Conformational changes in the GABAA receptor during channel gating and alcohol modulation

The overall goal of this thesis is to structurally characterize the conformational change in the GABAA receptor during alcohol modulation. This thesis investigated which residues are changed in the TM3 region of the GABAA 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 (GABAA) receptor during channel gating and allosteric modulation. In the present study, the surface accessibility and reaction rate of propyl- and hexylmethanethiosulfonate (PMTS and HMTS) to cysteine residues introduced into the third transmembrane segment (TM3) of the GABAA receptor α1 subunit were examined. GABA-induced currents in Xenopus oocytes expressing wild-type and cysteine-mutant GABAA 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 mutants to MTS reagents in the presence of GABA had significant effects on their GABA-induced currents, indicating that the water-cavity around A291C and Y294C residues expanded to F296C and V297C by a structural movement due to GABA-binding. The data show that GABAA receptor is a dynamic protein during alcohol-modulation and channel-gating. Furthermore, mutations (A291W or S270I) of the critical sites of alcohol-binding pocket reduced significantly the accessibility of HMTS to A295C or F296C residues during alcohol modulation, suggesting that the A291 or S270 residues to alcohol-binding pocket are important sites for alcohol-induced conformational changes.