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 8 chain and to a proposed cytoplasmic region (residues 350-358) of the (3 chain of the acetylcholine receptor from Torpedo californica. Binding of the COOHterminal 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 8 chain as well as residues 350-358 of the (3 chain are on the cytoplasmic surface. They strongly support a model in which each of the receptor subunits crosses the membrane five times and in which one transmembrane segment of each chain contributes to the formation of a central ion channel.The nicotinic acetylcholine receptor (AcChoR) is located in the postsynaptic membrane of cholinergic nerve terminals at neuromuscular junctions. It forms a gated ion channel that permits passage of 104 sodium ions per msec upon agonist binding (1-3). The receptor is a pentamer of four different subunits (a-8) with stoichiometry a2f8y8 (4-6). All of the subunits span the membrane and occupy quasi-equivalent positions around the ion channel as seen by electron microscopy (7). The apparent molecular weights of the mature subunits, based on NaDodSO4/PAGE, are 40,000 (a), 49,000 (,3), 60,000 (y), and 65,000 (8) (1, 2); based on the recently published cDNA sequences of the subunits, however, they are 53,649 (a), 56,060 (13), 58,053 (y), and 59,792 (8) before glycosylation or possible proteolytic processing (8-13). The receptor is glycosylated approximately eight times (14), so that the estimated total molecular weight is 295,000 (15). The deduced amino acid sequences reveal striking homology between the different subunits (6,13,15). This homology has led to the idea that each of the subunits contributes a similar peptide component to the formation of a central ion channel (3,15). The subject of this paper is the topology of the peptide chain within the membrane as it relates to the ion channel. Several models of peptide folding across the membrane, having either four, five, or six membrane crossings per subunit, have been suggested (10,12,13,(15)(16)(17)(18). Because the NH2 terminus of 8 is in the extracellular portion of the protein (19), models with four or six membrane crossings orient both NH2-and COOH termini on the extracellular side of the membrane. Models with five membrane crossings place the COOH terminus on the...
