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The level of transcripts encoding the skeletal muscle acetylcholine receptor (AChR) was determined during embryonic development in Xenopus laevis. cDNAs encoding the alpha, gamma, and delta subunits of the Xenopus AChR were isolated from Xenopus embryo cDNA libraries using Torpedo AChR cDNAs as probes. The Xenopus AChR cDNAs have greater than 60% amino acid sequence homology to their Torpedo homologues and hybridize to transcripts that are restricted to the somites of developing embryos. Northern blot analysis demonstrates that a 2.3-kb transcript hybridizes to the alpha subunit cDNA, a 2.4-kb transcript hybridizes to the gamma subunit cDNA, and that two transcripts, of 1.9 and 2.5 kb, hybridize to the delta subunit cDNA. RNase protection assays demonstrate that transcripts encoding alpha, gamma, and delta subunits are coordinately expressed at late gastrula and that the amount of each transcript increases in parallel with muscle-specific actin mRNA during the ensuing 12 h. After the onset of muscle activity the level of actin mRNA per somite remains relatively constant, whereas the level of alpha subunit and delta subunit transcripts decrease fourfold per somite and the level of gamma subunit transcript decreases greater than 50-fold per somite. The decrease in amount of AChR transcripts per somite, however, occurs when embryos are paralyzed with local anaesthetic during their development. These results demonstrate that AChR transcripts in Xenopus are initially expressed coordinately, but that gamma subunit transcript levels are regulated differently than alpha and delta at later stages. Moreover, these results demonstrate that AChR transcript levels in Xenopus myotomal muscle cells are not responsive to electrical activity and suggest that AChR transcript levels are influenced by other regulatory controls.

Acetylcholine receptor-rich membranes from the electric organ of Torpedo californica are enriched in the four different subunits of the acetylcholine receptor and in two peripheral membrane proteins at 43 and 300 kD. We produced monoclonal antibodies against the 300-kD protein and have used these antibodies to determine the location of the protein, both in the electric organ and in skeletal muscle. Antibodies to the 300-kD protein were characterized by Western blots, binding assays to isolated membranes, and immunofluorescence on tissue. In Torpedo electric organ, antibodies to the 300-kD protein stain only the innervated face of the electrocytes. The 300-kD protein is on the intracellular surface of the postsynaptic membrane, since antibodies to the 300-kD protein bind more efficiently to saponin-permeabilized, right side out membranes than to intact membranes. Some antibodies against the Torpedo 300-kD protein cross-react with amphibian and mammalian neuromuscular synapses, and the cross-reacting protein is also highly concentrated on the intracellular surface of the post-synaptic membrane.