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 a-chain of Torpedo california acetylc holine receptor (AChR) was mapped for regions that are accessible to b inding with antibodies against a panel of synthetic overlapping peptid es 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-rea ction with the two adjacent peptides that overlap the immunizing pepti de. With mbAChR, high antibody reactivity was obtained with antisera a gainst peptides alpha1-16, alpha89-104, alpha158-174, alpha262-276, an d alpha388-408. Lower, but significant, levels of reactivity were obta ined with antibodies against peptides alpha67-82, alpha78-93, alpha100 -115, and alpha111-126. On the other hand, free AChR bound high levels of antibodies against peptides alpha34-49, alpha78-93, alpha134-150, alpha170-186, and alpha194-210. It also bound moderate levels of antib odies against peptides alpha262-276 and alpha388-408. Low, yet signifi cant, levels of binding were exhibited by antibodies against peptides alpha45-60, alpha111-126, and alpha122-138. These binding studies, whi ch enabled a comparison of the accessible regions in mbAChR and free A ChR, revealed that the receptor undergoes considerable changes in conf ormation upon removal from the cell membrane. The exposed regions foun d here are discussed in relation to the functional sites of AChR (i.e. , the acetylcholine binding site, the regions that are recognized by a nti-AChR antibodies, T-cells and autoimmune responses and the regions that bind short and long neurotoxins).