Conformational Changes in the GABAA Receptor During Channel Gating and Alcohol Modulation

iv Acknowledgement I would like to give my deepest thanks to my mentor, Dr. R. Adron Harris. I have been thinking I have a good fortune to meet him. In the future, I want to be a professor in the academic field. He has been a standard model of academic professor to me. He has been always working on something, patient to me for obtaining data, and critical questions and suggestions during lab-meeting. I want to express my affections to my friends Jianwen Liu, Dr Rajani Maiya and Dr. Ingrid Lobo for discussing, arguing, chatting and sharing many things during my doctoral course. Also, I am giving thanks to my friends and colleagues, Dr. their help and encouragements. v I would like to thank Pastor Sungin Park and other members in YeRang Korean Church, Austin, Texas for their help and prayings. I would also like to express gratitudes to my parents, the late grandmother, parents-in-law and other family in Korea for their concern and support. Furthermore, I could not imagine a life without my wife, Seungkyung Yang. Finally, I want to confess special thanks to God for all ways bestowed on me. The overall goal of this thesis is to structurally characterize the conformational change in the GABA A receptor during alcohol modulation. This thesis investigated which residues are changed in the TM3 region of the GABA A receptor α1 subunit during alcohol modulation. The substituted cysteine accessibility method has proven useful for investigating structural changes of the γ-aminobutyric acid type A (GABA A) receptor during channel gating and allosteric modulation. In the present study, the surface accessibility and reaction rate of propyl-and hexyl-methanethiosulfonate (PMTS and HMTS) to cysteine residues introduced into the vii third transmembrane segment (TM3) of the GABA A receptor α1 subunit were examined. GABA-induced currents in Xenopus oocytes expressing wild-type and cysteine-mutant GABA A receptors were recorded before and after application of MTS reagents in the resting, GABA-or alcohol (ethanol or hexanol)-bound states. The results indicate that a water-filled cavity exists around the A291 and Y294 residues of TM3, in agreement with previous results. Furthermore, the data suggest that a conformational change produced by alcohols (200mM ethanol or 0.5mM hexanol) exposure induces the water-cavity surrounding the A291C and Y294C residues to extend deeper, causing the A295C and F296C residues to become accessible to the MTS reagents. In addition, exposure of the A291C, Y294C, F296C and V297C …