Abstract
Gap junctions, formed by connexin proteins, establish direct electrical and metabolic coupling between cells, enabling coordinated tissue responses. These channels universally respond to intracellular pH changes, closing under acidic conditions to limit the spread of cytotoxic signals during cellular stress, such as ischemia. Using cryo-electron microscopy (cryo-EM), we uncover insights into the structural mechanism of pH-gating in native lens connexin-46/50 (Cx46/50) gap junctions. Mild acidification drives lipid infiltration into the channel pore, displacing the N-terminal (NT) domain and stabilizing pore closure. Lipid involvement is shown to be both essential and fully reversible. Structural transitions involve an ensemble of gated states formed through non-cooperative NT domain movement as well as minor populations of a distinct destabilized open-state. These findings provide molecular insights into pH-gating dynamics, illustrating how structural changes may regulate gap junction function under cellular stress and linking Cx46/50 dysregulation to age-related cataract formation.
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Data availability
Cryo-EM density maps have been deposited to the Electron Microscopy Data Bank (EMDB) under accession numbers: EMD-73962; EMD-73957; EMD-73942; EMD-73965; EMD-73876; EMD-73900; EMD-73896; and EMD-73885. Coordinates for atomic models have been deposited to Protein Data Bank (PDB) under accession numbers: 9Z9X; 9Z9Y; 9Z9S; 9Z9W; 9Z9G; 9Z9H; 9ZA3; 9ZA4; 9Z7P; 9Z7W; 9Z8M; 9Z9B; 9Z8F; 9Z8L; 9Z81; and 9Z82. The original raw multi-frame micrographs have been deposited to EMPIAR under accession numbers: EMPIAR-13117; EMPIAR-13114; EMPIAR-13131; EMPIAR-13130; EMPIAR-13122; and EMPIAR-13116. Previously published models used for modeling and comparative analysis can be found at: 7JKC; 7JJP; 6UVT; 7QEQ; 8QA0; 7XKT; 8IYG; 7XNH; 7F92; 7F93; 7XQF; and 7XQB. The source data underlying Figs. 1i, 5b, 6e, 6f and 7b are provided as a Source Data file. Source data are provided with this paper.
Code availability
In-house code developed for the analysis of image processing meta data have been deposited to the Reichow Lab GitHub [https://github.com/reichow-lab] and Zenodo [https://zenodo.org/records/17634922] and [https://zenodo.org/records/17634918].
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Acknowledgements
We thank Dr. Thomas White for helpful discussions. We are grateful for the instrumentation access and training provided by Dr. Claudia López and the staff at the OHSU Multiscale Microscopy Core, the OHSU Advanced Computing Center (supported by NIH Grant S10OD034224), and the Pacific Northwest Cryo-EM Center (supported by NIH Grant R24GM154185). The research was funded by NIH grant R35GM124779 (to S.L.R.)
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J.M.J. designed and executed the nanodisc experiments. J.B.M. designed and executed the amphipol experiments. J.M.J. and J.B.M. collected the cryo-EM data. J.M.J. performed image processing and model building. J.M.J. and S.L.R. contributed to data analysis and prepared the initial manuscript. All authors contributed to the final revision of the manuscript.
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Jarodsky, J.M., Myers, J.B. & Reichow, S.L. Reversible lipid-mediated pH-gating of connexin-46/50 by cryo-EM. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68311-9
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DOI: https://doi.org/10.1038/s41467-026-68311-9
