AFM observation of single, functioning ionotropic glutamate receptors reconstituted in lipid bilayers

Nahoko Kasai, Chandra S. Ramanujan, Ichiro Fujimoto, Akiyoshi Shimada, John F. Ryan, Keiichi Torimitsu

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Background: Ionotropic glutamate receptors (iGluRs) are responsible for extracellular signaling in the central nervous system. However, the relationship between the overall structure of the protein and its function has yet to be resolved. Atomic force microscopy (AFM) is an important technique that allows nano-scale imaging in liquid. In the present work we have succeeded in imaging by AFM of the external features of the most common iGluR, AMPA-R (Α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor), in a physiological environment. Methods: Homomeric GluR3 receptors were over-expressed in insect cells, purified and reconstituted into lipid membranes. AFM images were obtained in a buffer from membranes immobilized on a mica substrate. Results: Using Au nanoparticle-conjugated antibodies, we show that proteins reconstitute predominantly with the N-terminal domain uppermost on the membrane. A tetrameric receptor structure is clearly observed, but it displays considerable heterogeneity, and the dimensions differ considerably from cryo-electron microscopy measurements. Conclusions: Our results indicate that the extracellular domains of AMPA-R are highly flexible in a physiological environment. General significance: AFM allows us to observe the protein surface structure, suggesting the possibility of visualizing real time conformational changes of a functioning protein. This knowledge may be useful for neuroscience as well as in pharmaceutical applications.

Original languageEnglish
Pages (from-to)655-661
Number of pages7
JournalBiochimica et Biophysica Acta - General Subjects
Volume1800
Issue number7
DOIs
Publication statusPublished - 2010 Jul 1
Externally publishedYes

Keywords

  • AMPA receptor
  • Atomic force microscopy
  • Membrane protein
  • Neuroreceptor

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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