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Structural mechanism of glutamate receptor activation and desensitization.

Meyerson JR, Kumar J, Chittori S, Rao P, Pierson J, Bartesaghi A, Mayer ML, Subramaniam S - Nature (2014)

Bottom Line: Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes.The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing.These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland 20892, USA.

ABSTRACT
Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.

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Cryo-electron microscopic imaging of GluK2 with 2S,4R-4-methylglutamate and 2D classesa,b, Representative cryo-EM image of GluK2 bound by the agonist 2S,4R-4-methylglutamate (leftmost panel), with the corresponding image power spectrum and CTF estimate showing signal beyond 8 Å resolution (rightmost panel, solid and dotted lines, respectively). The defocus value of the image is 3.7 μm. Scale bar is 100 nm. c, Two-dimensional classes of desensitized GluK2 particles subjected to single particle analysis. d, Gold-standard FSC plot (black line) for the GluK2 desensitized state density map showing a map resolution of 7.6 Å at an FSC value of 0.143. A plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, displays a resolution of 7.7 Å at an FSC value of 0.5, consistent with the gold-standard FSC curve.
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Figure 12: Cryo-electron microscopic imaging of GluK2 with 2S,4R-4-methylglutamate and 2D classesa,b, Representative cryo-EM image of GluK2 bound by the agonist 2S,4R-4-methylglutamate (leftmost panel), with the corresponding image power spectrum and CTF estimate showing signal beyond 8 Å resolution (rightmost panel, solid and dotted lines, respectively). The defocus value of the image is 3.7 μm. Scale bar is 100 nm. c, Two-dimensional classes of desensitized GluK2 particles subjected to single particle analysis. d, Gold-standard FSC plot (black line) for the GluK2 desensitized state density map showing a map resolution of 7.6 Å at an FSC value of 0.143. A plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, displays a resolution of 7.7 Å at an FSC value of 0.5, consistent with the gold-standard FSC curve.

Mentions: In the structure of the GluK2 desensitized state, determined at ~ 7.6 Å resolution (Fig. 4a, Extended Data Fig. 7 and 8), density was resolved for all α-helices in the ATD and LBD assemblies, and also for the M3 helix bundle, the upper segment of M1 and the pre-M1 cuff helix in the ion channel (Fig. 4b). The density map reveals preservation of 2-fold symmetry in the ATD layer while the LBD layer adopts a quasi 4-fold symmetric arrangement (Fig. 4c). To obtain a molecular model for the desensitized state, we fitted two copies of GluK2 ATD dimer assemblies (PDB ID: 3H6G) and four copies of a GluK2 subunit LBD glutamate complex (PDB ID: 3G3F). The resolution of our map is adequate to unambiguously show that in the desensitized state the ion channel adopts a closed conformation (Fig. 4b, panel vii) in which the M3 helices form a crossed bundle assembly with the pre-M1 helices wrapped around the outside of the channel, similar to that seen for GluA2cryst in its antagonist-bound closed state.


Structural mechanism of glutamate receptor activation and desensitization.

Meyerson JR, Kumar J, Chittori S, Rao P, Pierson J, Bartesaghi A, Mayer ML, Subramaniam S - Nature (2014)

Cryo-electron microscopic imaging of GluK2 with 2S,4R-4-methylglutamate and 2D classesa,b, Representative cryo-EM image of GluK2 bound by the agonist 2S,4R-4-methylglutamate (leftmost panel), with the corresponding image power spectrum and CTF estimate showing signal beyond 8 Å resolution (rightmost panel, solid and dotted lines, respectively). The defocus value of the image is 3.7 μm. Scale bar is 100 nm. c, Two-dimensional classes of desensitized GluK2 particles subjected to single particle analysis. d, Gold-standard FSC plot (black line) for the GluK2 desensitized state density map showing a map resolution of 7.6 Å at an FSC value of 0.143. A plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, displays a resolution of 7.7 Å at an FSC value of 0.5, consistent with the gold-standard FSC curve.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4199900&req=5

Figure 12: Cryo-electron microscopic imaging of GluK2 with 2S,4R-4-methylglutamate and 2D classesa,b, Representative cryo-EM image of GluK2 bound by the agonist 2S,4R-4-methylglutamate (leftmost panel), with the corresponding image power spectrum and CTF estimate showing signal beyond 8 Å resolution (rightmost panel, solid and dotted lines, respectively). The defocus value of the image is 3.7 μm. Scale bar is 100 nm. c, Two-dimensional classes of desensitized GluK2 particles subjected to single particle analysis. d, Gold-standard FSC plot (black line) for the GluK2 desensitized state density map showing a map resolution of 7.6 Å at an FSC value of 0.143. A plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, displays a resolution of 7.7 Å at an FSC value of 0.5, consistent with the gold-standard FSC curve.
Mentions: In the structure of the GluK2 desensitized state, determined at ~ 7.6 Å resolution (Fig. 4a, Extended Data Fig. 7 and 8), density was resolved for all α-helices in the ATD and LBD assemblies, and also for the M3 helix bundle, the upper segment of M1 and the pre-M1 cuff helix in the ion channel (Fig. 4b). The density map reveals preservation of 2-fold symmetry in the ATD layer while the LBD layer adopts a quasi 4-fold symmetric arrangement (Fig. 4c). To obtain a molecular model for the desensitized state, we fitted two copies of GluK2 ATD dimer assemblies (PDB ID: 3H6G) and four copies of a GluK2 subunit LBD glutamate complex (PDB ID: 3G3F). The resolution of our map is adequate to unambiguously show that in the desensitized state the ion channel adopts a closed conformation (Fig. 4b, panel vii) in which the M3 helices form a crossed bundle assembly with the pre-M1 helices wrapped around the outside of the channel, similar to that seen for GluA2cryst in its antagonist-bound closed state.

Bottom Line: Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes.The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing.These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland 20892, USA.

ABSTRACT
Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.

Show MeSH
Related in: MedlinePlus