Abstract
Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.
Highlights
Nicotinic acetylcholine (ACh) receptors are members of the pentameric ligand-gated ion channel family (Nemecz et al, 2016)
It has been shown to interact with ACh in a crystal structure of a nicotinic acetylcholine receptor (nAChR) homolog from Lymnaea stagnalis (Olsen et al, 2014) and with a-BTX when crystallized with either the a1 (Dellisanti et al, 2007) and a9 receptor subunits (Zouridakis et al, 2014) or a a7/AChBP chimera (Huang et al, 2013)
The present study shows that, contrary to previous assumptions, the a10 subunit contributes to the principal face of the ligand binding site in the heteromeric a9a10 nAChR
Summary
Nicotinic acetylcholine (ACh) receptors (nAChRs) are members of the pentameric ligand-gated ion channel family (Nemecz et al, 2016). At the interface of the extracellular domains of adjacent subunits lies the ACh binding site, formed by six noncontiguous regions (loops A–F). The components of the extracellular intersubunit binding sites are nonequivalent and their loops contribute differently to receptor function (Karlin, 2002)
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