Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain

J Biol Chem. 2009 Jun 5;284(23):15847-56. doi: 10.1074/jbc.M809343200. Epub 2009 Mar 13.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Binding Sites
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • Female
  • Glycine / metabolism
  • Humans
  • Kinetics
  • Ligands
  • Models, Molecular
  • Oocytes / physiology
  • Patch-Clamp Techniques
  • Protein Conformation
  • Receptors, Glycine / chemistry*
  • Receptors, Glycine / genetics
  • Receptors, Glycine / metabolism*
  • Xenopus laevis

Substances

  • DNA, Complementary
  • Ligands
  • Receptors, Glycine
  • glycine receptor alpha1
  • Glycine