Topological mapping of acetylcholine receptor: evidence for a model with five transmembrane segments and a cytoplasmic COOH-terminal peptide.

Antibodies were raised against two synthetic peptides whose sequences correspond respectively to the COOH-terminal end (residues 501-516) of the protein encoded by the gene for the delta chain and to a proposed cytoplasmic region (residues 350-358) of the beta chain of the acetylcholine receptor from Torpedo californica. Binding of the COOH-terminal antibody to the acetylcholine receptor in intact, receptor-rich vesicles was tested by radioimmunoassay and by precipitation with immobilized protein A. In both cases, binding was detected only after treatment of the vesicles with detergent, suggesting that the segment of the receptor that is recognized by this antibody is on the cytoplasmic side of the membrane. Electron microscopy of tissue from Torpedo electric organ labeled with colloidal gold-conjugated second antibodies established that both anti-receptor antibodies bind to the cytoplasmic surface of the postsynaptic membrane. These experiments give ultrastructural evidence that the COOH-terminal segment of the delta chain as well as residues 350-358 of the beta chain are on the cytoplasmic surface. They strongly support a model in which each of the receptor subunits crosses the membrane five times in which one transmembrane segment of each chain contributes to the formation of a central ion channel.