Electrophysiology-based investigations of G protein-coupled receptor pharmacology

Abstract: G protein-coupled receptors (GPCRs) constitute targets for ~34% of approved drugs. The muscarinic acetylcholine M2 receptor (M2R) activates G protein-coupled receptor inward rectifying potassium (GIRK) channels in the central nervous system and heart. Membrane potential modulates agonist potency at several GPCRs. However, the mechanism underlying the voltage sensitivity remains debated. A highly conserved aspartate residue (D2.5069) has been proposed to mediate the voltage-sensitivity of the M2R, although the low expression of D69 mutants has complicated further functional investigations. Dopamine D2 and D3 receptors (D2R and D3R) are pre- and postsynaptic inhibitory receptors in the central nervous system, involved in locomotion, cognition and endocrine functions. D2R antagonists and weak partial agonists are used clinically as antipsychotics but are associated with several side effects. Various strategies have been suggested to reduce the side-effect profile of novel antipsychotic drugs. One such strategy includes the selective targeting of non-canonical signaling pathways, e.g., the β-arrestin pathway, while leaving the classical, G protein pathway, undisturbed. Additionally, binding affinity and kinetics at the D2R, as well as ligand lipophilicity, have been suggested to be of significance in determining the side-effect liability of antipsychotics. In the thesis, M2R, D2R and D3R were investigated using two-electrode voltage-clamp in Xenopus laevis oocytes co-expressing the respective receptor and GIRK channels. M2R carrying a charge-neutralizing D69N mutation demonstrated a voltage-dependent shift of agonist-potency, similar to the wild type M2R. This finding is in line with a recent alternative hypothesis, which implicates three tyrosine residues in the M2R voltage sensor. The proposed β-arrestin-selective partial D2R agonist, UNC9994, was found to be a weak partial- and almost full agonist at D2R and D3R mediated GIRK activation, respectively. These findings are incongruent with β-arrestin-selectivity and suggest that the promising effects of UNC9994 in animal models of psychosis may be related, at least in part, to involvement of the D3R. Finally, the partial D2R agonist positron emission tomography ligand, SV-III-130, demonstrated an insurmountable, yet competitive, binding mechanism at the D2R. Mutations of residues in a secondary binding pocket, engaging the secondary pharmacophore, abolished the insurmountable binding. Kinetic models incorporating an irreversible, SV-III-130-bound state captured the experimentally observed data. Molecular dynamics simulations suggested that D2R extracellular linkers participate in an induced-fit binding mechanism. In summary, the thesis addresses the mechanism of voltage-dependent agonist-potency at GPCRs and contradicts earlier reports of a β-arrestin-selective action of the experimental antipsychotic, UNC9994, at the D2R. Finally, a two-step induced-fit binding mechanism was demonstrated for the aripiprazole analogue, SV-III-130, at the D2R. The findings may guide further mechanistic investigations and provide insights for the development of novel diagnostic and therapeutic GPCR ligands.