Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies.
A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross-reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR (alpha, beta, gamma, delta) cross-reacted with AChRs from each amphibian species. mAbs cross-reacting with Xenopus AChRs were, with one exception, a subset of the mAbs cross-reacting with Rana AChRs. The major difference detected between the two species was in binding by mAbs specific for the main immunogenic region (MIR) of the alpha-subunit. Whereas 22 of 33 anti-MIR mAbs tested cross-reacted with Rana AChRs, only one of these mAbs cross-reacted with Xenopus AChRs. Some (32) of the cross-reacting mAbs were tested for binding to AChRs in intact muscle. 21 of these mAbs bound to AChRs only when membranes were made permeable with saponin. Electron microscopy using immunoperoxidase or colloidal gold techniques revealed that these mAbs recognize cytoplasmic determinants and that mAbs that do not require saponin in order to bind AChRs in intact muscle recognize extracellular determinants. These results suggest that AChRs in skeletal muscle of Rana and Xenopus are composed of subunits corresponding to the alpha-, beta-, gamma-, and delta-subunits of AChRs from fish electric organs. The subunit specificity of mAbs whose binding was examined by electron microscopy suggests that parts of each subunit (alpha, beta, gamma, delta) are exposed on the cytoplasmic surface and that, as in AChRs from fish electric organs and mammalian muscle, the MIR on alpha-subunits of Rana AChRs is exposed on the extracellular surface.
D000818:Animals; D000911:Antibodies, Monoclonal; D002462:Cell Membrane / Q000032:analysis / Q000648:ultrastructure; D003429:Cross Reactions; D004557:Electric Organ / Q000032:analysis; D004593:Electrophorus; D007124:Immunoenzyme Techniques; D008854:Microscopy, Electron; D009132:Muscles / Q000032:analysis*; D011894:Rana pipiens; D011950:Receptors, Cholinergic / Q000032:analysis*; D013045:Species Specificity; D014101:Torpedo; D014981:Xenopus
D000911:Antibodies, Monoclonal; D011950:Receptors, Cholinergic