BPS2026 – Single-molecule imaging of AMPARs with noncompetitive inhibitors by high-speed AFM

Structural biology elucidates the three-dimensional atomic structures of proteins, illuminating their molecular mechanisms. Moreover, visualization of protein flexibility and dynamics improves our understanding of how proteins function. High-speed atomic force microscopy (HS-AFM) is a powerful technique for directly imaging flexible regions of single molecules under near-physiological conditions, with a temporal resolution of ∼ 100 millisecond at a nanometer resolution Ando T. et al. , Chem. Rev. 114, 3120–3188 (2014), Tsujioka S. et al., Sci. Adv. 9, eadh1069 (2023). α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptors (AMPARs) mediate fast excitatory synaptic transmission by localizing the postsynaptic density of glutamatergic synapses. AMPARs are important targets for antiepileptic drug development because of their central role in neuronal excitation. Recent studies have shown that noncompetitive AMPAR inhibitors, such as perampanel (PER), are clinically effective antiepileptic agents. However, the nanoscale dynamics of AMPARs during inhibition by noncompetitive antagonists remain poorly understood. In this study, we used HS-AFM to directly visualize the conformational dynamics of the GluA2 subunit of AMPARs complexed with TARP γ2 in lipid environments, in the presence and absence of inhibitors. HS-AFM videos of GluA2-TARP γ2 in resting and open states revealed fluctuations in large extracellular N-terminal domains (NTDs) dimers. In contrast, in the desensitized state, the two NTD dimers adopted a separate conformation with reduced fluctuations. Notably, we also observed individual NTD dimers transitioning into monomers. This NTD-dimer splitting facilitated intersubunit exchange between NTD dimers A. Sumino et al. , ACS Nano 18, 25018–25035 (2024). Furthermore, our findings indicate that the dimeric structure of the flexible NTDs frequently splits into monomers upon binding with PER. Together, these results underscore the significance of NTD dynamics for synaptic clustering of AMPARs and for inhibition mechanisms by noncompetitive antagonists.