Mapping the extracellular topography of the alpha-chain in free and in membrane-bound acetylcholine receptor by antibodies against overlapping peptides spanning the entire extracellular parts of the chain.

The extracellular surface of the alpha-chain of Torpedo california acetylcholine receptor (AChR) was mapped for regions that are accessible to binding with antibodies against a panel of synthetic overlapping peptides which encompassed the entire extracellular parts of the chain. The binding of the antipeptide antibodies to membrane-bound AChR (mbAChR) and to isolated, soluble AChR was determined. The specificity of each antiserum was narrowed down by determining the extent of its cross-reaction with the two adjacent peptides that overlap the immunizing peptide. With mbAChR, high antibody reactivity was obtained with antisera against peptides alpha 1-16, alpha 89-104, alpha 158-174, alpha 262-276, and alpha 388-408. Lower, but significant, levels of reactivity were obtained with antibodies against peptides alpha 67-82, alpha 78-93, alpha 100-115, and alpha 111-126. On the other hand, free AChR bound high levels of antibodies against peptides alpha 34-49, alpha 78-93, alpha 134-150, alpha 170-186, and alpha 194-210. It also bound moderate levels of antibodies against peptides alpha 262-276 and alpha 388-408. Low, yet significant, levels of binding were exhibited by antibodies against peptides alpha 45-60, alpha 111-126, and alpha 122-138. These binding studies, which enabled a comparison of the accessible regions in mbAChR and free AChR, revealed that the receptor undergoes considerable changes in conformation upon removal from the cell membrane. The exposed regions found here are discussed in relation to the functional sites of AChR (i.e., the acetylcholine binding site, the regions that are recognized by anti-AChR antibodies, T-cells and autoimmune responses and the regions that bind short and long neurotoxins).