Robert H. Fairclough

Subunit organization and structure of an acetylcholine receptor.

We have learned the positions of the alpha-subunits around the AChR rosette and the location of the toxin on the synaptic crest. A charge/hydrophobic character map of the 40 A X 30 A receptor surface that binds alpha-bungarotoxin has been constructed. A beta-structure domain surrounds the agonist binding site on the alpha-subunits, as predicted by amphipathic Fourier sequence analysis. The ion channel may be constructed from five amphipathic helices, which insert into the bilayer as the alpha2 beta gamma delta subunits come together. They form a water-filled channel on one side and interface with hydrophobic helices in each subunit on the other.

Location of terbium binding sites on acetylcholine receptor-enriched membranes☆

Acetylcholine receptor-enriched membranes bind 45 terbium cations per receptor. The Tb(III) X-ray scattering factor changes by as much as 30% over a 50 eV range about the L3 absorption edge. We exploit these changes to modulate the contribution of these ions to the X-ray diffraction pattern of oriented receptor-enriched membranes by varying the incident X-ray energy. Difference Fourier analysis of the meridional diffraction amplitudes at two X-ray energies revealed six localized regions of Tb(III) density across the membrane. Most significant is the finding of 18 Tb(III) ions near the entrance and 11 ions near the exit of the ion channel as well as 4 or 5 Tb(III) ions localized in the channel itself. This evidence strongly suggests the presence of anionic carboxylate side-chains on the channel lining.

Human × Human Hybridomas from Patients with Myasthenia Gravis: Possible Tools for Idiotypic Therapy for Myastheniaa

Hybridomas secreting monoclonal antibodies directed against the nicotinic acetylcholine receptor have been developed from rats with experimental autoimmune myasthenia gravis and from a patient with myasthenia gravis. Rat monoclonal antibodies were characterized by their ability to bind to electroblotted acetylcholine receptor subunits. Of 34 tested, 22 bound to the alpha subunit. Three bound to other subunits, and the remainder appeared to bind only to the native molecule. The human monoclonal antibodies were analyzed with respect to their binding to membrane-bound and solubilized acetylcholine receptor. Many bound with greater affinity to the membrane-bound form of the antigen. Two rat monoclonal antibodies capable of passively transferring experimental autoimmune myasthenia gravis, and with reactivities to the alpha subunit of the acetylcholine receptor, were employed to produce isogeneic monoclonal antiidiotypic antibodies. When they were injected prior to immunization with acetylcholine receptor, two of the antiidiotypic antibodies directed against framework determinants prevented the development of experimental autoimmune myasthenia gravis. This observation raises the possibility that the human monoclonal antibodies will be useful in the development of idiotypic treatment of the human disease.

Mapping functional domains on the acetylcholine receptor using monoclonal antibodies and small angle X-ray diffraction

To analyze the cholinergic bindingsite of the nicotinic acetylcholine receptor (AChR), with the long-term goal of identifying the primary-structure and tert iarystructure locations of this site, we have produced monoclonal antibodies (n~Abs) against two synthetic peptides from a candidate region of the alpha subunit. With the f i r s t peptide, al27-al45, as immunogen, 9 rat n~Abs were obtained that bound to free (unconjugated) peptide and 4 that bound to native AChR. Three of the anti-peptide n~Abs also bound native AChR. For the second peptide, which is more hydrophobic, a187=a205, the immunogen was peptide conjugated to keyhold limpet hemocyanin. The antigen employed in the antibody assay was peptide conjugated to bovine serum albumin. Two mAbs were obtained from this latter immunization, one that bound to conjugated peptide and to native AChR and one that bound only to native AChR. The binding to membrane-bound AChR of a mAb recognizing each peptide was studied in greater detail. For ~Ab KM3-7EG, that binds to the f i r s t peptide, mAb binding stoichiometry to the AChR was diminished by alpha bungarotoxin (aBgTx) and carbamylcholine (carb) without an apparent change in a f f in i ty , whereas n~Ab binding stoichiometry and af f in i ty were both decreased by d-tubocurarine {tubo). For the AChR-crossreactive n~Ab against the second peptide, mAb KNI-5G, aBgTx had no effect on the binding to AChR, while carb and tubo decreased mAb stoichiometry as well as the af f in i ty . In summary the two epitopes on the AChR recognized by the mJ~bs directed against each of the two peptides were both significantly altered by tubo, but were dif ferent ial ly affecteo by aBgTx and carb.

Monoclonal antibodies against two peptide sequences of the acetylcholine receptor distinguish between toxin binding and cholinergic ligand binding

To analyze the cholinergic bindingsite of the nicotinic acetylcholine receptor (AChR), with the long-term goal of identifying the primary-structure and tert iarystructure locations of this site, we have produced monoclonal antibodies (n~Abs) against two synthetic peptides from a candidate region of the alpha subunit. With the f i r s t peptide, al27-al45, as immunogen, 9 rat n~Abs were obtained that bound to free (unconjugated) peptide and 4 that bound to native AChR. Three of the anti-peptide n~Abs also bound native AChR. For the second peptide, which is more hydrophobic, a187=a205, the immunogen was peptide conjugated to keyhold limpet hemocyanin. The antigen employed in the antibody assay was peptide conjugated to bovine serum albumin. Two mAbs were obtained from this latter immunization, one that bound to conjugated peptide and to native AChR and one that bound only to native AChR. The binding to membrane-bound AChR of a mAb recognizing each peptide was studied in greater detail. For ~Ab KM3-7EG, that binds to the f i r s t peptide, mAb binding stoichiometry to the AChR was diminished by alpha bungarotoxin (aBgTx) and carbamylcholine (carb) without an apparent change in a f f in i ty , whereas n~Ab binding stoichiometry and af f in i ty were both decreased by d-tubocurarine {tubo). For the AChR-crossreactive n~Ab against the second peptide, mAb KNI-5G, aBgTx had no effect on the binding to AChR, while carb and tubo decreased mAb stoichiometry as well as the af f in i ty . In summary the two epitopes on the AChR recognized by the mJ~bs directed against each of the two peptides were both significantly altered by tubo, but were dif ferent ial ly affecteo by aBgTx and carb.