Epitope mapping of polyclonal and monoclonal antibodies against two alpha-bungarotoxin-binding alpha subunits from neuronal nicotinic receptors.

Recently, cDNAs for alpha subunits of two different neuronal alpha-bungarotoxin-binding proteins (alpha BgtBP) were isolated from chick brain, designated alpha BgtBP alpha 1 and alpha BgtBP alpha 2. These are now also referred to as subunits alpha 7 and alpha 8, respectively. Expression studies in Xenopus oocytes have indicated that alpha 7 subunits are able to form cation channels that are sensitive to nicotinic ligands, and therefore represent bona fide nicotinic acetylcholine receptor subunits. Polyclonal and monoclonal antibodies (mAbs) have been produced against: (i) affinity-purified chick brain alpha BgtBP; and (ii) fusion proteins containing the unique cytoplasmic sequences alpha 7(327-412) and alpha 8(293-435). Here, synthetic overlapping peptides corresponding to their deduced amino acid sequences are used to map the epitopes recognized by the different antibodies. The polyclonal response to affinity-purified alpha BgtBPs and the fusion proteins indicates that sequence segments 290-420 of both subunits contain several major and minor epitopes. mAbs selected for their ability to bind both native and denatured alpha BgtBPs isolated from chick brain also recognize subunit-specific sequential epitopes within the sequence segment 290-420. The epitopes recognized by the mAbs correspond to the minor epitopes defined using antisera. The mAbs characterized in these studies will provide useful probes for further studies of alpha BgtBP structure and histological localization.