Fast excitatory neurotransmission in the mammalian central nervous system is largely carried out by AMPA-sensitive ionotropic glutamate receptors. Localized within the postsynaptic density of glutamatergic spines, AMPA receptors are composed of heterotetrameric receptor assemblies associated with auxiliary subunits, the most common of which are transmembrane AMPA-receptor regulatory proteins (TARPs). The association of TARPs with AMPA receptors modulates the kinetics of receptor gating and pharmacology, as well as trafficking. Here we report the cryo-EM structure of the homomeric GluA2 AMPA receptor saturated with TARP γ2 subunits, showing how the TARPs are arranged with four-fold symmetry around the ion channel domain, making extensive interactions with the M1, M2 and M4 TM helices. Poised like partially opened ‘hands’ underneath the two-fold symmetric ligand binding domain (LBD) ‘clamshells’, one pair of TARPs are juxtaposed near the LBD dimer interface, while the other pair is near the LBD dimer-dimer interface. The extracellular ‘domains’ of TARP are positioned to not only modulate LBD ‘clamshell’ closure, but also to affect conformational rearrangements of the LBD layer associated with receptor activation and desensitization, while the TARP transmembrane (TM) domains buttress the ion channel pore.

Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA receptors are tetrameric assemblies composed of four subunits, GluA1–GluA4. Despite decades of study, the subunit composition, subunit arrangement, and molecular structure of native AMPA receptors remain unknown. Here we elucidate the structures of 10 distinct native AMPA receptor complexes by single-particle cryo–electron microscopy (cryo-EM). We find that receptor subunits are arranged nonstochastically, with the GluA2 subunit preferentially occupying the B and D positions of the tetramer and with triheteromeric assemblies comprising a major population of native AMPA receptors. Cryo-EM maps define the structure for S2-M4 linkers between the ligand-binding and transmembrane domains, suggesting how neurotransmitter binding is coupled to ion channel gating.

Summary 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/modulator complex harbors an ion-permeable channel gate, providing the first view of an activated AMPA receptor.

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/modulator complex harbors an ion-permeable channel gate, providing the first view of an activated AMPA receptor.

Ionotropic glutamate receptors in vertebrates are composed of three major subtypes-AMPA, kainate and NMDA receptors-and mediate the majority of fast excitatory neurotransmission at chemical synapses of the central nervous system. Among the three major families, native AMPA receptors function as complexes with a variety of auxiliary subunits, which in turn modulate receptor trafficking, gating, pharmacology and permeation. Despite the long history of structuremechanism studies using soluble receptor domains or intact yet isolated receptors, structures of AMPA receptor-auxiliary subunit complexes have not been available until recent breakthroughs in single-particle cryo-electron microscopy. Single particle cryo-EM studies have, in turn, provided new insights into the structure and organization of AMPA receptor-auxiliary protein complexes and into the molecular mechanisms of AMPA receptor activation and desensitization.

SummaryFast excitatory neurotransmission in the mammalian central nervous system is largely carried out by AMPA-sensitive ionotropic glutamate receptors. Localized within the postsynaptic density of glutamatergic spines, AMPA receptors are composed of heterotetrameric receptor assemblies associated with auxiliary subunits, the most common of which are transmembrane AMPAreceptor regulatory proteins (TARPs). The association of TARPs with AMPA receptors modulates the kinetics of receptor gating and pharmacology, as well as trafficking. Here we report the cryo-EM structure of the homomeric GluA2 AMPA receptor saturated with TARP γ2 subunits, showing how the TARPs are arranged with four-fold symmetry around the ion channel domain, making extensive interactions with the M1, M2 and M4 TM helices. Poised like partially opened 'hands' underneath the two-fold symmetric ligand binding domain (LBD) 'clamshells', one pair of TARPs are juxtaposed near the LBD dimer interface, while the other pair are near the LBD dimer-dimer interface. The extracellular 'domains' of TARP are positioned to not only modulate LBD 'clamshell' closure, but also to affect conformational rearrangements of the LBD layer associated with receptor activation and desensitization, while the TARP transmembrane (TM) domains buttress the ion channel pore.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/060046 doi: bioRxiv preprint first posted online Jun. 21, 2016; page 3Fast excitatory neurotransmission at chemical synapses of the brain underpins a spectrum of activities ranging from memory and learning, to speech and hearing, to movement and coordination. Ionotropic glutamate receptors (iGluRs) are a family of transmitter-gated ion channels comprised of three related subfamilies -AMPA, kainate and NMDA receptors -that mediate the majority of ionotropic excitatory signaling 1 X-ray crystal and single particle cryo-electron microscopy (cryo-EM) structures of AMPA receptors show that they are tetrameric assemblies consisting of three layers -the aminoAll rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. we found that whereas dodecyl maltopyranoside (DDM) leads to dissociation of the receptorAll rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/060046 doi: bioRxiv preprint first posted online Jun. 21, 2016; page 5 TARP complex, digitonin retains the complex integrity, allowing TARP to remain associated with receptor following solubilization and purification (Extended Data Fig. 1a). We proceeded to purify the native GluA2 receptor-full len...

SummaryAMPA receptors mediate the majority of 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 altered gating kinetics, receptor pharmacology and pore properties.Here we elucidate full-length GluA2-TARP γ2 complex structures in the presence of the partial agonist kainate or the full agonist quisqualate together with a positive allosteric modulator, and with quisqualate alone. We show how TARPs sculpt the ligand binding domain gating ring, enhancing kainate potency and diminishing the ensemble of desensitized states. The 4 TARPs encircle receptor ion channel, stabilizing M2 helices and pore loops, thus showing how TARPs alter receptor pore properties. Structural and computational analysis suggests the full agonist/modulator complex harbors an ionpermeable channel gate, thus providing the first view of an activated AMPA receptor.All rights reserved. No reuse allowed without permission.was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/158402 doi: bioRxiv preprint first posted online page 3In the mammalian central nervous system the majority of fast excitatory neurotransmission is initiated by -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive ionotropic glutamate receptors in complex with modulatory auxiliary subunits (Jackson and Nicoll, 2011;Traynelis et al., 2010). Transmembrane AMPA receptor regulatory proteins (TARPs) (Chen et al., 2000), the most widespread and well studied family of auxiliary proteins, alter AMPA receptor gating kinetics, ion channel properties and pharmacology (Milstein and Nicoll, 2008). The prototypical TARP, deemed stargazin or TARP 2, potentiates AMPA activity by decelerating deactivation and desensitization kinetics, facilitating recovery from desensitization, boosting the efficacy of partial agonists, and attenuating polyamine block (Milstein et al., 2007;Soto et al., 2007;Tomita et al., 2005).AMPA receptors have a modular architecture with synaptically localized amino terminal domains (ATDs) and ligand binding domains (LBDs), an ion channel forming transmembrane domain (TMD) and a largely unstructured cytoplasmic domain (CTD) (O'Hara et al., 1993;Soderling and Derkach, 2000;Stern-Bach et al., 1994;Wo and Oswald, 1995;Wollmuth and Sobolevsky, 2004). Extensive studies on isolated receptor domains and intact receptors have illuminated, at high resolution, how agonists induce local 'clamshell' closure of the LBDs and how the LBDs are arranged as nonequivalent pairs of A/C and B/D dimers within an overall 2-fold symmetric LBD 'gating ring' (Armstrong and Gouaux, 2000;Jin et al., 2009;Kuusinen et al., 1999;Sobolevsky et al., 2009;Sun et al., 2002). Although crystallograp...

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