title: Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain.
abstract: Understanding the activation mechanism of Cys loop ion channel receptors is key to understanding their physiological and pharmacological properties under normal and pathological conditions. The ligand-binding domains of these receptors comprise inner and outer beta-sheets and structural studies indicate that channel opening is accompanied by conformational rearrangements in both beta-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on alpha1 glycine receptors to compare changes mediated by the agonist, glycine, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner beta-sheet, we labeled residues in loop 2 and in binding domain loops D and E. At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes in the inner beta-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop receptors.
authors.affiliation: Queensland Brain Institute and School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Pless
authors.forename: Joseph W
authors.initials: JW
authors.lastname: Lynch
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D018009:Receptors, Glycine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D018076:DNA, Complementary; D008024:Ligands; D018009:Receptors, Glycine; D005998:Glycine