Acetylcholine Binding Protein as Model of α4β2 Nicotinic Acetylcholine Receptors - Structural and Functional Characterization

Line Aagot Hede Rohde

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels locatedin the cell membranes of neurons, where they mediates fast synaptic transmission.The nAChRs are involved in several psychiatric and neurodegenerative disorders and in nicotine addiction and thus important drug targets. Even though the receptors have been studied for decades, a lot of pieces are still missing in the puzzle to fully understand the receptors and their mechanism.

The overall goal in this study was to establish a strategy for screening and analysis of the structure-activity relationships of nAChR ligands and apply the strategy for analysis of α4β2 agonists. A series of α4β2 nAChR agonists were selected to investigate the structure-function relationship of the receptor using binding experiments, electrophysiology and x-ray crystallography. For the structural studies, the model protein, acetylcholine binding protein (AChBP), was used as structural surrogate for α4β2 nAChR.

A validation of AChBP as stuctural surrogate was done using binding studies. This showed a correlation in binding affinities between α4β2 nAChR and the acetylcholine binding protein from Lymnaea stagnalis (Ls-AChBP) for the α4β2 agonists and captured the rank order of the compounds. This correlation confirms the applicability of Ls-AChBP being a structural surrogate for the α4β2 nAChR.

Five compounds out of the α4β2 nAChR agonists were selected for functional and structural studies. The compounds were analyzed using voltage clamp electrophysiology on "high-sensitivity" α4β2 nAChR expressed in oocytes from Xenopus laevis. A concatenated construct (linking a α4 and β2 subunit) expressed together with α4 subunits, ensured a homogenous subpopulation of the receptor.

Finally, the agonists were successfully co-crystallized in complex with Ls-AChBP expressed in Sf9 cells using baculo virus expression, which in this study was found superior over the Pichia pastoris expression system when expression Ls-AChBP for crystallization. 

The five α4β2 nAChR agonists all had high potency (20 nM to 80 nM) and efficacies ranging from 20 % to 76 % measured on the high-sensitivity α4β2 nAChRs. Brominated compounds (NS3920 and NS3950) had higher efficacies, 62 % and 76 % respectively, than the corresponding compounds without bromine (41 % for both NS3531 and NS3573). A halogen bond observed in the crystal structures between the bromine and the protein is most likely responsible for the effect on efficacy and emphasizes the importance of the ligand-mediated contact between the subunits.

No variation in closure of the C-loop was observed for the five structures of α4β2 nAChR agonists in complex with Ls-AChBP, despite large variations in efficacy. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong inter-subunit bridge linking the primary and complementary binding interfaces. In addition to the ligands bound in the orthosteric sites, one ligand revealed additional binding sites, being potential allosteric modulatory sites.