| Summary: | Summary: Gap junction channels (GJCs) mediate intercellular communication and are gated by numerous conditions such as pH. The electron cryomicroscopy (cryo-EM) structure of Cx26 GJC at physiological pH recapitulates previous GJC structures in lipid bilayers. At pH 6.4, we identify two conformational states, one resembling the open physiological-pH structure and a closed conformation that displays six threads of density, that join to form a pore-occluding density. Crosslinking and hydrogen-deuterium exchange mass spectrometry reveal closer association between the N-terminal (NT) domains and the cytoplasmic loops (CL) at acidic pH. Previous electrophysiologic studies suggest an association between NT residue N14 and H100 near M2, which may trigger the observed movement of M2 toward M1 in our cryo-EM maps, thereby accounting for additional NT-CL crosslinks at acidic pH. We propose that these pH-induced interactions and conformational changes result in extension, ordering, and association of the acetylated NT domains to form a hexameric “ball-and-chain” gating particle. : During tissue injury, acidic pH blocks gap junction channel intercellular communication. Khan et al. use cryo-EM and mass spectrometry to show that acidification causes extension, ordering, and association of the N-terminal domains to form a hexameric gating particle that sterically occludes the human Cx26 gap junction channel pore. Keywords: connexins, gap junction channels, pH gating, cryo-EM, mass spectrometry, hydrogen deuterium exchange, crosslinking, membrane proteins, protein structure, conformational changes
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