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Related Experiment Videos

Glutamate receptor trafficking: endoplasmic reticulum quality control involves ligand binding and receptor function.

Stephanie J Mah1, Elizabeth Cornell, Nicholas A Mitchell

  • 1Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York 12208, USA.

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
|March 5, 2005
PubMed
Summary

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Specific amino acid mutations in glutamate receptors (GluRs) disrupt ligand binding and cause receptor retention in the endoplasmic reticulum. This confirms key residues are essential for GluR function and trafficking.

Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biochemistry

Background:

  • The agonist-binding site of glutamate receptors (GluRs) involves residues in the S1 and S2 domains.
  • Conserved residues in AMPA receptor binding sites are implicated in ligand interactions.

Purpose of the Study:

  • To confirm if conserved ligand-binding residues in AMPA receptors also function in GluR6 kainate receptors.
  • To investigate the impact of specific amino acid substitutions on GluR6 receptor function and trafficking.

Main Methods:

  • Site-directed mutagenesis of GluR6 at R523, T690, and E738.
  • Expression in human embryonic kidney 293 cells, Western blot analysis.
  • [3H]kainate binding assays and patch-clamp electrophysiology.
  • Immunocytochemistry, cell-surface biotinylation, Endoglycosidase-H digestion, ER marker colocalization.

Related Experiment Videos

  • Immunoprecipitation, native PAGE, and functional studies.

    • Main Results:

    • Mutations at R523, T690, and E738 abolished [3H]kainate binding and functional responses to glutamate or kainate.
    • Mutant receptors were retained intracellularly, failing to traffic to the cell surface.
    • Receptors showed immature glycosylation and ER retention, independent of gross protein folding errors or assembly defects.

    Conclusions:

    • Residues R523, T690, and E738 are directly involved in ligand binding to GluR6 receptors.
    • Nonfunctional GluRs are retained intracellularly by an ER quality control checkpoint, affecting trafficking and function.