article published yes Hadil Najjar author Sarah Weiß author Ferdinand Horvath author b0dc7f61-21a3-11ef-a9b4-e6ab1aa6f21e Valentina Hopl author Adéla Tiffner author Lorenz Höbarth author Julia Söllner author Maximilian Fröhlich author Magdalena Prantl author Nora Müller author Yuliia Nazarenko author Selina Harant author Lukas Weissenböck author Herwig Grabmayr author Matthias Sallinger author Lena Maltan author Linda V. Echefu author Tamara Radiskovic author Melanie Leopold author Sonja Lindinger author Christina Humer author Carmen Höglinger author Heinrich Krobath author Thomas Renger author Isabella Derler author AnSa department The Ca2+-release-activated Ca2+ (CRAC) channel Orai1 is activated by interaction with the Ca2+ sensor Stromal Interaction Molecule 1 (STIM1). Owing to the lack of structurally resolved Orai1/STIM1 complexes, the impact of their coupling on individual Orai1 transmembrane domain (TM) movements is unclear. This study investigates STIM1-independent and STIM1-dependent Orai1-TM dynamics using photocrosslinking unnatural amino acids (UAAs) at each individual TM position. We primarily identify CRAC-channel-like currents directly after UAA incorporation or additional UV-light irradiation at TM3 sites that interface with non-pore-lining TMs. Using UAAs combined with conventional site-directed mutagenesis and molecular dynamics simulations, we discover that pore opening involves a widening of interfaces formed by TM3 with non-pore-lining TMs. Orai1 mutants with a UAA in TM3 exhibit weaker STIM1-induced activation after UV exposure, possibly caused by a restricted widening of non-pore-lining TM interfaces. We demonstrate that photocrosslinking UAAs are excellent tools for improving our understanding of key determinants and ion channel dynamics modulating pore opening. https://research-explorer.ista.ac.at/download/19846/19868/2025_CellReportsPhysicalScience_Najjar.pdf application/pdfno https://creativecommons.org/licenses/by/4.0/ Elsevier2025 eng 2666-3864 00151657050000910.1016/j.xcrp.2025.102623 66 Najjar, H., Weiß, S., Horvath, F., Hopl, V., Tiffner, A., Höbarth, L., … Derler, I. (2025). STIM1-induced widening of non-pore-lining TM interfaces is crucial for Orai1 pore opening. <i>Cell Reports Physical Science</i>. Elsevier. <a href="https://doi.org/10.1016/j.xcrp.2025.102623">https://doi.org/10.1016/j.xcrp.2025.102623</a> H. Najjar <i>et al.</i>, “STIM1-induced widening of non-pore-lining TM interfaces is crucial for Orai1 pore opening,” <i>Cell Reports Physical Science</i>, vol. 6, no. 6. Elsevier, 2025. Najjar H, Weiß S, Horvath F, Hopl V, Tiffner A, Höbarth L, Söllner J, Fröhlich M, Prantl M, Müller N, Nazarenko Y, Harant S, Weissenböck L, Grabmayr H, Sallinger M, Maltan L, Echefu LV, Radiskovic T, Leopold M, Lindinger S, Humer C, Höglinger C, Krobath H, Renger T, Derler I. 2025. STIM1-induced widening of non-pore-lining TM interfaces is crucial for Orai1 pore opening. Cell Reports Physical Science. 6(6), 102623. H. Najjar, S. Weiß, F. Horvath, V. Hopl, A. Tiffner, L. Höbarth, J. Söllner, M. Fröhlich, M. Prantl, N. Müller, Y. Nazarenko, S. Harant, L. Weissenböck, H. Grabmayr, M. Sallinger, L. Maltan, L.V. Echefu, T. Radiskovic, M. Leopold, S. Lindinger, C. Humer, C. Höglinger, H. Krobath, T. Renger, I. Derler, Cell Reports Physical Science 6 (2025). Najjar H, Weiß S, Horvath F, et al. STIM1-induced widening of non-pore-lining TM interfaces is crucial for Orai1 pore opening. <i>Cell Reports Physical Science</i>. 2025;6(6). doi:<a href="https://doi.org/10.1016/j.xcrp.2025.102623">10.1016/j.xcrp.2025.102623</a> Najjar, Hadil, Sarah Weiß, Ferdinand Horvath, Valentina Hopl, Adéla Tiffner, Lorenz Höbarth, Julia Söllner, et al. “STIM1-Induced Widening of Non-Pore-Lining TM Interfaces Is Crucial for Orai1 Pore Opening.” <i>Cell Reports Physical Science</i>. Elsevier, 2025. <a href="https://doi.org/10.1016/j.xcrp.2025.102623">https://doi.org/10.1016/j.xcrp.2025.102623</a>. Najjar, Hadil, et al. “STIM1-Induced Widening of Non-Pore-Lining TM Interfaces Is Crucial for Orai1 Pore Opening.” <i>Cell Reports Physical Science</i>, vol. 6, no. 6, 102623, Elsevier, 2025, doi:<a href="https://doi.org/10.1016/j.xcrp.2025.102623">10.1016/j.xcrp.2025.102623</a>. 198462025-06-15T22:01:31Z2025-09-30T12:53:15Z