Differential Properties of a-Bungarotoxin and Myasthenic IgG Bound to Cloned a-Subunit of Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine receptor (nAChR) is a pentameric receptor composed of four subunit types (a 2 , /?, y, 5) (Conti-Tronconi et al. 1982; Hucho 1986). Several studies have shown that the acetylcholine binding sites are localized on the and they can be inhibited by snake venom a-neurotoxin. Experimental results obtained with synthetic peptides and antibodies against a-subunit suggest that the a-toxin binding site(s) may be distributed at least in five areas (Atassi et al. 1986; Kordossi et al. 1987). It remains unknown whether these five toxin-binding regions are distinct binding sites or whether they are faces of one binding site (Mulac-Jeričevič et al. 1987). Recombinant DNA technology has enabled the expression of complete a-subunit, in particular the a-bungarotoxin binding site in Escherichia coli transformants as could be moni­ tored simply by toxin overlays of colony blots (Gershoni 1987). The aim of our work was to investigate whether IgG isolated from myas­ thenic patients binds (Drachman 1987) to the cloned a-subunit of nAChR. This protein is in Escherichia coli expressed as unprocessed and represents the precur­ sor form of a-subunit. cDNA library from denervated rat muscle was prepared in Agtl 1, amplified in Escherichia coli strain Y1088. An universal oligonucleotide probe for acetyl­ choline receptor genes as described by Buonanno et al. (1986) was used for the first screening of the library. Y1090 cells were infected with positive charons containing appropriate inserts (about 2 kilobases) and the expression of recep­ tor proteins was monitored by binding of polyclonal antibodies against nAChR (of own provenance) and second antibody-l25 I protein A. The same protocol as with the polyclonal antibody against nAChR was used for the myasthenic IgG binding studies (Huynh et al. 1985). Figure 1 shows the ligand overlay of colony blots with (125 I) a-bungarotoxin giving a very strong and more selective signal (A) as compared to that of the polyclonal antibody (B). A much weaker signal was obtained from myasthenic IgG bound to the same replica (Fig. 2). The question has arisen whether myasthenic IgG has the same binding properties as do polyclonal antibodies and