Identification of cDNA clones coding for Torpedo marmorata acetylcholine receptor subunits and a model for the transmerabrane organization

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

“…We also raised antibodies to a synthetic peptide corresponding to residues 350-358t of the 8 chain to further map the topology of the receptor chains with respect to the membrane. This portion of the mature f3 chain also was localized to the cytoplasmic side of the membrane as previously suggested (10,12,13,15,16,18,20,21). There must, therefore, be an even number of transmembrane segments between residue 358 and the COOH terminus.…”

“…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)(9)(10)(11)(12)(13). The receptor is glycosylated approximately eight times (14), so that the estimated total molecular weight is 295,000 (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.…”

Antibodies to synthetic peptides as probes of acetylcholine receptor structure

“…Cleavage would therefore result in the generation of a new C-terminus. Models proposed for the insertion of the receptor chains in the membrane place the C-terminus outside [5,7,9] or inside [Ill the cell. If cleavage does occur, the new C-terminus would be internal on either model.…”

“…The cDNAs for all chains of T. californica [4 -71 and that for the alpha-chain of T. marmorata [8,9] have been cloned and the amino acid sequences deduced. However, the calculated molecular masses are 50, 54, 57 and 58 kDa respectively, neglecting the additional contribution of carbohydrate residues.…”