The habit of smoking tobacco products creates major health problems in the U.S. Because of the significant behavioral and psychological effects of nicotine, this research focuses on nicotinic acetylcholine receptors (nAChRs) from the central nervous system. The goals of this research are (1) to characterize the permeation of calcium ions through neuronal nAChRs, (2) determine how intracellular Ca regulates the activity of neuronal nAChRs, and (3) evaluate how mutations of the pore-lining amino acids change ion permeation, receptor desensitization, and pharmacological properties. The research will focus on the alpha7 neuronal nAChR because they are localized at presynaptic terminals and are sensitive to a-bungarotoxin, suggesting their importance in regulating neurotransmitter release. Several complimentary experimental approaches will be used. AChRs will be expressed in Xenopus oocytes to evaluate channel viability, macroscopic receptor desensitization rates, and pharmacological properties. Alpha7 nAChRs will be reconstituted from oocyte surface membranes into planar lipid bilayer where we can control the ionic and regulatory environment of the channels. Single-channel electrophysiological approaches will be used to study (a) the affinity of permeant ion interactions with the pore, (b) the permeation of Ca through the pore, and (c) the regulation of the channel activity by intracellular Ca. In addition, nAChRs will be synthesized in vitro using a rabbit reticulocyte lysate system supplemented with endoplasmic reticulum microsomes. PAGE, density gradients, and ligand binding techniques will be used to study the biochemical properties of the synthesized AChRs. The in vitro synthesized nAChRs will be reconstituted into planar lipid bilayers to characterize their functional properties in the absence of other membrane proteins that may act as allosteric regulators. The detailed analysis of the biochemical and functional properties of several forms of alpha7 nAChRs will provide valuable information about these nicotinic receptors, the molecular basis of nicotine addiction, and perhaps the basis for the death of cholinergic neurons in Alzheimer's disease.
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