium binding and kinetic data obtained for the two ligands. In this way we determined for a minimal model of the mechanism of action of the nAChR the complete set of rate constants and KD values involved. With these values available, we were able to simulate the rise and fall in the concentrations of individual receptor-ligand complexes and conformations occurring in the course of excitatory events at the electrocyte synapse. The membrane environment of the nAChR plays a decisive role with respect to the rates of conformational change of the nAChR occurring in the course of ligand interaction. Thus, artificial changes in membrane structure and composition can speed up by several orders of magnitude the rate of conformational change ("desensitization"). A proper structure of the surrounding membrane hence is a prerequisite for the physiological function of the membrane-embedded nAChR.

We have studied 20 monoclonal antibodies directed against both the solubilized and the membrane-bound receptor from Torpedo marmorata. We find the following: (i) Six of the antibodies compete with cholinergic ligands for receptor binding and, hence, are directed against the ligand binding regions. (ii) Of these six antibodies, two cross-react with receptor from Electrophorus electricus, rat myotubes, and chicken sympathetic ganglia. These two antibodies therefore define a preserved structure within the ligand binding regions. The other four antibodies bind to structures not common between the receptor preparations tested. (iii) From competition binding studies using internally 3H-labeled antibodies, nine nonoverlapping antigenic regions were defined at the surface of the receptor. Three of these regions overlap with the ligand binding regions. Since two of these three regions do not overlap with each other, two structurally distinct ligand binding regions must exist at the receptor. (iv) From competition binding studies with representative cholinergic ligands, the antibodies directed against the ligand binding regions can be subdivided into three groups: one group competes with all ligands tested; the second group competes with all ligands except the bismethonium compounds; the third group competes with all ligands except the bismethonium compounds and tubocurarine. The results are summarized in a model of the organization of ligand binding sites at the receptor: There are two ligand binding regions differing in their antigenic properties. Furthermore, either there exists separate sites for distinct groups of ligands within each of these binding regions or some ligands produce conformational changes of the receptor that reversibly abolish some antigenic sites. In any case, the cholinergic ligands must interact with the receptor by more and/or other structural determinants than are provided by the structure of acetylcholine.

Antibodies were raised against eight synthetic peptides matching preselected portions of the amino acid sequence of nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata. To increase the probability of obtaining antibodies specific for the exact sequence of the immunizing peptide, peptides of only five to seven amino acids in length were employed. Even under these limiting conditions some of the polyclonal rabbit immune sera showed cross-reactivity with other peptides and/or other sequence regions of the receptor. Further studies with polyclonal and monoclonal sera suggested that conformation and charge pattern rather than linear sequence are the essential determinants of antibody epitopes. Application of antibodies for topological studies therefore requires that the antibody specificity for a particular region of the antigen has been firmly established. Epitope mapping with the eight anti-peptide immune sera provides information on the accessibility to antibody of matching sequences within the receptor molecule. We find the sequence portions alpha 81-85, alpha 127-132, and alpha 190-195 to be freely accessible both at membrane-bound and at purified receptor. Binding of anti-alpha 387-392 serum does not prove accessibility of this region as the serum cross-reacts strongly with peptide fragments corresponding to the regions alpha 165-200 and beta 190-200 of nAChR from Torpedo californica. To permit binding of anti-alpha 137-142 immune serum, treatment of the receptor with endoglycosidase is required, showing that Asn-141 indeed is glycosylated in native nAChR. The homologous sequence of the other subunits differing only in one sequence position from alpha 137-142 is not accessible in native nAChR to antibody, indicating clear differences in folding of the receptor polypeptides. Sequence portions alpha 395-401 and alpha 161-166 must first be exposed by appropriate treatment to permit binding of respective serum. These results and previous epitope mapping studies by other laboratories are discussed with respect to the limited sequence specificity of antibodies.