Abstract

Neuronal bungarotoxin (NBT) is a highly selective, slowly reversible, competitive antagonist of the α3β2 neuronal nicotinic receptor. Contributions to NBT sensitivity are made by both the α3 and β2 subunits. We used a chimeric α subunit to demonstrate that the entire α3 contribution lies within sequence segment 84–215. Construction and analysis of a series of mutant α3 subunits identified seven amino acid residues (Thr143, Tyr184, Lys185, His186, Ile188, Gln198, Ser203) within this region that contribute to NBT sensitivity. Changing Thr143 to lysine, as in α2, resulted in a ∼1000-fold loss of NBT sensitivity. The effect on NBT sensitivity of changing each of the other six residues ranged from 1.8- to 40.5-fold. More extensive mutagenesis demonstrated that Thr143 serves as part of the consensus sequence for glycosylation at N141, and it is this glycosylation that is the determinant of NBT sensitivity. Only serine could substitute for threonine to maintain full NBT sensitivity, and changing Asn141 to alanine resulted in a ∼300-fold loss of NBT sensitivity. The chimera α2–181-α3, containing all identified determinants except the glycosylation site, formed receptors insensitive to 300 nmNBT. Installation of threonine to complete the glycosylation consensus site in this chimera conferred NBT sensitivity only 10-fold less than that of wild-type α3β2. These seven determinants of NBT sensitivity are located in close proximity to a series of conserved residues that are common features of all nicotinic receptor binding sites.