The effects of a myasthenic serum on the acetylcholine receptors of C2 myotubes. I. Immunological distinction between the two toxin-binding sites of the receptor.

We have examined the effect of a serum from a patient with myasthenia gravis on the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptors (AChRs) of a mouse muscle cell line, C2. After a 2-hr incubation, antibodies in the serum reduced toxin binding to C2 myotubes to a maximal extent of approximately 50%. The degradation of surface AChRs could account for the loss of only 5% of sites during the incubation; the remainder, therefore, must have been lost by blockage of binding. To investigate whether the antibodies blocked specifically one of the two toxin-binding sites that each AChR possesses, we used an analysis based on that of Sine, S. W., and P. Taylor, [1981) J. Biol. Chem. 255: 10144-10156). Although the two sites could not be distinguished by their rates of binding of alpha-BuTx, d-tubocurarine (dTC) inhibition of the initial rate of toxin binding revealed that the sites had affinities for dTC that differed by approximately 30-fold. Incubation with the myasthenic antibodies reduced the number of high affinity dTC sites, without affecting those of low affinity. We conclude that the two toxin-binding sites of the AChR are immunologically distinct.