Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain
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Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain. / Pless, Stephan Alexander; Lynch, Joseph W.
In: The Journal of Biological Chemistry, Vol. 284, No. 23, 05.06.2009, p. 15847-56.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain
AU - Pless, Stephan Alexander
AU - Lynch, Joseph W
PY - 2009/6/5
Y1 - 2009/6/5
N2 - 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.
AB - 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.
KW - Amino Acid Substitution
KW - Animals
KW - Binding Sites
KW - Cloning, Molecular
KW - DNA, Complementary
KW - Female
KW - Glycine
KW - Humans
KW - Kinetics
KW - Ligands
KW - Models, Molecular
KW - Oocytes
KW - Patch-Clamp Techniques
KW - Protein Conformation
KW - Receptors, Glycine
KW - Xenopus laevis
U2 - 10.1074/jbc.M809343200
DO - 10.1074/jbc.M809343200
M3 - Journal article
C2 - 19286654
VL - 284
SP - 15847
EP - 15856
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 23
ER -
ID: 122597818