Molecular determinants of α-conotoxin potency for inhibition of human and rat α6β4 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) containing α6 and β4 subunits are expressed by dorsal root ganglion neurons and have been implicated in neuropathic pain. Rodent models are often used to evaluate the efficacy of analgesic compounds, but species differences may affect the activity of some nAChR ligands. A previous candidate α-conotoxin-based therapeutic yielded promising results in rodent models, but failed in human clinical trials, emphasizing the importance of understanding species differences in ligand activity. Here, we show that human and rat α6/α3β4 nAChRs expressed in oocytes exhibit differential sensitivity to α-conotoxins. Sequence homology comparisons of human and rat α6β4 nAChR subunits indicated that α6 residues forming the ligand-binding pocket are highly conserved between the two species, but several residues of β4 differed, including a Leu-Gln difference at position 119. X-ray crystallography of α-conotoxin PeIA complexed with the acetylcholine-binding protein (AChBP) revealed that binding of PeIA orients Pro in close proximity to residue 119 of the AChBP complementary subunit. Site-directed mutagenesis studies revealed that Leu of human β4 contributes to higher sensitivity of human α6/α3β4 nAChRs to α-conotoxins, and structure-activity studies indicated that PeIA Pro is critical for high potency. Human and rat α6/α3β4 nAChRs displayed differential sensitivities to perturbations of the interaction between PeIA Pro and residue 119 of the β4 subunit. These results highlight the potential significance of species differences in α6β4 nAChR pharmacology that should be taken into consideration when evaluating the activity of candidate human therapeutics in rodent models.