Activation and Desensitization Mechanism of AMPA Receptor-TARP Complex by Cryo-EM

AMPA receptors mediate fast excitatory neurotransmission in the mammalian brain and transduce the binding of presynaptically released glutamate to the opening of a transmembrane cation channel. Within the postsynaptic density, however, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs), yielding a receptor complex with altered gating kinetics, pharmacology, and pore properties. Here, we elucidate structures of the GluA2-TARP γ2 complex in the presence of the partial agonist kainate or the full agonist quisqualate together with a positive allosteric modulator or with quisqualate alone. We show how TARPs sculpt the ligand-binding domain gating ring, enhancing kainate potency and diminishing the ensemble of desensitized states. TARPs encircle the receptor ion channel, stabilizing M2 helices and pore loops, illustrating how TARPs alter receptor pore properties. Structural and computational analysis suggests the full agonist and modulator complex harbors an ion-permeable channel gate, providing the first view of an activated AMPA receptor.

[1]  B. Wallace,et al.  HOLE: a program for the analysis of the pore dimensions of ion channel structural models. , 1996, Journal of molecular graphics.

[2]  Nikolaus Grigorieff,et al.  Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6 , 2015, eLife.

[3]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[4]  A. Aksimentiev,et al.  Exploring transmembrane transport through α -hemolysin with grid-steered molecular dynamics , 2007 .

[5]  E. Gouaux,et al.  Crystal structure and association behaviour of the GluR2 amino‐terminal domain , 2009, The EMBO journal.

[6]  Jan H. Jensen,et al.  PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions. , 2011, Journal of chemical theory and computation.

[7]  R. Nicoll,et al.  Regulation of AMPA receptor gating and pharmacology by TARP auxiliary subunits. , 2008, Trends in pharmacological sciences.

[8]  H. Adesnik,et al.  Stargazin modulates AMPA receptor gating and trafficking by distinct domains , 2005, Nature.

[9]  Vincent B. Chen,et al.  Correspondence e-mail: , 2000 .

[10]  Prashant Rao,et al.  Structural mechanism of glutamate receptor activation and desensitization , 2014, Nature.

[11]  S. Tomita,et al.  Autoinactivation of Neuronal AMPA Receptors via Glutamate-Regulated TARP Interaction , 2009, Neuron.

[12]  Jing Huang,et al.  CHARMM36 all‐atom additive protein force field: Validation based on comparison to NMR data , 2013, J. Comput. Chem..

[13]  M. Mayer,et al.  Inward rectification of both AMPA and kainate subtype glutamate receptors generated by polyamine-mediated ion channel block , 1995, Neuron.

[14]  K. Keinänen,et al.  Oligomerization and Ligand-binding Properties of the Ectodomain of the α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor Subunit GluRD* , 1999, The Journal of Biological Chemistry.

[15]  R. Nicoll,et al.  The Expanding Social Network of Ionotropic Glutamate Receptors: TARPs and Other Transmembrane Auxiliary Subunits , 2011, Neuron.

[16]  David N Mastronarde,et al.  Automated electron microscope tomography using robust prediction of specimen movements. , 2005, Journal of structural biology.

[17]  K. Schulten,et al.  Steered molecular dynamics simulation of the Rieske subunit motion in the cytochrome bc(1) complex. , 1999, Biophysical journal.

[18]  Aleksei Aksimentiev,et al.  Exploring transmembrane transport through alpha-hemolysin with grid-steered molecular dynamics. , 2007, The Journal of chemical physics.

[19]  T. Liljefors,et al.  Structural proof of a dimeric positive modulator bridging two identical AMPA receptor-binding sites. , 2007, Chemistry & biology.

[20]  M. Mayer,et al.  Mechanism of glutamate receptor desensitization , 2002, Nature.

[21]  M A Rogawski,et al.  Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Mayer,et al.  Mechanism of activation and selectivity in a ligand-gated ion channel: structural and functional studies of GluR2 and quisqualate. , 2002, Biochemistry.

[23]  W. C. Still,et al.  Semianalytical treatment of solvation for molecular mechanics and dynamics , 1990 .

[24]  E. Gouaux,et al.  Measurement of Conformational Changes accompanying Desensitization in an Ionotropic Glutamate Receptor , 2006, Cell.

[25]  E. Gouaux,et al.  Structure and Dynamics of AMPA Receptor GluA2 in Resting, Pre-Open, and Desensitized States , 2014, Cell.

[26]  A. Kristensen,et al.  Evaluation of PhTX-74 as Subtype-Selective Inhibitor of GluA2-Containing AMPA Receptors , 2014, Molecular Pharmacology.

[27]  D. Agard,et al.  MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy , 2017, Nature Methods.

[28]  Eric Gouaux,et al.  X-ray structures of AMPA receptor–cone snail toxin complexes illuminate activation mechanism , 2014, Science.

[29]  U. Dirnagl,et al.  ZK200775: a phosphonate quinoxalinedione AMPA antagonist for neuroprotection in stroke and trauma. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[30]  N. Grigorieff,et al.  CTFFIND4: Fast and accurate defocus estimation from electron micrographs , 2015, bioRxiv.

[31]  Adrian A Canutescu,et al.  SCWRL and MolIDE: computer programs for side-chain conformation prediction and homology modeling , 2008, Nature Protocols.

[32]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[33]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[34]  E. Gouaux,et al.  Mechanisms for Activation and Antagonism of an AMPA-Sensitive Glutamate Receptor Crystal Structures of the GluR2 Ligand Binding Core , 2000, Neuron.

[35]  Kai Zhang,et al.  Gctf: Real-time CTF determination and correction , 2015, bioRxiv.

[36]  L. Vyklický,et al.  Hippocampal neurons exhibit cyclothiazide-sensitive rapidly desensitizing responses to kainate , 1993, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[37]  H. Monyer,et al.  A molecular determinant for submillisecond desensitization in glutamate receptors. , 1994, Science.

[38]  R. Nicoll,et al.  TARP Subtypes Differentially and Dose-Dependently Control Synaptic AMPA Receptor Gating , 2007, Neuron.

[39]  Randy J. Read,et al.  Acta Crystallographica Section D Biological , 2003 .

[40]  Sjors H.W. Scheres,et al.  RELION: Implementation of a Bayesian approach to cryo-EM structure determination , 2012, Journal of structural biology.

[41]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[42]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[43]  Terri L. Gilbert,et al.  The ligand-binding domain in metabotropic glutamate receptors is related to bacterial periplasmic binding proteins , 1993, Neuron.

[44]  Klaus Schulten,et al.  Molecular dynamics-based refinement and validation for sub-5 Å cryo-electron microscopy maps , 2016, eLife.

[45]  B. Brooks,et al.  Constant pressure molecular dynamics simulation: The Langevin piston method , 1995 .

[46]  P. Biggin,et al.  Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes , 2016, Neuron.

[47]  S. Heinemann,et al.  Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins , 1994, Neuron.

[48]  Mark Ellisman,et al.  Contribution of the Global Subunit Structure and Stargazin on the Maturation of AMPA Receptors , 2010, The Journal of Neuroscience.

[49]  C. Ptak,et al.  Dynamics of Cleft Closure of the GluA2 Ligand-binding Domain in the Presence of Full and Partial Agonists Revealed by Hydrogen-Deuterium Exchange* , 2013, The Journal of Biological Chemistry.

[50]  P. Seeburg,et al.  RNA editing in brain controls a determinant of ion flow in glutamate-gated channels , 1991, Cell.

[51]  M Radermacher,et al.  DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy. , 2009, Journal of structural biology.

[52]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[53]  L. Wollmuth,et al.  The Outer Pore of the Glutamate Receptor Channel Has 2-Fold Rotational Symmetry , 2004, Neuron.

[54]  M. Mayer,et al.  Structural basis for partial agonist action at ionotropic glutamate receptors , 2003, Nature Neuroscience.

[55]  Hemant D. Tagare,et al.  The Local Resolution of Cryo-EM Density Maps , 2013, Nature Methods.

[56]  E. C. Twomey,et al.  Elucidation of AMPA receptor–stargazin complexes by cryo–electron microscopy , 2016, Science.

[57]  Mark Farrant,et al.  Stargazin attenuates intracellular polyamine block of calcium-permeable AMPA receptors , 2007, Nature Neuroscience.

[58]  E. Gouaux,et al.  X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor , 2009, Nature.

[59]  Leonardo G. Trabuco,et al.  Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. , 2008, Structure.

[60]  A. Sali,et al.  Modeling of loops in protein structures , 2000, Protein science : a publication of the Protein Society.

[61]  E. Gouaux,et al.  Architecture of fully occupied GluA2 AMPA receptor–TARP complex elucidated by cryo-EM , 2016, Nature.

[62]  R. Oswald,et al.  Unraveling the modular design of glutamate-gated ion channels , 1995, Trends in Neurosciences.

[63]  Narayanan Eswar,et al.  Protein structure modeling with MODELLER. , 2008, Methods in molecular biology.

[64]  E. Campbell,et al.  Crystal Structure of a Mammalian Voltage-Dependent Shaker Family K+ Channel , 2005, Science.

[65]  S. Heinemann,et al.  Ca2+ permeability of KA-AMPA--gated glutamate receptor channels depends on subunit composition , 1991, Science.

[66]  Robert M Stroud,et al.  TARP Auxiliary Subunits Switch AMPA Receptor Antagonists into Partial Agonists , 2007, Science.

[67]  T. Darden,et al.  A smooth particle mesh Ewald method , 1995 .

[68]  S. Subramaniam,et al.  Structural basis of kainate subtype glutamate receptor desensitization , 2016, Nature.

[69]  R. Dingledine,et al.  Glutamate Receptor Ion Channels: Structure, Regulation, and Function , 2010, Pharmacological Reviews.

[70]  Youxing Jiang,et al.  The open pore conformation of potassium channels , 2002, Nature.

[71]  D. K. Patneau,et al.  Stargazin Modulates Native AMPA Receptor Functional Properties by Two Distinct Mechanisms , 2005, The Journal of Neuroscience.

[72]  L. Wollmuth,et al.  Structure and gating of the glutamate receptor ion channel , 2004, Trends in Neurosciences.

[73]  Dane M. Chetkovich,et al.  Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms , 2000, Nature.

[74]  David A. Agard,et al.  Anisotropic Correction of Beam-induced Motion for Improved Single-particle Electron Cryo-microscopy , 2016, bioRxiv.

[75]  P. Usherwood,et al.  Block of open channels of recombinant AMPA receptors and native AMPA/kainate receptors by Adamantane derivatives , 1997, The Journal of physiology.

[76]  Sunjeev K Kamboj,et al.  Intracellular spermine confers rectification on rat calcium‐permeable AMPA and kainate receptors. , 1995, The Journal of physiology.

[77]  Sebastian Pascarelle,et al.  Unusual spectral energy distribution of a galaxy previously reported to be at redshift 6.68 , 2000, Nature.

[78]  M. Klein,et al.  Constant pressure molecular dynamics algorithms , 1994 .