Molecular determinants of {alpha}-conotoxin potency for inhibition of human and rat {alpha}6{beta}4 nicotinic acetylcholine receptors [Molecular Biophysics] (Journal of Biological Chemistry)

Nicotinic acetylcholine receptors (nAChRs) containing ?6 and ?4 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 ?-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 ?6/?3?4 nAChRs expressed in Xenopus laevis oocytes exhibit differential sensitivity to ?-conotoxins. Sequence homology comparisons of human and rat ?6?4 nAChR subunits indicated that ?6 residues forming the ligand-binding pocket are highly conserved between the two species, but several residues of ?4 differed, including a Leu-Gln difference at position 119. X-ray crystallography of ?-conotoxin PeIA complexed with the Aplysia californica acetylcholine-binding protein (AChBP) revealed that binding of PeIA orients Pro13 in close proximity to residue 119 of the AChBP complementary subunit. Site-directed mutagenesis studies revealed that Leu119 of human ?4 contributes to higher sensitivity of human ?6/?3?4 nAChRs to ?-conotoxins, and structure-activity studies indicated that PeIA Pro13 is critical for high potency. Human and rat ?6/?3?4 nAChRs displayed differential sensitivities to perturbations of the interaction between PeIA Pro13 and residue 119 of the ?4 subunit. These results highlight the potential significance of species differences in ?6?4 nAChR pharmacology that should be taken into consideration when evaluating the activity of candidate human therapeutics in rodent models.