Abstract

Ca2+-dependent repair of plasma membrane breaches is essential for animal cell viability. An initial passive influx of extracellular Ca2+ triggers the formation of a protein plug that rapidly seals breaches. However, the mechanism of extracellular Ca2+ requirement for subsequent repair remains undefined. EHD2 protein stabilizes the plasma membrane caveolae, which sustain membrane repair, and maintains high surface levels of the caveolae-resident Ca2+ channel Orai1. We establish the requirement of both Orai1 and EHD2 for repair of plasma membrane lesions induced by mechanical injury or by a model bacterial pore-forming toxin. We demonstrate rapid EHD2 recruitment and Orai1-mediated Ca2+ entry at plasma membrane sites of localized mechanical stimulus, the latter requiring EHD2 and CAV1. EHD2 and Orai1 are necessary for mechanosensitive YAP/TAZ-TEAD activation and positive feedback for CAV1 expression that promotes membrane repair. Our studies establish EHD2 and Orai1 as novel components of mammalian plasma membrane repair and mechanoadaptation. ### Competing Interest Statement KAS is an employee of CalciMedica Inc., which is advancing Orai1 inhibitors into clinic. United States Department of Defense, https://ror.org/0447fe631, W81XWH-17-1-0616, W81XWH-20-1-0058, W81XWH-20-1-0546, HT94252410337, HT9425-23-1-0052 NIH Common Fund, https://ror.org/001d55x84, R21CA297629, R21CA241055, R03CA253193, P20 GM121316 Nebraska Department of Health and Human Services, https://ror.org/03m7azy07, 18123-Y3 Fred and Pamela Buffett Cancer Center, https://ror.org/03n63wv08, P30CA036727