Identification of a cytoplasmic region of the Torpedo nicotinic acetylcholine receptor α-subunit by epitope mapping

Analysis of the binding of monoclonal antibodies (mAbs) by Torpedo nicotinic acetylcholine receptor (AChR) has demonstrated that a region of the α-subunit between α-156 and α-179 is exposed on the cytoplasmic surface of the nicotinic post-synaptic membrane. A panel of mAbs was produced that recognized sodium dodecyl sulfate-denatured subunits of the Torpedo AChR. Antibodies recognizing a-subunit were distinguished in terms of their ability to bind α-subunit fragments generated by Staphylococcus aureus V8 protease: an 18-kDa fragment beginning at Val-46, a 20-kDa fragment beginning at Ser-173/Ser-162, and a 10-kDa fragment beginning at Asn-339. Three mAbs, selected for binding to each of the V8-protease α-subunit fragments, respectively, were characterized in detail. The location of epitopes recognized by both antiV8-18 and anti-V8-20 mAbs was determined to be within α-156 to α-179 by isolation of small immunoreactive peptides from proteolytic digests of the α-subunit, while the mAb reactive to V8-10 was bound to an epitope within α-339 to α-386. Quantitative evaluation of binding of the anti-V8-18 and anti-V8-20 mAbs to overlapping synthetic peptides corresponding to α-147 to α-179 localized the epitopes to distinct portions of this region. Further screening of the panel of mAbs using these synthetic peptides revealed three additional mAbs that bind in this region. The mAbs that bound the three distinct V8-protease a-subunit fragments were shown to bind to native AChR by indirect immunofluorescence on frozen sections of Torpedo electric organ. Binding to the native AChR was to the cytoplasmic surface of the AChR since the mAbs could bind to AChR in native vesicles, in which the AChR is oriented right-side-out, only after permeabilization of the vesicles by alkaline treatment or after scrambling of the orientation of the AChR by solubilization and reconstitution into liposomes. The location of the mAb-binding sites at the cytoplasmic surface of the AChR was visualized directly by freeze-etch immunoelectron microscopy. The identification of α-156 and α-179 as containing a cytoplasmic exposed sequence implies the existence of two non-hydrophobic transmembrane sequences between the site of N-glycosylation (Asn-141) and Cys-192, a site alkylated by the cholinergic affinity labels.