GLYCOSYLATION SITES SELECTIVELY INTERFERE WITH ALPHA-TOXIN BINDING TOTHE NICOTINIC ACETYLCHOLINE-RECEPTOR

Sequence analysis reveals unique features in the alpha-subunit of nico tinic acetylcholine receptors from the alpha-toxin-resistant cobra and mongoose. Included are N-linked glycosylation signals just amino-term inal to the Tyr(190), Cys(192)-Cys(193) region of the Ligand binding d omain, substitution of Trp(187) and Phe(189) by non-aromatic residues and alteration of the proline sequence Pro(194)-X-X-Pro(197). Glycosyl ation signals were inserted into the toxin sensitive mouse alpha-subun it by the mutations F189N and W187N/F189T. The F189N alpha-subunit, wh en trans-fected with beta, gamma and delta, showed a 140-fold loss of alpha-bungarotoxin affinity, whereas the W187N/F189T double mutation e xhibited a divergence in alpha-toxin affinities at the two sites, one class showing a 600-fold and the other showing an 11-fold reduction. T he W187N mutant and the double mutant F189N/S198A lacking the requisit e glycosylation signals exhibited little alteration in affinity, as di d the P194L and P197H mutations. The glycosylation sites had little or no influence on binding of toxins of intermediate (alpha-conotoxin, 1 500 Da) or small mass (lophotoxin, 500 Da) and of the agonist, carbamy lcholine. The two sites for the binding of alpha-conotoxin M1 have wid ely divergent dissociation constants of 2.1 and 14,800 nM. Expression of alpha/gamma- and alpha/delta-subunit pairs indicated that the high and low affinity sites are formed by the alpha/delta and alpha/delta c ontacts, respectively.