Probing Binding Interactions of Agonists with the α6β2 Nicotinic Acetylcholine Receptor

Abstract

The nicotinic acetylcholine receptor (nAChR) is a ligand gated ion channel and a member of the Cys-loop family, which also contains receptors for serotonin, glycine, and GABA. There are twelve known neuronal subunits which pentamerize in different combinations to form various subtypes, each with a unique function, pharmacology and distribution in the brain. The α6β2-containing subtypes are found mainly in dopaminergic neurons and are thus important targets in the study of Parkinson's disease and addiction. Advanced knowledge of the binding site, which sits at the interface of α6 and β2, could lead toward design of agonists that specifically target this subtype. The α6L9'sβ2_(LFM/AAQA)L9's (α6β2‡) construct enabled heterologous expression and activation of a pure and stoichiometrically controlled population of α6β2 receptors in X. laevis oocytes. Currents were high enough in the α6β2‡ system to tolerate nonsense suppression-based non-canonical amino acid mutagenesis, which allowed for structure function studies between the receptor and several agonists. Initial structure-function studies probed for a cation-π interaction between agonists and the indole side chain of α6 TrpB. This is accomplished using an analog-tryptophan series whereby the negative electrostatic potential on the surface the indole ring was incrementally decreased via electron-withdrawing substituents such as fluorine and measuring the effect that has on binding. Results show that ACh makes a cation-π interaction, but surprisingly nicotine and TC-299423 do not. However, the latter two agonists do make a hydrogen bond interaction with the carbonyl of TrpB. This conclusion was made by substituting the adjacent amino acid with an alpha-hydroxy acid and measuring the corresponding shift in binding due to that amide-to-ester backbone mutation. The result was further quantified for nicotine in a double-mutant cycle analysis utilizing N’-methylnicotinium, which showed the interaction to have a coupling energy of ∼2 kcal/mol.