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The biochemistry, ultrastructure, and subunit assembly mechanism of AMPA receptors.

Nakagawa T - Mol. Neurobiol. (2010)

Bottom Line: The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex.The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing.Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. nakagawa@ucsd.edu

ABSTRACT
The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity.

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Different conformations of native AMPA-R. The AMPA-R particles adopt different conformations in the presence of various drugs. Unliganded, NBQX (30 µM) treated, and glutamate (1 mM) + CTZ (330 µM) treated particles, respectively, adopt conformation shown in left. In this conformation, the two NTD dimers are close to each other. The structural difference between the three conditions was not detectable at the resolution of the study. Right box—particles that are treated with glutamate (1 mM) adopt conformations in which the two NTD dimers are separated. Each NTD dimer is indicated by the short arrows. The images were reproduced from [66]
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Fig3: Different conformations of native AMPA-R. The AMPA-R particles adopt different conformations in the presence of various drugs. Unliganded, NBQX (30 µM) treated, and glutamate (1 mM) + CTZ (330 µM) treated particles, respectively, adopt conformation shown in left. In this conformation, the two NTD dimers are close to each other. The structural difference between the three conditions was not detectable at the resolution of the study. Right box—particles that are treated with glutamate (1 mM) adopt conformations in which the two NTD dimers are separated. Each NTD dimer is indicated by the short arrows. The images were reproduced from [66]

Mentions: The first insights into the ultrastructure of the intact full-length AMPA-Rs were provided by electron microscopy [66–69]. In 2001, the first negative stain single particle EM projection structures of the GluA2 tetramer expressed and purified from Sf9 cells were reported [68]. This study provided the overall dimensions of the particles (approximately 11 × 14 × 17 nm) and the pseudo-2-fold symmetry of the receptor. The 3D EM density map of GluA2 tetramer expressed and purified from Sf9 cells demonstrated overall 2-fold symmetry of the receptor but consistency was not described with the known crystal structures [69]. The possible discrepancy between the EM structure and the crystal structures of the related domains prompted the need to accumulate more data in the field to understand the structure of the intact full-length AMPA-Rs. In 2005, a method to purify homogeneous particles of native AMPA-Rs from rat brain was developed and the purified receptors were further analyzed using single particle EM [66]. At the resolution of about 30 Å, the cryo-negative stained single particle 3D EM structures of the native AMPA-R containing auxiliary subunits stargazin/TARP and the native AMPA-Rs without the stargazin/TARP were reported [66, 67] (Fig. 2). The EM structure of native AMPA-Rs purified from rat brain [66, 67] looked very different from those of GluR2 homotetramers expressed and purified from Sf9 insect cells [69, 70]. Of particular importance, however, is that the sizes and shapes of the individual globular densities observed in the EM density map of the native AMPA-Rs were consistent with those of the crystal structures of the GluA2 LBD and GluA2 NTD [66, 71]. The structure also was consistent with the proposed dimer-of-dimers assembly of the subunits. The electron microscopy studies of native AMPA-Rs also revealed the global conformational changes of the receptor upon desensitization [66, 67] (Fig. 3). The detail of this study is discussed in the later section.Fig. 2


The biochemistry, ultrastructure, and subunit assembly mechanism of AMPA receptors.

Nakagawa T - Mol. Neurobiol. (2010)

Different conformations of native AMPA-R. The AMPA-R particles adopt different conformations in the presence of various drugs. Unliganded, NBQX (30 µM) treated, and glutamate (1 mM) + CTZ (330 µM) treated particles, respectively, adopt conformation shown in left. In this conformation, the two NTD dimers are close to each other. The structural difference between the three conditions was not detectable at the resolution of the study. Right box—particles that are treated with glutamate (1 mM) adopt conformations in which the two NTD dimers are separated. Each NTD dimer is indicated by the short arrows. The images were reproduced from [66]
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2992128&req=5

Fig3: Different conformations of native AMPA-R. The AMPA-R particles adopt different conformations in the presence of various drugs. Unliganded, NBQX (30 µM) treated, and glutamate (1 mM) + CTZ (330 µM) treated particles, respectively, adopt conformation shown in left. In this conformation, the two NTD dimers are close to each other. The structural difference between the three conditions was not detectable at the resolution of the study. Right box—particles that are treated with glutamate (1 mM) adopt conformations in which the two NTD dimers are separated. Each NTD dimer is indicated by the short arrows. The images were reproduced from [66]
Mentions: The first insights into the ultrastructure of the intact full-length AMPA-Rs were provided by electron microscopy [66–69]. In 2001, the first negative stain single particle EM projection structures of the GluA2 tetramer expressed and purified from Sf9 cells were reported [68]. This study provided the overall dimensions of the particles (approximately 11 × 14 × 17 nm) and the pseudo-2-fold symmetry of the receptor. The 3D EM density map of GluA2 tetramer expressed and purified from Sf9 cells demonstrated overall 2-fold symmetry of the receptor but consistency was not described with the known crystal structures [69]. The possible discrepancy between the EM structure and the crystal structures of the related domains prompted the need to accumulate more data in the field to understand the structure of the intact full-length AMPA-Rs. In 2005, a method to purify homogeneous particles of native AMPA-Rs from rat brain was developed and the purified receptors were further analyzed using single particle EM [66]. At the resolution of about 30 Å, the cryo-negative stained single particle 3D EM structures of the native AMPA-R containing auxiliary subunits stargazin/TARP and the native AMPA-Rs without the stargazin/TARP were reported [66, 67] (Fig. 2). The EM structure of native AMPA-Rs purified from rat brain [66, 67] looked very different from those of GluR2 homotetramers expressed and purified from Sf9 insect cells [69, 70]. Of particular importance, however, is that the sizes and shapes of the individual globular densities observed in the EM density map of the native AMPA-Rs were consistent with those of the crystal structures of the GluA2 LBD and GluA2 NTD [66, 71]. The structure also was consistent with the proposed dimer-of-dimers assembly of the subunits. The electron microscopy studies of native AMPA-Rs also revealed the global conformational changes of the receptor upon desensitization [66, 67] (Fig. 3). The detail of this study is discussed in the later section.Fig. 2

Bottom Line: The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex.The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing.Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA. nakagawa@ucsd.edu

ABSTRACT
The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity.

Show MeSH
Related in: MedlinePlus