alpha-neurotoxin binding to the human nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) of striated muscle is an integral membrane protein consisting of five subunits of stoichiometry a:, p, E and 6, in which the E replaces the embryonic y form. T h e genes for Torpedo and human subunits have been cloned ([ 1-31; D. Beeson, unpublished work) and the a-subunit shows > 80% sequence similarity with its Torpedo counterpart. Nevertheless, some differences in binding of snake a-neurotoxins to the human AChR have been reported, particularly for the short toxin. erabutoxin B 14, 51. Moreover, the long toxin ' ?'I-a-bungarotoxin ( "'1-a-BuTx) binds to the synthetic sequence representing amino acids 185106 o f the Torpedo a-subunit, but poorly or not at all to the equivalent human peptide 161. We have compared the binding of "'I-a-BuTx t o human and Torpedo AChR by Scatchard analysis. Initial studies used purified Torpedo and denervated human AChR with filtration on Whatman DE81 discs. T h e results suggested a difference in affinity of about two-fold, but the K,, values (7brpedo l l . X k 5 . 7 x lo -" M; human 2 3 . 0 k 6 . 6 ~ lo-" M) were a little higher than normally reported for Torpedo AChR. Here we have used precipitation of the I2'l-a-BuTxAChR complexes with monoclonal antibodies [ 7, 81 to measure binding t o both denervated and normal muscle AChR (in Triton X-100 extracts of muscle) as well as purified Torpedo AChR. We have also investigated the ability of the short neurotoxin erabutoxin A (Era A ; kind gift of Professor R. Hider), which differs at only one amino acid from erabutoxin B, to inhibit binding of "'I-a-BuTx to each AChR preparation.