Nicotine addiction is still a major problem in American society. Sixteen percent of adults report smoking regularly, resulting in 96 billion dollars in healthcare costs per year. Parkinson’s disease is incurable, with 6,000 new diagnoses each year - it is the 14th leading cause of death in the US. Parkinson’s disease and nicotine addiction are disorders of the dopamine pathway, making α6β2-containing subtypes of the nicotinic acetylcholine receptor (nAChR), which are ligand gated ion channels localized to dopaminergic neurons, an attractive target. Advanced knowledge of the binding site, which sits at the interface of α6 and β2, could lead to design of agonists which specifically target that subtype. When α6L9’Sβ2LFM/AAQAL9’S (α6β2‡) mRNA is injected in X. laevis oocytes, heterologous expression and activation of a pure and stoichiometrically controlled population of receptors is observed. Currents are high enough in the α6β2‡ system to tolerate nonsense suppression-based non-canonical amino acid mutagenesis, which allows for structure-function studies between the receptor and agonists. Initial structure-function studies probed for a cation-π interaction between TrpB and several agonists. This was accomplished using a series of substituted tryptophans. Electron-withdrawing groups such as fluorine were incrementally added to the indole side chain, and effects on binding were compared to ab initio calculated expected binding energies. Results show that acetylcholine makes a cation-π interaction, while nicotine and TC-299423 do not. They do however make a hydrogen bond to TrpB’s backbone carbonyl. The amide backbone was mutated to an ester bond via an alpha-hydroxy acid substitution at the amino acid adjacent to TrpB making the carbonyl a weaker hydrogen bond acceptor, and a corresponding loss in function was observed. The hydrogen bond to nicotine was then quantified with a double-mutant cycle analysis utilizing N’-methylnicotinium and the amide-ester mutation. It showed the interaction to have a coupling energy of ~2 kcal/mol. Further studies of this important pharmacophore are underway.

Probing binding interactions of agonists at a nicotinic acetylcholine receptor subtype important to addiction and Parkinson's disease

251st National Meeting (2016)

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