Significance Gloeobacter violaceus ligand-gated ion channel (GLIC) is a proton-gated, cation-selective channel, a Cys-loop family member of the pentameric ligand-gated ion channels (pLGICs). In a pursuit to provide evidence of function-related conformational changes of a pLGIC in membrane, at ambient temperature and pressure and of the very same unlabeled molecules, we used high-speed atomic force microscopy (HS-AFM) combined with a buffer exchange system to visualize reversible changes of the supramolecular arrangement and conformations of GLIC during pH changes: a structural titration experiment. Reference-free classification of the molecules assigns conformations to states during the pH titration. We find large conformational changes with an unexpected closed-state structure with tightened extracellular domains. Furthermore, we provide evidence for the short-term existence of asymmetric channels at early stages of activation.