title: Probing the Structural Mechanism of Partial Agonism in Glycine Receptors Using the Fluorescent Artificial Amino Acid, ANAP.
abstract: The efficacy of an agonist at a pentameric ligand-gated ion channel is determined by the rate at which it induces a conformational change from the resting closed state to a preopen ("flip") state. If the ability of an agonist to promote this isomerization is sufficiently low, then it becomes a partial agonist. As partial agonists at pentameric ligand-gated ion channels show considerable promise as therapeutics, understanding the structural basis of the resting-flip-state isomerization may provide insight into therapeutic design. Accordingly, we sought to identify structural correlates of the resting-flip conformational change in the glycine receptor chloride channel. We used nonsense suppression to introduce the small, fluorescent amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP), into specific sites in the extracellular and transmembrane domains. Then, under voltage-clamp conditions in Xenopus oocytes, we simultaneously quantified current and fluorescence responses induced by structurally similar agonists with high, medium, and low efficacies (glycine, β-alanine, and taurine, respectively). Analyzing results from nine ANAP-incorporated sites, we show that glycine receptor activation by agonists with graded efficacies manifests structurally as correspondingly graded movements of the β1-β2 loop, the β8-β9 loop, and the Cys-loop from the extracellular domain and the TM2-TM3 linker in the transmembrane domain. We infer that the resting-flip transition involves an efficacy-dependent molecular reorganization at the extracellular-transmembrane domain interface that primes receptors for efficacious opening.
authors.affiliation: Queensland Brain Institute, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Ming S
authors.initials: MS
authors.lastname: Soh
authors.affiliation: Queensland Brain Institute, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Argel
authors.initials: A
authors.lastname: Estrada-Mondragon
authors.affiliation: Queensland Brain Institute, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Nela
authors.initials: N
authors.lastname: Durisic
authors.affiliation: Queensland Brain Institute, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Angelo
authors.initials: A
authors.lastname: Keramidas
authors.affiliation: Queensland Brain Institute, The University of Queensland , Brisbane, Queensland 4072, Australia.|School of Biomedical Sciences, The University of Queensland , Brisbane, Queensland 4072, Australia.
authors.forename: Joseph W
authors.identifier: 0000-0002-1227-2252
authors.initials: JW
authors.lastname: Lynch
mesh_terms: D000596:Amino Acids / Q000494:pharmacology*; D011487:Protein Conformation; D018009:Receptors, Glycine / Q000819:agonists* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D000596:Amino Acids; D018009:Receptors, Glycine