title: Conformational changes in extracellular loop 2 associated with signal transduction in the glycine receptor.
abstract: Ligand-gated ion channels efficiently couple neurotransmitter binding to the opening of an intrinsic ion channel to generate the post-synaptic potentials that are characteristic of fast synaptic transmission. In the Cys-loop family of ligand-gated ion channels, the ligand-binding site is approximately 60 Å above the channel gate. Structural modelling of related proteins and mutagenesis studies led to the hypothesis that loops 2 and 7 of the extracellular domain may couple ligand binding to receptor activation. Mutating loop 2 residues of the glycine receptor to cysteine reveals an alternating pattern of effect upon receptor function. Mutations A52C, T54C and M56C produced a threefold right-shift in EC(50) . In contrast, a 30-fold right-shift was seen for mutations E53C, T55C and D57C. Loop 2 conformational changes associated with ligand binding were assessed by measuring the rate of covalent modification of substituted cysteines by charged methane thiosulfonate reagents. We show for the first time state-dependent differences in the rate of reaction. A52C and T54C are more accessible in the resting state and M56C is more accessible in the activated state. These results demonstrate that loop 2 does undergo a conformational change as part of the mechanism that couples ligand binding to channel opening.
authors.affiliation: School of Medical Sciences, The University of New South Wales, Sydney, New South Wales, Australia.
authors.forename: Jennie M E
authors.initials: JM
authors.lastname: Cederholm
authors.forename: Nathan L
authors.initials: NL
authors.lastname: Absalom
authors.forename: Silas
authors.initials: S
authors.lastname: Sugiharto
authors.forename: Renate
authors.initials: R
authors.lastname: Griffith
authors.forename: Peter R
authors.initials: PR
authors.lastname: Schofield
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Lewis
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002461:Cell Line, Transformed; D003545:Cysteine / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005998:Glycine / Q000494:pharmacology; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics; D008024:Ligands; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics / Q000502:physiology; D016297:Mutagenesis, Site-Directed / Q000379:methods; D018408:Patch-Clamp Techniques / Q000379:methods; D011487:Protein Conformation; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D018009:Receptors, Glycine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014162:Transfection / Q000379:methods
chemical_list: D008024:Ligands; D018009:Receptors, Glycine; D013439:Sulfhydryl Reagents; D003545:Cysteine; D005998:Glycine