TY - JOUR T1 - Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations JF - Life Science Alliance JO - Life Sci. Alliance DO - 10.26508/lsa.202101011 VL - 4 IS - 8 SP - e202101011 AU - Urška Rovšnik AU - Yuxuan Zhuang AU - Björn O Forsberg AU - Marta Carroni AU - Linnea Yvonnesdotter AU - Rebecca J Howard AU - Erik Lindahl Y1 - 2021/08/01 UR - https://www.life-science-alliance.org/content/4/8/e202101011.abstract N2 - Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here, we report cryo-electron microscopy (cryo-EM) structures of the proton-activated Gloeobacter violaceus ligand-gated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the β1–β2 and M2–M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryo-EM classes further indicated a low-pH population with an expanded pore. These results allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family. ER -