Noetzel, Meredith2009-03-062009-03-062009-01https://hdl.handle.net/11299/48064University of Minnesota Ph.D. dissertation. January 2009. Major: Neuroscience. Advisor: Dr. Esam El-Fakahany. 1 computer file (PDF); xiii, 248 pages, appendix A.Muscarinic acetylcholine receptors are part of the G protein-coupled superfamily of receptors. There are five subtypes of muscarinic receptors (M1-M5). These receptors are therapeutic targets for in a number of diseases, including Alzheimer's disease and schizophrenia. The orthosteric (primary) binding domain of muscarinic receptors is highly conserved across subtypes making it difficult to develop agonists or antagonists that bind with selectivity at a particular subtype. However, there are allosteric (secondary) binding sites on muscarinic receptors that are thought to be less well conserved between subtypes, which may lead to the development of drugs that bind in a subtype-selective manner. One potential drug of interest is xanomeline. Previous research has found that it binds in a unique manner to muscarinic receptors; it binds in a reversible manner at the orthosteric site and in a wash-resistant manner at an allosteric site. Furthermore, xanomeline is thought to be an M1/M4 selective agonist. The goal of the current research was to characterize the effects of prolonged exposure of the various receptor subtypes to xanomeline and determine the mechanisms through which these effects occur. To do this Chinese hamster ovary cells expressing the various individual muscarinic receptors were exposed to xanomeline for brief and long-term time periods and radioligand binding and functional assays were performed. Xanomeline was shown to bind in a reversible and wash-resistant manner at the M1-M4 receptor subtypes. Although the effects of acute xanomeline exposure were similar across subtypes, long-term treatment with xanomeline resulted in differential effects among subtypes. More detailed experiments were conducted at the M1 and M3 receptor subtypes to elucidate the mechanisms of the long-term effects of xanomeline. Wash-resistant xanomeline binding was able to modulate the functional properties of the receptor. The consequences of the long-term effects of wash-resistant xanomeline binding are dependent on activation of the receptor through the orthosteric binding site. It appears that these effects are a result of receptor internalization and allosteric modulation of the receptor. Thus, my research demonstrates that it is important to determine both the acute and long-term drug interactions with the receptor as part of the drug development process.en-USMuscarinic ReceptorsPI HydrolysisRadioligand BindingXanomelineNeuroscienceThesis or Dissertation