Molecular Interaction of a-Conotoxin RgIA with the Rat a9a10 Nicotinic Acetylcholine Receptor

The a9a10 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 a-conotoxin (a-CTx) RgIA, one of the few a9a10 selective ligands identified to date, is 300-fold less potent on human versus rat a9a10 nAChR. This species difference was conferred by only one residue in the (2), rather than (1), binding region of the a9 subunit. In light of this unexpected discovery, we sought to determine other interacting residues with a-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 a9(1) face and two residues on the a10(2) face of the a9a10 nAChR. However, mutations of these residues had little or no effect on toxin block of the a9a10 nAChR. In contrast, mutations of homologous residues in the opposing nAChR subunits (a10 Ε197, P200 and a9 T61, D121) resulted in 19to 1700-fold loss of toxin activity. Based on the crystal structure of the extracellular domain (ECD) of human a9 nAChR, we modeled the rat a9a10 ECD and its complexes with a-CTx RgIA and acetylcholine. Our data support the interaction of a-CTx RgIA at the a10/a9 rather than the a9/a10 nAChR subunit interface, and may facilitate the development of selective ligands with therapeutic potential.