Zk200775

(S)-AMPA (Sigma-Aldrich, MO) and ZK 200775, a competitive AMPA receptor antagonist (Tocris Bioscience, MO) were dissolved in artificial cerebrospinal fluid (aCSF) consisting of (in mM): 148 NaCl, 2.7 KCl, 1.2 CaCl₂, and 0.85 MgCl₂, pH adjusted to 7.0–7.5.

To achieve heteromeric AMPAR expression, FLAG-tagged GluA1 and GluA2-TARPγ8-eGFP tandem plasmids were co-transfected into HEK-Expi293^TM cells at a ratio of 1:1. To prevent AMPA-mediated excitotoxicity, AMPAR antagonists ZK200775 (2 nM, Tocris, Cat# 2345) and kynurenic acid (0.1 mM, Sigma, Cat# K335-5G) were added to the culture medium. 36-44 hours post-transfection, cells were harvested and lysed for 3 hours in lysis buffer containing: 25 mM Tris pH 8, 150 mM NaCl, 0.6% digitonin (w/v) (Sigma, Cat# 300410-5 G), 5 μM NBQX, 1 mM PMSF, 1× Protease Inhibitor (Roche, Cat# 05056489001). Insoluble material was then removed by ultracentrifugation (131,000 × g, 1 h, rotor 45-50 Ti) and the clarified lysate incubated with anti-GFP beads for 3 h. After washing with glyco-diosgenin (GDN) (Anatrace, Cat# GDN101) buffer (25 mM Tris pH 8, 150 mM NaCl, 0.02% GDN) the protein was eluted from the beads by digestion with 0.01 mg/ml 3 C protease at 4 °C overnight. Eluted fractions were incubated with ANTI-FLAG M2 affinity gel (Sigma, Cat# A2220) for 1.5 h and washed 3 times with GDN buffer. Finally, the complex was eluted using 0.15 mg/ml 3×FLAG peptide (Millipore Cat# F4799) in GDN buffer. Eluted fractions were pooled and concentrated to ~2.5 mg/ml for cryo-EM grid preparation.

Cryo-EM grids were prepared using an FEI Vitrobot Mark IV. For the resting state, protein was incubated with 300 μM ZK200775 (Tocris, catalogue no. 2345) for at least 30 min on ice before freezing. For the active state, GluA1/γ3 homomeric complex, protein was first incubated with 300 μM cyclothiazide (Tocris, catalogue no. 0713) for at least 30 min on ice and then quickly mixed with 1 M l-glutamate stock solution to a final concentration of 100 mM before loading onto the grids. For desensitized structures, 10 mM quisqualate (Tocris, catalogue no. 0188) was quickly added to protein to a final concentration of 1 mM before loading. Quantifoil Au 1.2/1.3 (300 mesh) or Quantifoil Cu 1.2/1.3 (300 mesh) grids were glow-discharged for 30 s before use. A 3 μl sample was applied to the grids, blotted for 4.5–6 s at 4 °C with 100% humidity and plunge-frozen in liquid ethane.
All cryo-EM data were collected on an FEI Titan Krios operated at 300 kV, equipped with a K3 detector (Gatan) and a GIF Quantum energy filter (slit width 20 eV). Videos at 1.5–2.5 μm underfocus were taken in counting mode with a pixel size of 1.06 Å per pixel or 0.826 Å per pixel. A combined total dose of 50 e/Ų was applied with each exposure and 50 frames were recorded for each video. All datasets were collected using EPU2.