Abstract
The ten types of nicotinic acetylcholine receptor α-subunits show substantial sequence homology, yet some types confer high affinity for α-bungarotoxin, whereas others confer negligible affinity. Combining sequence alignments with structural data reveals three residues unique to α-toxin-refractory α-subunits that coalesce within the 3D structure of the α4β2 receptor and are predicted to fit between loops I and II of α-bungarotoxin. Mutating any one of these residues, Lys189, Ile196 or Lys153, to the α-toxin-permissive counterpart fails to confer α-bungarotoxin binding. However, mutating both Lys189 and Ile196 affords α-bungarotoxin binding with an apparent dissociation constant of 104 nM, while combining mutation of Lys153 reduces the dissociation constant to 22 nM. Analogous residue substitutions also confer high affinity α-bungarotoxin binding upon α-toxin-refractory α2 and α3 subunits. α4β2 receptors engineered to bind α-bungarotoxin exhibit slow rates of α-toxin association and dissociation, and competition by cholinergic ligands typical of muscle nicotinic receptors. Receptors engineered to bind α-bungarotoxin co-sediment with muscle nicotinic receptors on sucrose gradients, and mirror single channel signatures of their α-toxin-refractory counterparts. Thus the inability of α-bungarotoxin to bind to neuronal nicotinic receptors arises from three unique and interdependent residues that coalesce within the receptor's 3D structure.