eprintid: 20543 rev_number: 8 userid: 2543 dir: disk0/00/02/05/43 datestamp: 2014-02-13 20:49:08 lastmod: 2016-11-15 14:17:28 status_changed: 2014-02-13 20:49:08 type: thesis_degree metadata_visibility: show contact_email: ddmowrey@gmail.com item_issues_count: 0 eprint_status: archive creators_name: Mowrey, David creators_email: dam127@pitt.edu creators_id: DAM127 title: Allosteric Modulation of Cys-loop Receptors ispublished: unpub divisions: sch_med_Computational_Systems_Biology full_text_status: public keywords: allostery, anesthetic, Cys-loop receptor, nicotinic acetylcholine receptor, glycine receptor, protein structure, protein dynamics abstract: The Cys-loop receptor superfamily includes the GABAA, GABAC, glycine, and serotonin receptors as well as the nicotinic acetylcholine receptors (nAChRs). Cys-loop receptors are important drug targets for Parkinson’s disease, Alzheimer’s disease, and nicotine addiction. They are also targets of general anesthetics. Understanding the mechanisms of allosteric modulation for Cys-loop receptors has implications for the design of novel therapeutics for the treatment of pain, inflammation, and neurological disease. I employed a combination of computational and experimental approaches to understand allosteric modulation of these receptors. Four major contributions resulted from my graduate research: 1) NMR structures of the transmembrane (TM) domains of the α7 and α4β2 nAChRs as well as the α1 glycine receptor were resolved to provide a scaffold for rationalizing drug-binding sites and drug action. While all structures revealed the typical four-helix bundle, differences were observed which could affect drug binding and allosteric modulation. 2) Computational and experimental results showed that the general volatile anesthetic halothane bound to both α7 and α4β2 nAChRs, despite different sensitivities of these receptors to halothane. NMR data also revealed that volatile anesthetics halothane and isoflurane bound to the EC end of the β2 TM domain, but only at the IC end of the α7 TM domain. 3) We not only revealed the drug binding sites but also determined that the binding site at the EC end of the TM domain is functionally relevant. 4) Several factors critical to allosteric modulation in Cys-loop receptors were identified. Applying the perturbation-based Markovian transmission model to GLIC, we identified signaling pathways of agonist-induced channel gating. Using NMR, we identified a link between protein dynamics changes and allosteric modulation. Molecular dynamics simulations suggested that asymmetric binding of the anesthetic propofol to GLIC facilitated the transition from an open- to a closed-channel structure. The study provides evidence that ligand-induced asymmetry facilitates conformational transitions. date: 2014-02-13 date_type: published pages: 235 institution: University of Pittsburgh refereed: TRUE etdcommittee_type: committee_chair etdcommittee_type: committee_member etdcommittee_type: committee_member etdcommittee_type: thesis_advisor etdcommittee_name: Bahar, Ivet etdcommittee_name: Zuckerman, Daniel etdcommittee_name: Kurnikova, Maria etdcommittee_name: Tang, Pei etdcommittee_email: bahar@pitt.edu etdcommittee_email: ddmmzz@pitt.edu etdcommittee_email: kurnikova@cmu.edu etdcommittee_email: tangp@anes.upmc.edu etdcommittee_id: BAHAR etdcommittee_id: DDMMZZ etdcommittee_id: etdcommittee_id: etd_defense_date: 2014-01-30 etd_approval_date: 2014-02-13 etd_submission_date: 2014-02-10 etd_release_date: 2014-02-13 etd_access_restriction: immediate etd_patent_pending: FALSE thesis_type: dissertation degree: PhD citation: Mowrey, David (2014) Allosteric Modulation of Cys-loop Receptors. Doctoral Dissertation, University of Pittsburgh. (Unpublished) document_url: http://d-scholarship-dev.library.pitt.edu/20543/1/Mowrey_Thesis_02112014.pdf