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• W89608766 abstract "Abstract The GABA-A receptor (GABA-AR), the GABA-C receptor (GABA-CR) and the glycine receptor (GlyR) are anion-permeable members of the Cys-loop ligand-gated ion channel (LGIC) receptor superfamily, which also includes the nicotinic acetylcholine receptor (nAChR) and the serotonin type-3 receptor (5-HT3R). GABA-ARs and GlyRs play important roles in fast synaptic inhibitory transmission. The aims of my project are to investigate the nature of conformational changes induced by agonists, antagonists and allosteric modulators in GABA-AR α, β and γ subunits, the nature of conformational changes induced by desensitization in the α1 GlyR, and to compare conformational changes induced by glycine and ivermectin in the α1 GlyR using the voltage clamp fluorometry (VCF) method. VCF involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist binding induces large conformational changes in binding domain loops C and F. However, it is controversial as to whether these conformational changes are essential for gating. The first part of project involved using VCF to investigate the roles of loops C and F in gating the α1β2γ2 GABAA-R. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting directly with attached fluorophores at the same site. Here we show that ligands binding to the β2α1 subunit interface GABA binding site produce conformational changes at the adjacent subunit interface. This is most likely due to agonist-induced loop C closure directly altering loop F conformation at the adjacent α1β2 subunit interface. However, as antagonists and agonists produce identical α1 subunit loop F conformational changes, these conformational changes appear unimportant for gating. Finally, we demonstrate that TM2-TM3 loops from adjacent β2 subunits in α1β2 receptors can dimerize via K24’C disulfides in the closed state. This result implies unexpected conformational mobility in this crucial part of the gating machinery. Ligand-gated channels display a phenomenon termed desensitization, which is the progressive fading of ionic current in the prolonged presence of agonist. This process involves a conformational change that disengages the binding site from the channel gate. Despite the physiological and pathological importance of desensitization, little is known about the underlying conformational changes in Cys-loop ion channel receptors. So the second part of my project is we employed VCF to identify conformational changes that occur with a similar time course as the desensitization rate in both slow and fast desensitizing α1 GlyRs. We compared the rate of current and fluorescence changes at nine extracellular sites in both slow and fast desensitizing glycine receptors. As labels attached to three sites at the interface between ligand-binding and transmembrane domains reported VII fluorescence responses that changed in parallel with the current desensitization rate, we concluded that they reported conformational changes associated with desensitization. These labeled sites included A52 in loop 2, Q219C in the pre-M1 and M227C in the TM1 domain. At each site, activation and desensitization were accompanied by physically distinct conformational changes. Since activation is mediated by a specific reorganisation of molecular interactions at this interface, we propose that desensitization is mediated by a distinct set of conformational changes that prevents this reorganisation from correctly occurring, thereby preventing channel activation. As we showed above, our studies indicate several domains are important for activation, but we still do not have direct evidence that structural rearrangements of loop C and loop F may trigger channel activation. In the third part of my project, we hypothesised that if the pore can be induced to open without agonist occupation of the ligand-binding pocket, then conformational changes initiated by movement of the pore helices will propagate in reverse to the extracellular domains. This would reveal those regions near the ligand-binding site that are allosterically linked to the pore gate. Because ivermectin binds in transmembrane domain and directly open the GlyR channel pore, it is an ideal agonist for this purpose. Using VCF, we compared glycine- and ivermectin-induced conformational changes at the different labelled site in the ligand-binding domain. Fluorescentlylabelled residues in binding loops C (N203), D (Q67) and F (V178) revealed fluorescence changes in response to both ivermectin and glycine, suggesting that the conformational changes we observed in this domain must be allosterically linked to conformational changes in the pore-lining helices. We also showed that a double mutant (A288G L233W) GlyR in which ivermectin inhibited GlyR currents also resulted in ivermectin-mediated conformational changes at the glycine-binding site. These results imply a direct allosteric linkage between loop C, loop F and loop D and the transmembrane domain, and suggest that glycine-mediated conformational changes in these loops are important for initiating channel opening. Taken together, the results of this thesis have provided novel insights into the activation and desensitization mechanisms of Cys-loop receptor family members." @default.
• W89608766 created "2016-06-24" @default.
• W89608766 creator A5046225712 @default.
• W89608766 date "2011-08-01" @default.
• W89608766 modified "2023-09-27" @default.
• W89608766 title "Investigating GABA-A and Glycine Receptor Structure and Function" @default.
• W89608766 hasPublicationYear "2011" @default.
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