Glycosylation sites selectively interfere with α-toxin binding to the nicotinic acetylcholine receptor

Sequence analysis reveals unique features in the α-subunit of nicotinic acetylcholine receptors from the α-toxin-resistant cobra and mongoose. Included are N-linked glycosylation signals just amino-terminal to the Tyr¹⁹⁰, Cys¹⁹²-Cys¹⁹³ region of the ligand binding domain, substitution of Trp¹⁸⁷ and Phe¹⁸⁹ by non-aromatic residues and alteration of the proline sequence Pro¹⁹⁴-X-X-Pro¹⁹⁷. Glycosylation signals were inserted into the toxin-sensitive mouse α-subunit by the mutations F189N and W187N/F189T. The F189N α-subunit, when transfected with β, γ and δ, showed a 140-fold loss of α-bungarotoxin affinity, whereas the W187N/F189T double mutation exhibited a divergence in α-toxin affinities at the two sites, one class showing a 600-fold and the other showing an 11- fold reduction. The W187N mutant and the double mutant F189N/S191A lacking the requisite glycosylation signals exhibited little alteration in affinity, as did the P194L and P197H mutations. The glycosylation sites had little or no influence on binding of toxins of intermediate (α-conotoxin, 1500 Da) or small mass (lophotoxin, 500 Da) and of the agonist, carbamylcholine. The two sites for the binding of α-conotoxin M1 have widely divergent dissociation constants of 2.1 and 14,800 nM. Expression of α/γ- and α/δ-subunit pairs indicated that the high and low affinity sites are formed by the α/δ and α/γ contacts, respectively.