Two Neuronal Nicotinic Acetylcholine Receptors, α4β4 and α7, Show Differential Agonist Binding Modes

Nyssa L Puskar,Xinan Xiu,Henry A Lester,Dennis A Dougherty

doi:10.1074/jbc.m110.206565

Nyssa L Puskar, Xinan Xiu + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m110.206565

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Abstract

Nicotinic acetylcholine receptors (nAChRs) are pentameric, neurotransmitter-gated ion channels responsible for rapid excitatory neurotransmission in the central and peripheral nervous systems, resulting in skeletal muscle tone and various cognitive effects in the brain. These complex proteins are activated by the endogenous neurotransmitter ACh as well as by nicotine and structurally related agonists. Activation and modulation of nAChRs has been implicated in the pathology of multiple neurological disorders, and as such, these proteins are established therapeutic targets. Here we use unnatural amino acid mutagenesis to examine the ligand binding mechanisms of two homologous neuronal nAChRs: the α4β4 and α7 receptors. Despite sequence identity among the residues that form the core of the agonist-binding site, we find that the α4β4 and α7 nAChRs employ different agonist-receptor binding interactions in this region. The α4β4 receptor utilizes a strong cation-π interaction to a conserved tryptophan (TrpB) of the receptor for both ACh and nicotine, and nicotine participates in a strong hydrogen bond with a backbone carbonyl contributed by TrpB. Interestingly, we find that the α7 receptor also employs a cation-π interaction for ligand recognition, but the site has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist. ACh participates in a cation-π interaction with TyrA, whereas epibatidine participates in a cation-π interaction with TyrC2.

Highlights

Nicotinic acetylcholine receptors2 belong to the Cys loop superfamily of neurotransmitter-gated ion channels, which includes GABAA and GABAC, glycine, and serotonin type 3 (5-HT3) receptors
The ␣7 receptor employs a cation-␲ interaction for ligand recognition, but surprisingly, we find that the locus has moved to a different aromatic amino acid of the agonist-binding site depending on the agonist
Challenges in Studying Neuronal Nicotinic acetylcholine receptors (nAChRs) with Unnatural Amino Acids—The nonsense suppression methodology for incorporating unnatural amino acids into receptors and ion channels expressed in Xenopus oocytes has proven to be broadly applicable, including studies of serotonin (5-HT3) receptors, GABA receptors, glycine receptors, Kϩ and Naϩ channels, and G protein-coupled receptors such as the D2 dopamine and M2 muscarinic ACh receptors [13, 33,34,35,36,37]

Summary

Introduction

Nicotinic acetylcholine receptors (nAChRs) belong to the Cys loop superfamily of neurotransmitter-gated ion channels, which includes GABAA and GABAC, glycine, and serotonin type 3 (5-HT3) receptors These transmembrane proteins are essential for proper rapid synaptic transmission in the central and peripheral nervous systems [1]. The cationic moiety of ACh interacts with a cluster of aromatic amino acids These aromatic residues were first identified by photoaffinity labeling and mutagenesis experiments of the full receptor and subsequently located by the acetylcholine-binding protein crystal structures [1, 8]. Neuronal nAChRs have variable stoichiometries formed from various combinations of ␣ and ␤ subunits [11] This large collection of closely related receptors— current estimates are that as many as 25 nAChR subtypes are active in humans—presents special challenges to drug discovery efforts [3]. We extend our studies of the principal component of the agonist-binding site to two

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