Molecular Interaction of α-Conotoxin RgIA with the Rat α9α10 Nicotinic Acetylcholine Receptor

The alpha 9 alpha 10 nicotinic acetylcholine receptor (nAChR) was first identified in the auditory system, where it mediates synaptic transmission between efferent olivocochlear cholinergic fibers and cochlea hair cells. This receptor gained further attention due to its potential role in chronic pain and breast and lung cancers. We previously showed that alpha-conotoxin (alpha-CTx) RgIA, one of the few alpha 9 alpha 10 selective ligands identified to date, is 300-fold less potent on human versus rat alpha 9 alpha 10 nAChR. This species difference was conferred by only one residue in the (-), rather than (+), binding region of the alpha 9 subunit. In light of this unexpected discovery, we sought to determine other interacting residues with alpha-CTx RgIA. A previous molecular modeling study, based on the structure of the homologous molluscan acetylcholine-binding protein, predicted that RgIA interacts with three residues on the alpha 9(+) face and two residues on the alpha 10(-) face of the alpha 9 alpha 10 nAChR. However, mutations of these residues had little or no effect on toxin block of the alpha 9 alpha 10 nAChR. In contrast, mutations of homologous residues in the opposing nAChR subunits (alpha 10 E197, P200 and alpha 9 T61, D121) resulted in 19- to 1700-fold loss of toxin activity. Based on the crystal structure of the extracellular domain (ECD) of human alpha 9 nAChR, we modeled the rat alpha 9 alpha 10 ECD and its complexes with alpha-CTx RgIA and acetylcholine. Our data support the interaction of alpha-CTx RgIA at the alpha 10/alpha 9 rather than the alpha 9/alpha 10 nAChR subunit interface, and may facilitate the development of selective ligands with therapeutic potential.