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Description
Drug addiction and neurological disorders have long been linked to nicotinic acetylcholine receptors (nAChRs). The nAChRs are implicated in diseases including schizophrenia, drug addiction (specifically nicotine addiction), Alzheimer’s, and Parkinson’s diseases. They are composed of an extracellular domain (ECD), which contains the ligand binding site, a transmembrane domain which contains the ion-channel gate and forms the pore, and an intracellular domain (ICD) which is generally thought to play a role in receptor regulation and trafficking. Very little is currently understood about the unresolved ICD. Among the 16 nAChR subunits that yield a vast array of stoichiometric subtypes, the mammalian neuronal nAChRs contain two predominant compositions: the homomeric α7-nAChR and the heteromeric α4β2*-nAChR, where * denotes the possibility of an additional accessory subunit. Although the general pentameric arrangement is α-β-α-β-α/β, a vast stoichiometric variability exists and leads to unique localization, for which the mechanism remains unclear, of the specific nAChR subtype variants. For α4β2*-nAChRs, a distinction in the localization between (α4)3(β2)2, (α4)2(β2)3, and (α4)2(β2)2* subtypes is not possible using current biochemical techniques. Unfortunately, many years of pharmacological study also have not yielded robust therapies for the maledictions caused by a dysfunction of nAChRs. There is still a profound lack of information about the ICD and the regulation/trafficking of these receptors. Targeting their regulatory proteins may combat neurodegenerative disorders better than targeting the receptors themselves. Therefore this proposal aims to study and elucidate the mechanisms of regulation of nAChRs.
Summary of project results
The project aimed to study the mechanism of functional regulation on nicotinic acetylcholine receptors through their domain located inside the cell, with the hope that targeting the regulatory systems of these receptors may form the basis for developing new therapeutics in the future against neurological disorders such as Alzheimer’s disease, schizophrenia, and Parkinson’s disease, in addition to drug addiction. These disorders have long been linked to the cholinergic system, and specifically nicotinic acetylcholine receptors, and this family of receptors is involved in all major functions of the central nervous system. Understanding the mechanisms of regulation of nicotinic acetylcholine receptors, an aspect that has thus far remained elusive, is the key to developing efficient therapeutics for neurological disorders.
The project aimed to develop small single-domain antibodies, nanobodies, against specific receptor stoichiometries to answer questions about regulatory differences and develop an understanding of regulatory mechanisms. In addition, the created nanobodies, useful tools for future projects, were planned to be used to properly localize given stoichiometries in the brain, creating a translational bridge between the biochemical mechanisms of regulation to the neurobiological system composition.
The project created proteins composed of an intracellular domain of nicotinic acetylcholine receptors linked to a soluble homologous protein. Two compositions were targeted, the homomeric α7- and heteromeric α4β2-nicotinic acetylcholine receptors. Chimeric proteins created in this project were produced with an α7-chimera as a soluble homomer and an α4-chimera co-expressed with a β2-chimera to produce a heteromeric composition. These chimeras were created with the hope of isolating soluble proteins which interact with the intracellular domain of these nicotinic acetylcholine receptor subunits. There were technical issues with using these soluble linked-proteins as tools to identify proteins which interact with the intracellular domain, thus the project pivoted to focus on the single-domain antibodies. The project created a synthetic nanobody library that may be used to screen and isolate nanobodies against any target. This library was used to develop nanobodies for the most prolifically expressed heteromeric nicotinic acetylcholine receptor, the α4β2-subtype, with specific focus on the α4 subunit and especially the α4/α4-subunit interface, since nanobodies for the predominant homomeric α7-nicotinic acetylcholine receptor had already been published.
The project launched the career of a young principal investigator. It also instilled a passion for science to the associated female master’s student, whom as a result decided to pursue her PhD and continue in an academic scientific career. This resulted in her acceptance into the program and multiple applications to different scholarships/grants to pursue her ideas.
In addition, the project created the synthetic nanobody library which is beneficial to the project’s promoting institution as other investigators may now effortlessly collaborate with the principal investigator’s group to develop other nanobodies of interest. This benefit also extends to external investigators which wish to collaborate. The impact of the created nanobodies is still being assessed with further grant applications being submitted to enable their characterization as tools for future study of nicotinic acetylcholine receptors, useful to the entire pentameric ligand-gated ion channel family field of study.