First Images of a Glutamate Receptor Ion Channel:  Oligomeric State and Molecular Dimensions of GluRB Homomers

Abstract

We have expressed, purified, and characterized glutamate receptor ion channels (GluR) assembled as homomers of the subunit GluRB. For the first time, single-milligram quantities of biochemically homogeneous GluR have been obtained. The protein exhibits the expected pharmacological profile and a high specific activity for ligand binding. Density-gradient centrifugation reveals a uniform oligomeric assembly and a molecular mass suggesting that the channel is a tetramer. On the basis of electron microscopic images, the receptor appears to form an elongated structure that is visualized in several orientations. The molecular dimensions of the molecule are approximately 11 x 14 x 17 nm, and solvent-accessible features can be seen; these may contribute to formation of the ion-conducting pathway of the channel. The channel dimensions are consistent with an overall 2-fold symmetric assembly, suggesting that the tetrameric receptor may be a dimer of dimers.

The Three-dimensional Structure of an Ionotropic Glutamate Receptor Reveals a Dimer-of-dimers Assembly

“…Although there have been reports that GluRB homomers can be retained in the endoplasmic reticulum (ER) as dimers in human tissue, 17,18 hydrodynamic studies 15 and contouring experiments with the EM model confirm a tetrameric assembly of the insect-cell expressed homomers described here. Moreover, similar hydrodynamic behavior 10 and EM projection images (not shown) …”

“…Based on our ability to purify tetrameric GluRB homomers, 15 we present here the first three-dimensional (3D) structure of an AMPA receptor, obtained by electron microscopy of negatively stained specimens and single-particle image analysis. The molecular envelope provides direct structural evidence for the overall 2-fold symmetry of the channel and suggests a probable orientation relative to the lipid bilayer.…”

Molecular aspects of glutamate dysregulation: implications for schizophrenia and its treatment

“…Ionotropic glutamate receptors-Ionotropic glutamate receptors are named after their distinguishing ligands and classified into NMDA receptors, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, and kainate receptors (Hollmann & Heinemann, 1994;Ozawa et al, 1998). Molecular cloning has shown that all ionotropic glutamate receptors consist of four subunits, which form a functional receptor with an ion permeable pore (Clements & Westbrook, 1991;Rosenmund et al, 1998;Safferling et al, 2001) (Figs. 1A, 1B).…”

Nano-biointerfaces for Detection and Control of Biological Information

“…14,15 Cryo-electron microscopy (cryo-EM) of single intact iGluRs (at 2 nm resolution) also indicates that the receptor may have a dimer-of-dimers configuration; it measures 17 © 11 © 14 nm, with the largest dimension corresponding to longitudinal extent along the two-fold symmetry axis. 16,17 Another group found that the iGluR conformation changed with stimulation, and that the orientation of NTDs is also changed substantially by desensitization. 18 Although the above methods can be used to examine detailed iGluR structural information, they do not allow structural measurements under physiological conditions.…”

“…16,17 Nakagawa et al studied heterotetrameric iGluR purified from rat brains and obtained quite different results: two distinct structural types were identified -one with an extended extracellular configuration measuring 11 nm above the TMD, the second being a distinctly splayed structure in which the NTD dimers were well separated: considerable heterogeneity was observed in the latter case, and the height of the extracellular domains was as small as 4-5 nm, essentially the height of the LBD. 18 The small height measured with AFM may be due in part to a tip-protein interaction causing the protein to be driven deeper into the membrane.…”