A COMBINED ELISA-IMMUNOELECTRON MICROSCOPIC APPROACH FOR TOPOLOGICAL MAPPING OF MEMBRANE-PROTEIN EPITOPES - APPLICATION TO THE NICOTINIC ACETYLCHOLINE-RECEPTOR

Identification of epitope localization on either side of the lipid mem brane by immunoelectron microscopy constitutes an intrinsic powerful m ethod of structure determination for membrane proteins. We have develo ped a method allowing measurement and observation, under almost identi cal experimental conditions, of the binding of monoclonal antibodies ( MAb) to membrane-bound acetylcholine receptor from Torpedo marmorata e lectric tissue. This method, based on ELISA and electron microscopy of negatively stained specimens, was developed with MAb of known epitope specificity. With native membrane fragments, we found that MAb bound to extracellular epitopes in a stoichiometric manner, whereas almost n o binding was detected for intracellular epitopes. The treatment based on tissue homogenization in the presence of Zn2+ ions and sucrose res ulted in the formation of large, stable openings, rendering accessible about 25% of intracellular epitopes. Electron microscopic observation s showed a clear distinction between antibody binding to either intrac ellular or extracellular epitopes, both with native and Zn2+-treated m embranes. In addition, the binding of one antibody directed against an extracellular epitope was strikingly dependent on the packing density of acetylcholine receptor molecules, thus enabling us to further dist inguish between two levels of accessibility for extracellular epitopes . The method presented here is of general application for studies of e pitope mapping in membrane proteins.