Ligand gated ion channels (LGIC) are cell surface protein channels whose actions are gated by various ligands. They belong to the Cys-loop receptor super family and are permeable to cations (nicotinic acetylcholine receptors (nAChRs), serotonin type 3 receptors (5-HT3Rs)) and anions (GABA and glycine receptors). The 5-HT3 type A receptors are homomeric LGIC receptors that play important roles in regulating pain, cognition, and contribute to multiple neurological disorders including depression, epilepsy, anxiety, learning, and attention deficiency disorders. 3-Chlorophenyl guanidine (3-CPG) and its analogs are 5-HT3A receptor agonists and negative allosteric modulators (NAM) of nACh α7 receptors that show efficacy in models of depression and nociception. Using two electrode voltage clamp electrophysiology and mutagenesis, we found that the guanidine moiety of 3-CPG interacts with W183 and the aryl group interacts with Y143 of the 5-HT3AR; thus, these amino acids appear to form a component of the binding site for these compounds. This binding site was further evaluated by various analogs of 3-CPG. Bulky analogs seem to impart steric hindrance in movement of the C-loop region of the binding site and produce antagonist like responses. This supports the hypothesis that the degree of C-loop closure is at least partially responsible for the ability of the compound to either stimulate (agonist) or inhibit the receptor (antagonist). We also found that a small para substitution makes 3-CPG derivatives behave as partial agonists compared to the antagonist properties of bulky 3-CPG derivatives. Restricting the movement of the guanidine moiety converted 3-CPG to an antagonist, possibly due to decreased interaction with W183 or binding in a different orientation. Conformationally constrained analogs, via ring closure, also led to a new group of potential antidepressive ligands called quinazolines which interact with Y143 and Y153. It remains unclear if interaction of quinazolines with the E-loop was direct or just functional. This thesis addresses several primary objectives including the role of C-loop closure in receptor function, the identification of interacting amino acids and structural features of mono-guanidine that mediate partial agonist effects.
|Advisor:||Schulte, Marvin K.|
|Commitee:||Harvison, Peter J., Issa, Amalia M., Tchao, Ruy|
|School:||University of the Sciences in Philadelphia|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 79/04(E), Dissertation Abstracts International|
|Keywords:||3-cholorophenyl guanidine, Cys-loop, Ion channel, Ligand gated ion channel, Serotonin|
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