Central to synaptic transmission are the family of ionotropic neurotransmitter receptors, which are responsible for the rapid responses to neurotransmitters in nerve and muscle. In this proposal, a class of receptors that are members of this family, the neuronal nicotinic acetylcholine receptors (AChRS), will be studied. As the site where nicotine binds in the brain, these receptors are responsible for nicotine addiction and may also play a role in neurodegenerative diseases such as Alzheimers disease. Neuronal AChRs are composed of a number of subtypes as classified by their diverse pharmacology and distribution. Further evidence of the diversity has been the cloning of a large number neuronal AChR subunit isoforms. While neuronal AChRs are found throughout the central and peripheral nervous system, neither the subunit composition nor the function of single nAChR subtype is known. The neuronal alpha-bungarotoxin binding receptor (BuTxR), a neuronal AChR subtype, has been selected for study because recent studies suggest that these receptors are a Ca2+ influx pathway, which at presynaptic sites stimulates neurotransmitter release and underlie nicotine addiction. The first objective is to examine whether BuTxRs are homomers composed only of alpha7 subunits or heteromers composed of subunits in addition to alpha. In addition, the number of BuTx sites per BuTxR will be determined. The next objective is to begin to define the function of BuTxRs. Towards this objective, we will test how permeable BuTxRs are to Ca+2 and if Ca2+ entering through BuTxRs contributes to secretion in PC12 cells. We will also address why so few BuTxRs functional in our undifferentiated PC12 cells and test the hypothesis that the number of functional BuTxRs are regulated. The last part of the grant is concerned with questions about BuTxR expression. Heterologous expression of alpha7 homomers in mammalian cells is poor to nonexistent. In contrast, alpha7/5HT3 chimeric subunits readily express as homomers in mammalian cells. Using alpha7/5HT3 chimeric subunits, we will determine alpha7 subunit regions that prevent homomer formation. In PC12 cells,different subunit processing at folding events will be characterized and their effect on BuTxR expression tested. Finally, we will further characterize intracellular BuTxR subunit pools, which appear to be precursors of the cell-surface BuTxRs.
Showing the most recent 10 out of 14 publications
