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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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Structural changes accompanying opening of GluA2a, Representative 2D class averages of GluA2em in the active state after initial classification of 31,637 projection images. b, GluA2em active state structure shown in isosurface representation, fitted with ATD dimers (PDB ID: 3KG2) and glutamate-bound LBD dimers (PDB ID: 1FTJ) with the transmembrane region covered by micellar density. c, Density maps for a single subunit showing the visible difference between the antagonist-bound open cleft conformation (top) and the glutamate-bound closed cleft conformation (bottom) of the LBD “clamshell”; the right-hand panel shows the corresponding coordinate fits. d, Ribbon and cylinder diagrams for GluA2 coordinates fit to the closed (magenta) and active (blue) states reveal a ~ 7 Å downward displacement of the ATD in the active state (top), with proximal and distal subunit LBD dimer assemblies viewed perpendicular to (middle) and parallel to (bottom) the membrane. Black dashed lines show the approximate planar interface between subunits in the dimer assembly. e, Isosurface views of LBD tetramer region density maps fit with LBD dimers in closed (left) and active (right) states. Colored dots identify the locations of Cα atoms for Val395 (upper lobe) and Ala665 (lower lobe). f, Movement of the S1-M2 linker (Lys505), M3-S2 linker (Glu634), and S2-M4 linker (Gly771) shows how LBD tetramer movements drive channel opening; arrows show the direction of movement from closed to active states.
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Figure 2: Structural changes accompanying opening of GluA2a, Representative 2D class averages of GluA2em in the active state after initial classification of 31,637 projection images. b, GluA2em active state structure shown in isosurface representation, fitted with ATD dimers (PDB ID: 3KG2) and glutamate-bound LBD dimers (PDB ID: 1FTJ) with the transmembrane region covered by micellar density. c, Density maps for a single subunit showing the visible difference between the antagonist-bound open cleft conformation (top) and the glutamate-bound closed cleft conformation (bottom) of the LBD “clamshell”; the right-hand panel shows the corresponding coordinate fits. d, Ribbon and cylinder diagrams for GluA2 coordinates fit to the closed (magenta) and active (blue) states reveal a ~ 7 Å downward displacement of the ATD in the active state (top), with proximal and distal subunit LBD dimer assemblies viewed perpendicular to (middle) and parallel to (bottom) the membrane. Black dashed lines show the approximate planar interface between subunits in the dimer assembly. e, Isosurface views of LBD tetramer region density maps fit with LBD dimers in closed (left) and active (right) states. Colored dots identify the locations of Cα atoms for Val395 (upper lobe) and Ala665 (lower lobe). f, Movement of the S1-M2 linker (Lys505), M3-S2 linker (Glu634), and S2-M4 linker (Gly771) shows how LBD tetramer movements drive channel opening; arrows show the direction of movement from closed to active states.

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)

Structural changes accompanying opening of GluA2a, Representative 2D class averages of GluA2em in the active state after initial classification of 31,637 projection images. b, GluA2em active state structure shown in isosurface representation, fitted with ATD dimers (PDB ID: 3KG2) and glutamate-bound LBD dimers (PDB ID: 1FTJ) with the transmembrane region covered by micellar density. c, Density maps for a single subunit showing the visible difference between the antagonist-bound open cleft conformation (top) and the glutamate-bound closed cleft conformation (bottom) of the LBD “clamshell”; the right-hand panel shows the corresponding coordinate fits. d, Ribbon and cylinder diagrams for GluA2 coordinates fit to the closed (magenta) and active (blue) states reveal a ~ 7 Å downward displacement of the ATD in the active state (top), with proximal and distal subunit LBD dimer assemblies viewed perpendicular to (middle) and parallel to (bottom) the membrane. Black dashed lines show the approximate planar interface between subunits in the dimer assembly. e, Isosurface views of LBD tetramer region density maps fit with LBD dimers in closed (left) and active (right) states. Colored dots identify the locations of Cα atoms for Val395 (upper lobe) and Ala665 (lower lobe). f, Movement of the S1-M2 linker (Lys505), M3-S2 linker (Glu634), and S2-M4 linker (Gly771) shows how LBD tetramer movements drive channel opening; arrows show the direction of movement from closed to active states.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4199900&req=5

Figure 2: Structural changes accompanying opening of GluA2a, Representative 2D class averages of GluA2em in the active state after initial classification of 31,637 projection images. b, GluA2em active state structure shown in isosurface representation, fitted with ATD dimers (PDB ID: 3KG2) and glutamate-bound LBD dimers (PDB ID: 1FTJ) with the transmembrane region covered by micellar density. c, Density maps for a single subunit showing the visible difference between the antagonist-bound open cleft conformation (top) and the glutamate-bound closed cleft conformation (bottom) of the LBD “clamshell”; the right-hand panel shows the corresponding coordinate fits. d, Ribbon and cylinder diagrams for GluA2 coordinates fit to the closed (magenta) and active (blue) states reveal a ~ 7 Å downward displacement of the ATD in the active state (top), with proximal and distal subunit LBD dimer assemblies viewed perpendicular to (middle) and parallel to (bottom) the membrane. Black dashed lines show the approximate planar interface between subunits in the dimer assembly. e, Isosurface views of LBD tetramer region density maps fit with LBD dimers in closed (left) and active (right) states. Colored dots identify the locations of Cα atoms for Val395 (upper lobe) and Ala665 (lower lobe). f, Movement of the S1-M2 linker (Lys505), M3-S2 linker (Glu634), and S2-M4 linker (Gly771) shows how LBD tetramer movements drive channel opening; arrows show the direction of movement from closed to active states.
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