Structural basis for α-bungarotoxin insensitivity of neuronal nicotinic acetylcholine receptors

The ten types of nicotinic acetylcholine receptor alpha-subunits show substantial sequence homology, yet some types confer high affinity for alpha-bungarotoxin, whereas others confer negligible affinity. Combining sequence alignments with structural data reveals three residues unique to alpha-toxin-refractory alpha-subunits that coalesce within the 3D structure of the alpha 4 beta 2 receptor and are predicted to fit between loops I and II of alpha-bungarotoxin. Mutating any one of these residues, Lys189, Ile196 or Lys153, to the alpha-toxin-permissive counterpart fails to confer alpha-bungarotoxin binding. However, mutating both Lys189 and Ile196 affords alpha-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 alpha-bungarotoxin binding upon alpha-toxin-refractory alpha 2 and alpha 3 subunits. alpha 4 beta 2 receptors engineered to bind alpha-bungarotoxin exhibit slow rates of alpha-toxin association and dissociation, and competition by cholinergic ligands typical of muscle nicotinic receptors. Receptors engineered to bind alpha-bungarotoxin co-sediment with muscle nicotinic receptors on sucrose gradients, and mirror single channel signatures of their alpha-toxin-refractory counterparts. Thus the inability of alpha-bungarotoxin to bind to neuronal nicotinic receptors arises from three unique and interdependent residues that coalesce within the receptor's 3D structure.