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

Abstract

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 19-to 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.

Cloning, synthesis, and characterization of αO-conotoxin GeXIVA, a potent α9α10 nicotinic acetylcholine receptor antagonist

“…α-Conotoxins are competitive blockers of nAChRs, and their blocking of α9α10 nAChRs occurs by occupation of a site at the α10/α9 subunit interface (16,33,36). There are no characterized radioligands for α9α10 nAChRs or cell lines that robustly express α9α10 nAChRs to perform radioligand competition binding assays.…”

Inhibition of α9α10 nicotinic acetylcholine receptors prevents chemotherapy-induced neuropathic pain

“…This targeting specificity provided a plausible mechanistic rationale for the failed clinical development: a striking "affinity gap" of the Vc1.1 peptide for the human vs. rodent α9α10 nAChR, with the human receptor having orders of magnitude lower potency (10)(11)(12).…”

Molecular Basis for Differential Sensitivity of α-Conotoxin RegIIA at Rat and Human Neuronal Nicotinic Acetylcholine Receptors

“…Evidence that a-conotoxins can exhibit considerably different potencies on homologous nAChR subtypes of different species comes from a few recent reports (Azam and McIntosh, 2012;Yu et al, 2013;Azam et al, 2015). Here, we report novel species-and receptor subtype-specific differences between a3b2 and a3b4 nAChR sensitivity to a-conotoxin RegIIA.…”