Expression of Water-soluble, Ligand-binding Concatameric Extracellular Domains of the Human Neuronal Nicotinic Receptor α4 and β2 Subunits in the Yeast Pichia pastoris GLYCOSYLATION IS NOT REQUIRED FOR LIGAND BINDING

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that are responsible for cell communication via the neurotransmitter acetylcholine. The predominant nAChR subtype in the mammalian brain with a high affinity for nicotine is composed of alpha 4 and beta 2 subunits. This nAChR subtype is responsible for addiction to nicotine and is thought to be implicated in Alzheimer and Parkinson diseases and therefore presents an important target for drug design. In an effort to obtain water-soluble, ligand-binding domains of the human alpha 4 beta 2 nAChR for structural studies, we expressed the extracellular domains (ECDs) of these subunits in the eukaryotic expression system Pichia pastoris. The wild-type ECDs and their mutants containing the more hydrophilic Cys-loop from the snail acetylcholine- binding protein (individually expressed or coexpressed) did not demonstrate any specific interaction with ligands. We then linked the mutated ECDs with the 24-amino acid peptide (AGS)(8) and observed that the beta 2-24-alpha 4 ECD concatamer, but not the alpha 4-24-beta 2 one, exhibited very satisfactory water solubility and ligand binding properties. The I-125-epibatidine and [H-3] nicotine bound to beta 2-24-alpha 4 with dissociation constants (K-d) of 0.38 and 19 nM, respectively, close to the published values for the intact alpha 4 beta 2 AChR. In addition, I-125-epibatidine binding was blocked by nicotine, cytisine, acetylcholine, and carbamylcholine with inhibition constants (K-i) of 20.64, 3.24, 242, and 2,254 nM, respectively. Interestingly, deglycosylation of the concatamer did not affect its ligand binding properties. Furthermore, the deglycosylated beta 2-24-alpha 4 ECD existed mainly in monomeric form, thus forming an appropriate material for structural studies and possibly for pharmacological evaluation of novel alpha 4 beta 2 nAChR-specific agonists.