Conformational changes in extracellular loop 2 associated with signal transduction in the glycine receptor

• Published in: Journal of neurochemistry 2010-12, Vol.115 (5), p.1245-1255
• Main Authors: Cederholm, Jennie M.E, Absalom, Nathan L, Sugiharto, Silas, Griffith, Renate, Schofield, Peter R, Lewis, Trevor M
• Format: Article
• Language: English
• Subjects: Aminoacid receptors (glycine, glutamate, gaba); Binding sites; Binding Sites - genetics; Biological and medical sciences; Cell Line, Transformed; Cell receptors; Cell structures and functions; Cellular biology; channel gating; Channel opening; Cysteine; Cysteine - genetics; Dose-Response Relationship, Drug; extracellular domain; Fundamental and applied biological sciences. Psychology; Glycine - pharmacology; Glycine receptor; Glycine receptors; Humans; Ion Channel Gating - genetics; Ion channels; Ion channels (ligand-gated); Ligands; loop 2; Membrane Potentials - drug effects; Membrane Potentials - genetics; Methane; Models, Biological; Models, Molecular; Molecular and cellular biology; Molecular Sequence Data; Monoamines receptors (catecholamine, serotonine, histamine, acetylcholine); Mutagenesis; Mutagenesis - genetics; Mutagenesis - physiology; Mutagenesis, Site-Directed - methods; Mutation; Neurochemistry; Neurotransmitters; Patch-Clamp Techniques - methods; Protein Conformation; Protein Structure, Tertiary - genetics; Protein Structure, Tertiary - physiology; Proteins; Receptor mechanisms; Receptors, Glycine - chemistry; Receptors, Glycine - genetics; Receptors, Glycine - metabolism; Signal transduction; Signal Transduction - genetics; Signal Transduction - physiology; Sulfhydryl Reagents - pharmacology; Synaptic transmission; Transfection - methods
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/j.1471-4159.2010.07021.x

J. Neurochem. (2010) 115, 1245-1255. 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 EC₅₀. 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.