Architecture of fully occupied Glua2 Ampa receptor – TARP complex elucidated by single particle cryo-electron microscopy

Zhao, Yan; Chen, Shanshuang; Yoshioka, Craig; Baconguis, Isabelle; Gouaux, Eric

doi:10.1101/060046

Cited by 3 publications

(2 citation statements)

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“…In particular, digitonin stabilizes AMPAR–Stg complex (Zhao et al . ). Neither of these detergents produced significant improvements in complex stability of C528L, L789F or A793F (Fig.…”

Section: Resultsmentioning

confidence: 97%

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Engineering defined membrane‐embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin

Hawken

Zaika

Nakagawa

2017

The Journal of Physiology

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During excitatory synaptic transmission, various structurally unrelated transmembrane auxiliary subunits control the function of AMPA receptors (AMPARs), but the underlying mechanisms remain unclear. We identified lipid-exposed residues in the transmembrane domain (TMD) of the GluA2 subunit of AMPARs that are critical for the function of AMPAR auxiliary subunits, stargazin (Stg) and cornichon 3 (CNIH3). These residues are essential for stabilizing the AMPAR-CNIH3 complex in detergents and overlap with the contacts made between GluA2 TMD and Stg in the cryoEM structures. Mutating these residues had opposite effects on gating modulation and complex stability when Stg- and CNIH3-bound AMPARs were compared. Specifically, in detergent the GluA2-A793F formed an unstable complex with CNIIH3 but in the membrane the GluA2-A793F-CNIH3 complex expressed a gain of function. In contrast, the GluA2-A793F-Stg complex was stable, but had diminished gating modulation. GluA2-C528L destabilized the AMPAR-CNIH3 complex but stabilized the AMPAR-Stg complex, with overall loss of function in gating modulation. Furthermore, loss-of-function mutations in this TMD region cancelled the effects of a gain-of-function Stg carrying mutation in its extracellular loop, demonstrating that both the extracellular and the TMD elements contribute independently to gating modulation. The elements of AMPAR functionally recruited by auxiliary subunits are, therefore, located not only in the extracellular domains but also in the lipid accessible surface of the AMPAR. The TMD surface we defined is a potential target for auxiliary subunit-specific compounds, because engineering of this hotspot induces opposing functional outcomes by Stg and CNIH3. The collection of mutant-phenotype mapping provides a framework for engineering AMPAR gating using auxiliary subunits.

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“…In particular, digitonin stabilizes AMPAR–Stg complex (Zhao et al . ). Neither of these detergents produced significant improvements in complex stability of C528L, L789F or A793F (Fig.…”

Section: Resultsmentioning

confidence: 97%

“…; Zhao et al . ). The intein approach reported here has further advantages over the traditional GluA2–Stg fusion constructs because the two entities are expressed separately and fold at much higher efficiencies than the GluA2–Stg fusing (data not shown).…”

Section: Discussionmentioning

confidence: 97%

Engineering defined membrane‐embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin

Hawken

Zaika

Nakagawa

2017

The Journal of Physiology

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Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

Poulsen

Poshtiban

Klippenstein

et al. 2018

Preprint

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Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission 11 throughout the nervous system. Conformational changes of the transmembrane domain 12 (TMD) underlying ion channel activation and desensitization remain poorly understood.13 Here, we explored the dynamics of the TMD of AMPA-type iGluRs using genetically-14 encoded unnatural amino acid (UAA) photo-crosslinkers, p-benzoyl-L-phenylalanine 15 (BzF) and p-azido-L-phenylalanine (AzF). We introduced UAAs at sites throughout the 16 TMD of the GluA2 receptor and characterized these mutants in patch-clamp recordings, 17 exposing them to glutamate and UV light. This approach revealed a range of optical 18 effects on the activity of mutant receptors. We found evidence that an interaction 19 between the Pre-M1 and the M4 TMD helix was essential for normal activation and 20 desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter, had 21 extraordinarily broad effects on gating and desensitization. This observation suggests 22 coupling to other parts of the receptor and like in other tetrameric channels, selectivity 23 filter gating.

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Measurements of the timescale and conformational space of AMPA receptor desensitization

Salazar

Mischke

Plested

2019

Preprint

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8 Ionotropic glutamate receptors (iGluRs) are ligand gated ion channels that mediate excitatory 9 synaptic transmission in the central nervous system (CNS). Desensitization of the AMPA-10 subtype following glutamate binding appears critical for brain function, and involves 11 rearrangement of the ligand binding domains (LBDs). Recently, several full-length structures 12 of iGluRs in putative desensitized states were published. These structures indicate movements 13 of the LBDs that might be trapped by cysteine crosslinks and metal bridges. We found that 14 cysteine mutants at the interface between subunits A and C, and lateral zinc bridges (between 15 subunits C & D or A & B) can trap freely-desensitizing receptors in a spectrum of states with 16 different stabilities. Consistent with close approach of subunits during desensitization 17 processes, introduction of bulky amino acids at the A-C interface produced a receptor with slow 18 recovery from desensitization. Further, in wild-type GluA2 receptors, we detected population 19 of stable desensitized state with a lifetime around 1 second. Using mutations that progressively 20 stabilise deep desensitize states (E713T & Y768R), we were able to selectively protect receptors 21 from crosslinks at both the diagonal and lateral interfaces. Ultrafast perfusion enabled us to 22 perform chemical modification in less than 10 ms, reporting movements associated to 23 desensitization on this timescale within LBD dimers in resting receptors. These observations 24 suggest small disruptions of quaternary structure are sufficient for fast desensitization, and 25 that substantial rearrangements likely correspond to stable desensitized states that are adopted 26 relatively slowly, on a timescale much longer than physiological receptor activation. 28Significance statement 29 iGluRs are central components of fast synaptic transmission in the brain. iGluR desensitization occurs as a 30 natural consequence of receptor activation and can reduce the response of an excitatory synapse. AMPA receptor 31 desensitization also appears necessary for proper brain development. Molecular structures of iGluRs in putative 32 desensitized states predict a range of movements during desensitization. In the present study, we performed a 33 series of crosslinking experiments on mutant receptors that we subjected to similar desensitizing conditions 34 over time periods from milliseconds to minutes. These experiments allowed us to count desensitized 35 configurations and rank them according to their stabilities. These data show that large-scale rearrangements 36 occur during long glutamate exposures that are probably not seen in healthy brain tissue, whereas smaller 37 changes in structure probably suffice for desensitization at synapses. 39crosslinked very stably between A & C subunits by the A665C disulfide bond (Lau et al., 2013). Using 79 a fast perfusion system, we used several approaches on mutant and wild-type receptors to count 80 conformational states attained during desensitization. Comparing pot...

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Architecture of fully occupied Glua2 Ampa receptor – TARP complex elucidated by single particle cryo-electron microscopy

Cited by 3 publications

References 44 publications

Engineering defined membrane‐embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin

Engineering defined membrane‐embedded elements of AMPA receptor induces opposing gating modulation by cornichon 3 and stargazin

Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

Measurements of the timescale and conformational space of AMPA receptor desensitization

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