The goals of this study are to discover the cytoplasmic interactome of the a7 nicotinic acetylcholine receptor (nAChR) subtype and how this interactome is altered by chronic nicotine exposure. Although many aspects of the nAChR have been well studied, the intracellular protein interactions of these receptors are still unknown. Learning about these cytoplasmic protein interactions would generate a better understanding of this receptor's physiology and pathology. The a7 nAChR subtype has been linked to many pathological conditions including seizure disorders, Alzheimer's disease, diffuse lewy body dementia, and nicotine addiction. Understanding how the native a7 nAChR interactome is influenced by nicotine could identify drug targets for smoking cessation therapy as well as provide insight into nicotine's beneficial aspects such as cognitive enhancement and protection against Alzheimer's disease. The numerous pathological conditions linked to chronic smoking are frequently insidioust and over time drastically reduce a patient's quality of life. Smoking cessation is key to preventing these pathologies 'from becoming fulminant.
The specific aims of this project employ the isolation of receptors by a specific toxin and cutting edge mass spectrometry to generate an in vivo picture of the a7 nAChR interactome. The experimental models will be wildtype mice and a7 nAChR knockout mice will function as a control. The first specific aim of this project is to identify the native interactome of a7 nAChRs. Whole brains extracted from wildtype and a7 knockout mice will be homogenized. a7 nAChR subtypes will be purified from the homogenate with a-bungarotoxin (Bgtx) beads. The a7 nAChR and any interacting proteins bound to it can be released from the Bgtx by carbachol elution. The proteins derived from Bgtx isolation will be degraded by proteolytic enzymes and analyzed by mass spectrometers sensitive to molecules at attomolar concentrations. The mass spectrometers available to us are extremely specialized instruments with the highest mass accuracy and resolution available. These instruments can also perform tandem mass spectrometry, a technique that generates peptide amino acid sequence information to facilitate accurate protein identification. Tandem mass spectrometry can also be used to asses the phosphorylation status of peptides. The second specific aim will be to determine how the a7 nAChR interactome is influenced by chronic nicotine exposure. The protocol used in specific aim 1 will be repeated on wildtype and a7 nAChR knockouts mice that have been exposed to nicotine for 5 months. The interactome data gathered from the nicotine exposed mice will be compared to the unexposed mice to determine if any difference exists.