Intersubunit contacts governing assembly of the mammalian nicotinic acetylcholine receptor.

Through specific intersubunit contacts, the four subunits of the nicotinic acetylcholine receptor assemble into an alpha 2 beta gamma delta pentamer. The specificity of subunit association leads to formation of proper ligand binding sites and to transport of assembled pentamers to the cell surface. To identify determinants of subunit association, we constructed chimeric subunits, transfected them into HEK 293 cells, and studied their association with wild-type subunits. We used beta gamma chimeras to determine sequences that associate with the alpha subunit to form a ligand binding site and found residues 21-131 of the gamma subunit sufficient to form the site. Residues 51-131 of the beta subunit do not form a binding site, but do promote surface expression of pentamers; of these residues, R117 is key for surface expression. We studied formation of tetramers by alpha and gamma subunits and dimers by alpha and delta subunits, and used gamma delta chimeras to identify sequences that result in either dimers or tetramers. The conserved residues I145 and T150 of the gamma subunit promote alpha gamma alpha gamma tetramer formation, whereas the corresponding residues in the delta subunit, K145 and K150, allow only alpha delta dimer formation.

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

Sequence analysis reveals unique features in the alpha-subunit of nicotinic acetylcholine receptors from the alpha-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 alpha-subunit by the mutations F189N and W187N/F189T. The F189N alpha-subunit, when transfected with beta, gamma and delta, showed a 140-fold loss of alpha-bungarotoxin affinity, whereas the W187N/F189T double mutation exhibited a divergence in alpha-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 (alpha-conotoxin, 1500 Da) or small mass (lophotoxin, 500 Da) and of the agonist, carbamylcholine. The two sites for the binding of alpha-conotoxin M1 have widely 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/gamma contacts, respectively.