A Conserved Arginine with Non-Conserved Function is a Key Determinant of Agonist Selectivity in Alpha7 Nicotinic Acetylcholine Receptors

Abstract

The α7 and α4β2 subtypes are the most abundant nicotinic acetylcholine receptors (nAChR) in the mammalian brain, and also the most commonly targeted nAChR subtypes in drug discovery programs for brain disorders. Activation of α4β2 nAChR by agonists, particularly partial agonists, is a valid strategy to intervene therapeutically in nicotine addiction. However, the development of subtype specific agonists remains challenging, mainly due to the high degree of sequence homology coupled to the conservation of function in the nAChR family. Smoking cessation drugs such as varenicline or cytisine partially activate α4β2 nAChR but also behave as full agonists at other nAChR such as the α7 subtype, which may underlie some of the off-target effects of these compounds. Here, by combining molecular dynamics simulations with mutagenesis and whole-cell and single-channel current recordings, we determined the structural underpinning of the selectivity of 10-methylcytisine, a compound that shows high-affinity for α4β2 nAChR but has negligible selectivity for the α7 subtype. We identify a conserved arginine residue (α7R101) in the β3-strand that impairs agonist binding in α7 nAChR. in α4β2, the equivalent arginine residue establishes an inter-subunit electrostatic interaction with an aspartate residue in loop B that is necessary for functional expression, whereas in the α7 subtype, the aspartate residue is exchanged for glycine residue, making the side chain of arginine highly mobile. This enables α7R101 to interact with loop C residues, which influences agonist binding and channel function. We conclude that the high mobility of the arginine residue in the α7 nAChR subtype affects agonist function by influencing agonist binding and the pathway communicating agonist binding to the ion channel. The findings have implications for rational design of subtype-selective cholinergic agents.