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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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Open state GluA2 density map quality and resolutiona,b, Density map of glutamate bound GluA2em in the open state with coordinates for ATD dimers (PDB ID: 3KG2) and glutamate-bound LBD dimers (PDB ID: 1FTJ) fit separately into the map. In panel (b) the density map is shown at a higher contour than (a) to highlight closeness of fit between X-ray domain coordinates and the density map. c, Secondary structural features from ATD chains B/D of the density map corresponding to regions marked in panel (b). Roman numerals identify helices 5, 6, 7 and the ATD lower domain beta sheet. d, Gold-standard FSC plot (black line) for the GluA2em open state density map showing a map resolution of 12.8 Å at an FSC value of 0.143, and a plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, which displays a resolution of 12.7 Å at an FSC value of 0.5, consistent with the gold-standard FSC curve.
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Figure 9: Open state GluA2 density map quality and resolutiona,b, Density map of glutamate bound GluA2em in the open state with coordinates for ATD dimers (PDB ID: 3KG2) and glutamate-bound LBD dimers (PDB ID: 1FTJ) fit separately into the map. In panel (b) the density map is shown at a higher contour than (a) to highlight closeness of fit between X-ray domain coordinates and the density map. c, Secondary structural features from ATD chains B/D of the density map corresponding to regions marked in panel (b). Roman numerals identify helices 5, 6, 7 and the ATD lower domain beta sheet. d, Gold-standard FSC plot (black line) for the GluA2em open state density map showing a map resolution of 12.8 Å at an FSC value of 0.143, and a plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, which displays a resolution of 12.7 Å at an FSC value of 0.5, consistent with the gold-standard FSC curve.

Mentions: To determine structural changes that occur with transition to the active state, purified GluA2 was pre-mixed with 0.5 mM LY451646, a potent allosteric modulator that prevents entry into the desensitized state16. After equilibration for 30 minutes, a saturating concentration of glutamate (100 mM) was added to activate ion channel gating, followed by immediate plunge freezing. Under these conditions there is very high occupancy of the open state17, and the activation of sub-conductance states which are prominent at low agonist concentrations is reduced18. Analysis of molecular images obtained from AMPA receptors in the active state revealed the presence of well-defined 2D class averages (Fig. 2a), allowing reconstruction of the structure to a resolution of ~ 12 Å with a set of images of similar size and quality to that used to obtain the structure of the closed state (Extended Data Fig. 4). The slightly lower resolution suggests that despite the presence of glutamate at a high concentration, the active state may be more conformationally variable than the closed state, perhaps due to the occurrence of sub-conductance states, or transient excursions to a closed state. Nevertheless, the ATD and LBD domains were fit without ambiguity (Fig. 2b) supported by identification of secondary structure elements (Extended Data Fig. 4). Because ATD and LBD crystal structures provide information at atomic resolution, combining this with the quaternary constraints provided by cryo-EM density maps allows interpretation of structural changes in the full-length receptor at resolutions higher than the nominal resolution of the density map. Although the TMD is not resolved with sufficient detail to allow interpretation of the conformation of TM helices, rigid body fits of ATD dimers and glutamate-bound LBD dimer crystal structures are sufficiently well-constrained by the density map to allow a molecular interpretation of the activation mechanism under conditions where GluA2 has a high open probability (Fig. 2b).


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)

Open state GluA2 density map quality and resolutiona,b, Density map of glutamate bound GluA2em in the open state with coordinates for ATD dimers (PDB ID: 3KG2) and glutamate-bound LBD dimers (PDB ID: 1FTJ) fit separately into the map. In panel (b) the density map is shown at a higher contour than (a) to highlight closeness of fit between X-ray domain coordinates and the density map. c, Secondary structural features from ATD chains B/D of the density map corresponding to regions marked in panel (b). Roman numerals identify helices 5, 6, 7 and the ATD lower domain beta sheet. d, Gold-standard FSC plot (black line) for the GluA2em open state density map showing a map resolution of 12.8 Å at an FSC value of 0.143, and a plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, which displays a resolution of 12.7 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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Show All Figures
getmorefigures.php?uid=PMC4199900&req=5

Figure 9: Open state GluA2 density map quality and resolutiona,b, Density map of glutamate bound GluA2em in the open state with coordinates for ATD dimers (PDB ID: 3KG2) and glutamate-bound LBD dimers (PDB ID: 1FTJ) fit separately into the map. In panel (b) the density map is shown at a higher contour than (a) to highlight closeness of fit between X-ray domain coordinates and the density map. c, Secondary structural features from ATD chains B/D of the density map corresponding to regions marked in panel (b). Roman numerals identify helices 5, 6, 7 and the ATD lower domain beta sheet. d, Gold-standard FSC plot (black line) for the GluA2em open state density map showing a map resolution of 12.8 Å at an FSC value of 0.143, and a plot (red line) of the FSC between the experimentally obtained cryo-EM density map and a map computed from the fitted coordinates, which displays a resolution of 12.7 Å at an FSC value of 0.5, consistent with the gold-standard FSC curve.
Mentions: To determine structural changes that occur with transition to the active state, purified GluA2 was pre-mixed with 0.5 mM LY451646, a potent allosteric modulator that prevents entry into the desensitized state16. After equilibration for 30 minutes, a saturating concentration of glutamate (100 mM) was added to activate ion channel gating, followed by immediate plunge freezing. Under these conditions there is very high occupancy of the open state17, and the activation of sub-conductance states which are prominent at low agonist concentrations is reduced18. Analysis of molecular images obtained from AMPA receptors in the active state revealed the presence of well-defined 2D class averages (Fig. 2a), allowing reconstruction of the structure to a resolution of ~ 12 Å with a set of images of similar size and quality to that used to obtain the structure of the closed state (Extended Data Fig. 4). The slightly lower resolution suggests that despite the presence of glutamate at a high concentration, the active state may be more conformationally variable than the closed state, perhaps due to the occurrence of sub-conductance states, or transient excursions to a closed state. Nevertheless, the ATD and LBD domains were fit without ambiguity (Fig. 2b) supported by identification of secondary structure elements (Extended Data Fig. 4). Because ATD and LBD crystal structures provide information at atomic resolution, combining this with the quaternary constraints provided by cryo-EM density maps allows interpretation of structural changes in the full-length receptor at resolutions higher than the nominal resolution of the density map. Although the TMD is not resolved with sufficient detail to allow interpretation of the conformation of TM helices, rigid body fits of ATD dimers and glutamate-bound LBD dimer crystal structures are sufficiently well-constrained by the density map to allow a molecular interpretation of the activation mechanism under conditions where GluA2 has a high open probability (Fig. 2b).

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