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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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The relationship between the EM and crystal structure of AMPA-R. a The cryo-negative stain EM structure (left) and the crystal structure of tetrameric GluA2cryst (PDB:3KG2) are shown at the same scale. Comparison of the two structures (as shown below in b and c) suggests that the NTD–LBD linker is flexible (see text for detail). The red arrows indicate the pivot points that locate the positions of the linkers that connect the NTD and LBD. b The NTD tetramer from the crystal structure of GluR2cryst (green) was placed into the EM density map of the native AMPA-R shown in (a) (represented here in mesh). Four different views are shown. The size and shape of the NTD density in the EM map is consistent with the dimer-of-dimers organization of the NTDs in the crystal structure of GluA2cryst. However, in order to place the NTD crystal into the EM density map, the crystal needs to be displaced and tilted along the pivot axis shown in (a). c The crystal structure of GluR2cryst (green) was placed into the EM density map of the native AMPA-R shown in (a) (represented here in mesh). Four different views are shown. The arrangements of the NTDs relative to the rest of the structure are the main differences between the global domain arrangements between the two structures. The images were produced using UCSF Chimera
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Fig5: The relationship between the EM and crystal structure of AMPA-R. a The cryo-negative stain EM structure (left) and the crystal structure of tetrameric GluA2cryst (PDB:3KG2) are shown at the same scale. Comparison of the two structures (as shown below in b and c) suggests that the NTD–LBD linker is flexible (see text for detail). The red arrows indicate the pivot points that locate the positions of the linkers that connect the NTD and LBD. b The NTD tetramer from the crystal structure of GluR2cryst (green) was placed into the EM density map of the native AMPA-R shown in (a) (represented here in mesh). Four different views are shown. The size and shape of the NTD density in the EM map is consistent with the dimer-of-dimers organization of the NTDs in the crystal structure of GluA2cryst. However, in order to place the NTD crystal into the EM density map, the crystal needs to be displaced and tilted along the pivot axis shown in (a). c The crystal structure of GluR2cryst (green) was placed into the EM density map of the native AMPA-R shown in (a) (represented here in mesh). Four different views are shown. The arrangements of the NTDs relative to the rest of the structure are the main differences between the global domain arrangements between the two structures. The images were produced using UCSF Chimera

Mentions: The size and shape of the globular features seen in the EM density map were consistent with the known crystal structures of the related domains that were in the PDB. The crystal structure of the GluA2 NTD tetramer can be placed into the densities corresponding to the NTDs of the EM structure of native AMPA-R void of stargazin/TARPs [71]. The consistency between the crystal structure of the tetrameric GluA2 NTD and the low-resolution EM density map suggested that the inter-domain contacts seen in the crystal may be preserved in the full-length tetrameric receptor. The arrangements of the two NTD dimers in the crystal structure of the tetrameric GluA2 NTD [71] and the crystal structure of the tetrameric GluA2cryst [75] are indistinguishable, and thus the global arrangements of the NTDs in the GluA2cryst tetramer are in agreement with the low-resolution EM map (Fig. 5b). However, when compared side by side, it is immediately recognized that the EM structure is shorter than the GluR2cryst structure (Fig. 5a). The densities that correspond to the NTD dimer in the EM structure are tilted in one direction, whereas in the X-ray structure they are standing upright (Fig. 5a; the pivot axis of the tilt is indicated by the red arrows). The difference in the arrangements of the NTDs in the two structures contributes to the different heights and the difference in the overall symmetry (Fig. 5c). This tilt is the major cause of asymmetry that was observed in the EM structure. The linker that connects the LBD and the NTD in GluR2cryst is engineered such that it is six amino acids shorter and two predicted glycosylation sites [26, 136] are removed. Sobolevsky et al. [75] reported that these modifications were necessary to obtain crystals. Proteins with flexible conformation are more difficult to crystallize, and thus it makes sense if the linker that connects the LBD and NTD is flexible in the native AMPA-Rs that have a longer glycosylated linker. But simplifying the linker would reduce the structural complexity and conformational variety intrinsic to the NTD–LBD connection. Consistently, the NTD dimers were arranged in a variety of angles relative to the LBDs in the projection structures of the negative stained EM images of the native AMPA-Rs from brain [66, 67].Fig. 5


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

Nakagawa T - Mol. Neurobiol. (2010)

The relationship between the EM and crystal structure of AMPA-R. a The cryo-negative stain EM structure (left) and the crystal structure of tetrameric GluA2cryst (PDB:3KG2) are shown at the same scale. Comparison of the two structures (as shown below in b and c) suggests that the NTD–LBD linker is flexible (see text for detail). The red arrows indicate the pivot points that locate the positions of the linkers that connect the NTD and LBD. b The NTD tetramer from the crystal structure of GluR2cryst (green) was placed into the EM density map of the native AMPA-R shown in (a) (represented here in mesh). Four different views are shown. The size and shape of the NTD density in the EM map is consistent with the dimer-of-dimers organization of the NTDs in the crystal structure of GluA2cryst. However, in order to place the NTD crystal into the EM density map, the crystal needs to be displaced and tilted along the pivot axis shown in (a). c The crystal structure of GluR2cryst (green) was placed into the EM density map of the native AMPA-R shown in (a) (represented here in mesh). Four different views are shown. The arrangements of the NTDs relative to the rest of the structure are the main differences between the global domain arrangements between the two structures. The images were produced using UCSF Chimera
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2992128&req=5

Fig5: The relationship between the EM and crystal structure of AMPA-R. a The cryo-negative stain EM structure (left) and the crystal structure of tetrameric GluA2cryst (PDB:3KG2) are shown at the same scale. Comparison of the two structures (as shown below in b and c) suggests that the NTD–LBD linker is flexible (see text for detail). The red arrows indicate the pivot points that locate the positions of the linkers that connect the NTD and LBD. b The NTD tetramer from the crystal structure of GluR2cryst (green) was placed into the EM density map of the native AMPA-R shown in (a) (represented here in mesh). Four different views are shown. The size and shape of the NTD density in the EM map is consistent with the dimer-of-dimers organization of the NTDs in the crystal structure of GluA2cryst. However, in order to place the NTD crystal into the EM density map, the crystal needs to be displaced and tilted along the pivot axis shown in (a). c The crystal structure of GluR2cryst (green) was placed into the EM density map of the native AMPA-R shown in (a) (represented here in mesh). Four different views are shown. The arrangements of the NTDs relative to the rest of the structure are the main differences between the global domain arrangements between the two structures. The images were produced using UCSF Chimera
Mentions: The size and shape of the globular features seen in the EM density map were consistent with the known crystal structures of the related domains that were in the PDB. The crystal structure of the GluA2 NTD tetramer can be placed into the densities corresponding to the NTDs of the EM structure of native AMPA-R void of stargazin/TARPs [71]. The consistency between the crystal structure of the tetrameric GluA2 NTD and the low-resolution EM density map suggested that the inter-domain contacts seen in the crystal may be preserved in the full-length tetrameric receptor. The arrangements of the two NTD dimers in the crystal structure of the tetrameric GluA2 NTD [71] and the crystal structure of the tetrameric GluA2cryst [75] are indistinguishable, and thus the global arrangements of the NTDs in the GluA2cryst tetramer are in agreement with the low-resolution EM map (Fig. 5b). However, when compared side by side, it is immediately recognized that the EM structure is shorter than the GluR2cryst structure (Fig. 5a). The densities that correspond to the NTD dimer in the EM structure are tilted in one direction, whereas in the X-ray structure they are standing upright (Fig. 5a; the pivot axis of the tilt is indicated by the red arrows). The difference in the arrangements of the NTDs in the two structures contributes to the different heights and the difference in the overall symmetry (Fig. 5c). This tilt is the major cause of asymmetry that was observed in the EM structure. The linker that connects the LBD and the NTD in GluR2cryst is engineered such that it is six amino acids shorter and two predicted glycosylation sites [26, 136] are removed. Sobolevsky et al. [75] reported that these modifications were necessary to obtain crystals. Proteins with flexible conformation are more difficult to crystallize, and thus it makes sense if the linker that connects the LBD and NTD is flexible in the native AMPA-Rs that have a longer glycosylated linker. But simplifying the linker would reduce the structural complexity and conformational variety intrinsic to the NTD–LBD connection. Consistently, the NTD dimers were arranged in a variety of angles relative to the LBDs in the projection structures of the negative stained EM images of the native AMPA-Rs from brain [66, 67].Fig. 5

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