UC San Diego

Receptors play a vital role in the transduction of cell to cell signaling. They are important proteins with recognition capacity in signal transduction, particularly in neurons. Cholinergic neurons in the central and peripheral nervous systems are defined by storage, release, and stimulation of the neurotransmitter acetylcholine. At the synaptic cleft, the release of acetylcholine from presynaptic neurons triggers activation through channel opening of the nicotinic acetylcholine receptors (nAChRs) in the postsynaptic membrane. Previous research implicates the nAChRs in diseases including schizophrenia, drug addiction (specifically nicotine addiction), Alzheimer's and Parkinson's diseases. The work presented here uses a comprehensive approach including structural studies, protein engineering, functional studies, and protein characterization to gain insight into the distinguishing characteristics that may contribute to ligand selectivity and structural components that contribute to receptor function. The main aim of this work was to create a soluble template of the ligand binding domain of human alpha-7-nAChRs to understand pharmacological selectivity, while also creating an efficient tool for therapeutic development. In order to validate this study, methods to test the functional activity of nAChRs were developed and in the process a novel calcium FRET sensor based assay was developed as a cost-effective tool for drug screening and functional ligand characterization on nAChRs and other ligand-gated, calcium-permeable, ion channels. To process the information generated by the developed assay, a novel data storage system in the form of a database was created. The creation of a soluble template fully identical to the ligand binding domain of human alpha-7-nAChR was not successful, but the protein generated served to identify structurally a glycosylation site on the receptor as well as proved to be a better binding homologue for alpha-7- nAChRs than any other proteins currently available. Overall, the mutational conversion of a soluble homologue to the human alpha-7-nAChR gave insight into the structural understanding of the extracellular domain of nAChRs. This template provides a better model to create lead compounds as therapeutics for diseases associated with the nAChRs