Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

J Neurochem. 2009 Mar;108(6):1585-94. doi: 10.1111/j.1471-4159.2009.05930.x. Epub 2009 Jan 24.

Abstract

Ligand binding to Cys-loop receptors produces either global conformational changes that lead to activation or local conformational changes that do not. We found that the fluorescence of a fluorophore tethered to R271C in the extracellular M2 region of the alpha1 glycine receptor increases during glycine activation but not during ivermectin activation. This prompted the hypothesis that this signal reports a glycine-mediated conformational change not essential for activation. We tested this by investigating whether the fluorescence signal depended on whether the fluorophore was attached to a glycine-free or a glycine-bound subunit. Agonist-free subunits were created by incorporating T204A and R65K mutations, which disrupted glycine binding to both (+) and (-) subunit interfaces. In heteromeric receptors comprising wild-type and R65K,T204A,R271C triple-mutant subunits, the fluorescence response exhibited a drastically reduced glycine sensitivity relative to the current response. Two conclusions can be drawn from this. First, because the labeled glycine-free subunits were activated by glycine binding to neighboring wild-type subunits, our results provide evidence for a cooperative activation mechanism. However, because the fluorescent label on glycine-free subunits does not reflect movements at the channel gate, we conclude that glycine binding also produces a local non-concerted conformational change that is not essential for receptor activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation
  • Amino Acid Sequence
  • Amino Acids / genetics
  • Animals
  • Binding Sites / genetics
  • Biophysical Phenomena
  • Electric Stimulation
  • Female
  • Gene Expression / genetics
  • Humans
  • Membrane Potentials / drug effects
  • Membrane Potentials / physiology
  • Microinjections / methods
  • Models, Biological
  • Mutation
  • Oocytes
  • Patch-Clamp Techniques
  • Protein Binding / genetics
  • Protein Subunits / genetics
  • Protein Subunits / metabolism*
  • RNA, Messenger / genetics
  • Receptors, Glycine / agonists
  • Receptors, Glycine / chemistry*
  • Receptors, Glycine / genetics
  • Receptors, Glycine / metabolism*
  • Xenopus laevis

Substances

  • Amino Acids
  • Protein Subunits
  • RNA, Messenger
  • Receptors, Glycine