Abstract
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 Xenopus laevis 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 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 β4 contributes to higher sensitivity of human α6/α3β4 nAChRs to α-conotoxins, and structure-activity studies indicated that PeIA Pro13 is critical for high potency. Human and rat α6/α3β4 nAChRs displayed differential sensitivities to perturbations of the interaction between PeIA Pro13 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.
Highlights
Nicotinic acetylcholine receptors containing ␣6 and 4 subunits are expressed by dorsal root ganglion neurons and have been implicated in neuropathic pain
When tested on human and rat ␣6/␣34 Nicotinic acetylcholine receptors (nAChRs) heterologously expressed in Xenopus oocytes, we found that all three ␣-Ctxs showed higher potency on the human homolog
The IC50 value for PnIA was ϳ9-fold lower on the rat ␣6/␣34Q119L mutant (Fig. 11A and Table 7), in contrast to Compounds expected to have analgesic properties are often tested in rodent models of pain with the expectation that the results will be translatable to humans
Summary
Nicotinic acetylcholine receptors (nAChRs) containing ␣6 and 4 subunits are expressed by dorsal root ganglion neurons and have been implicated in neuropathic pain. Human and rat ␣6/␣34 nAChRs displayed differential sensitivities to perturbations of the interaction between PeIA Pro and residue 119 of the 4 subunit These results highlight the potential significance of species differences in ␣64 nAChR pharmacology that should be taken into consideration when. ␣-Ctx Vc1.1 [23, 24] is an example of a ligand developed as a treatment for neuropathic pain that showed promising results in rodent models but failed to produce similar levels of analgesia in human clinical trials. Through structure–activity studies of PeIA, site-directed mutagenesis of ␣6 and 4 subunits, X-ray crystallography, and NMR studies of PeIA, we have identified important molecular determinants of ␣-Ctx potency for human and rat ␣64 nAChRs. The information obtained from these studies offers important insights into the pharmacology of ␣64 nAChRs and might facilitate the development of selective ligands that interact with this potential neuropathic pain target
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