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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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Conformational ensemble of desensitized GluA2a, Representative desensitized state GluA2em 2D class averages from initial classification of 35,083 projection images. Selected class-averages that illustrate the range of observed conformations are highlighted. b, Segmented isosurface representations of three distinct desensitized state GluA2em structures, with the ATD and LBD layers identified in blue and orange, respectively. c, Top views of ATD and LBD layers for the three GluA2em desensitized states (middle columns) flanked by those from the active state (left), and restored active state (right).
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Figure 3: Conformational ensemble of desensitized GluA2a, Representative desensitized state GluA2em 2D class averages from initial classification of 35,083 projection images. Selected class-averages that illustrate the range of observed conformations are highlighted. b, Segmented isosurface representations of three distinct desensitized state GluA2em structures, with the ATD and LBD layers identified in blue and orange, respectively. c, Top views of ATD and LBD layers for the three GluA2em desensitized states (middle columns) flanked by those from the active state (left), and restored active state (right).

Mentions: AMPA and kainate receptors exhibit rapid and nearly complete desensitization of ion flux within milliseconds after glutamate binding21. To trap GluA2em in the desensitized state, we incubated the purified protein with 1 mM quisqualate, a full agonist with a Kd of ~ 20 nM and 10-fold higher affinity than glutamate22. Analysis of cryo-electron microscopic images revealed evidence of substantial conformational heterogeneity (Fig. 3a) precluding determination of a single desensitized state 3D structure. Three-dimensional classification enabled separation of three dominant classes at nominal resolutions of 21 Å, 23 Å and 26 Å, with variable degrees of displacement between ATD dimers compared to the closed and active states (Fig. 3b and Extended Data Fig. 5). In all three classes, the LBD layer separates into four lobes of density, with different degrees of separation between the proximal and distal LBD subunits, strikingly different from the “dimer-of-dimers” structure found in the closed and active states. This variability in ATD and LBD conformation is further illustrated in top views that capture the extent of the quaternary structural change in the three desensitized states as compared to the active state (Fig. 3c). It is likely that the three desensitized states are subsets of an even larger spectrum that includes additional weakly populated conformational variants. Nevertheless, our findings establish that desensitization results in separation of the LBD dimers into a quasi 4-fold arrangement, coupled with conformational heterogeneity in the ATD layer not observed for either the closed or active states.


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)

Conformational ensemble of desensitized GluA2a, Representative desensitized state GluA2em 2D class averages from initial classification of 35,083 projection images. Selected class-averages that illustrate the range of observed conformations are highlighted. b, Segmented isosurface representations of three distinct desensitized state GluA2em structures, with the ATD and LBD layers identified in blue and orange, respectively. c, Top views of ATD and LBD layers for the three GluA2em desensitized states (middle columns) flanked by those from the active state (left), and restored active state (right).
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Related In: Results  -  Collection

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Figure 3: Conformational ensemble of desensitized GluA2a, Representative desensitized state GluA2em 2D class averages from initial classification of 35,083 projection images. Selected class-averages that illustrate the range of observed conformations are highlighted. b, Segmented isosurface representations of three distinct desensitized state GluA2em structures, with the ATD and LBD layers identified in blue and orange, respectively. c, Top views of ATD and LBD layers for the three GluA2em desensitized states (middle columns) flanked by those from the active state (left), and restored active state (right).
Mentions: AMPA and kainate receptors exhibit rapid and nearly complete desensitization of ion flux within milliseconds after glutamate binding21. To trap GluA2em in the desensitized state, we incubated the purified protein with 1 mM quisqualate, a full agonist with a Kd of ~ 20 nM and 10-fold higher affinity than glutamate22. Analysis of cryo-electron microscopic images revealed evidence of substantial conformational heterogeneity (Fig. 3a) precluding determination of a single desensitized state 3D structure. Three-dimensional classification enabled separation of three dominant classes at nominal resolutions of 21 Å, 23 Å and 26 Å, with variable degrees of displacement between ATD dimers compared to the closed and active states (Fig. 3b and Extended Data Fig. 5). In all three classes, the LBD layer separates into four lobes of density, with different degrees of separation between the proximal and distal LBD subunits, strikingly different from the “dimer-of-dimers” structure found in the closed and active states. This variability in ATD and LBD conformation is further illustrated in top views that capture the extent of the quaternary structural change in the three desensitized states as compared to the active state (Fig. 3c). It is likely that the three desensitized states are subsets of an even larger spectrum that includes additional weakly populated conformational variants. Nevertheless, our findings establish that desensitization results in separation of the LBD dimers into a quasi 4-fold arrangement, coupled with conformational heterogeneity in the ATD layer not observed for either the closed or active states.

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