Two-pore channel-2 controls calmodulin-dependent STIM1 inactivation

Two-pore channel-2 controls calmodulin-dependent STIM1 inactivation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Two-pore channel-2 controls calmodulin-dependent STIM1 inactivation Nicolas Demaurex, Subo Lee, Raphael Néré, Kyung-Sun Park, Kyu-Sang Park This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8701585/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Lysosomes and the endoplasmic reticulum (ER) are Ca 2+ stores that interact to generate Ca 2+ signals regulating fundamental cellular processes. NAADP-sensitive TPC2 channels on lysosomes induce local Ca 2+ elevations that sensitize ER Ca 2+ release channels, triggering global Ca 2+ signals. The ensuing ER Ca 2+ depletion activates STIM1-gated Orai1 channels that mediate store-operated Ca 2+ entry (SOCE) to sustain long-lasting Ca 2+ signals. How TPC2 channels interact with STIM1 to integrate distinct intra and extracellular cues is unclear. Here, we show that TPC2 activation inhibits SOCE by enforcing rapid and persistent Ca 2+ -CaM-dependent inactivation of the STIM-Orai activating region (SOAR). The TPC2 agonists NAADP and TPC2-A1-N abrogated SOCE in multiple cell lines and enhanced the slow Ca 2+ dependent inactivation (SCDI) of STIM1-gated Orai1 channels. TPC2 engagement triggered lysosomal Ca 2+ release and mobilized ER Ca2+ stores independently of inositol trisphosphate receptors but prevented RFP-STIM1 recruitment to the TIRF plane by thapsigargin and disassembled RFP-STIM1 clusters forming after store depletion, preventing and acutely reversing SOCE. These effects persisted in STIM1 mutants truncated after the SOAR and were prevented by genetic or pharmacological invalidation of TPC2, Calmodulin (CaM) inhibition, and cytosolic Ca 2+ chelation. We conclude that Ca 2+ ions released by TPC2 channels on lysosomes regulate CaM-dependent STIM1 inactivation. Biological sciences/Cell biology/Cell signalling/Calcium signalling Biological sciences/Cell biology/Organelles/Lysosomes Full Text Additional Declarations There is NO Competing Interest. Supplementary Files TPC2controlscalmodulindependentSTIM1inactivationGraphicalabstract.pdf graphical abstract Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8701585","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":583624212,"identity":"c5f0f704-9dfd-4002-bdb6-b779bd51667b","order_by":0,"name":"Nicolas 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Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8701585/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8701585/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Lysosomes and the endoplasmic reticulum (ER) are Ca\u003csup\u003e2+\u003c/sup\u003e stores that interact to generate Ca\u003csup\u003e2+\u003c/sup\u003e signals regulating fundamental cellular processes. NAADP-sensitive TPC2 channels on lysosomes induce local Ca\u003csup\u003e2+\u003c/sup\u003e elevations that sensitize ER Ca\u003csup\u003e2+\u003c/sup\u003e release channels, triggering global Ca\u003csup\u003e2+\u003c/sup\u003e signals. The ensuing ER Ca\u003csup\u003e2+\u003c/sup\u003e depletion activates STIM1-gated Orai1 channels that mediate store-operated Ca\u003csup\u003e2+\u003c/sup\u003e entry (SOCE) to sustain long-lasting Ca\u003csup\u003e2+\u003c/sup\u003e signals. How TPC2 channels interact with STIM1 to integrate distinct intra and extracellular cues is unclear. Here, we show that TPC2 activation inhibits SOCE by enforcing rapid and persistent Ca\u003csup\u003e2+\u003c/sup\u003e-CaM-dependent inactivation of the STIM-Orai activating region (SOAR). The TPC2 agonists NAADP and TPC2-A1-N abrogated SOCE in multiple cell lines and enhanced the slow Ca\u003csup\u003e2+\u003c/sup\u003e dependent inactivation (SCDI) of STIM1-gated Orai1 channels. TPC2 engagement triggered lysosomal Ca\u003csup\u003e2+\u003c/sup\u003e release and mobilized ER Ca2+ stores independently of inositol trisphosphate receptors but prevented RFP-STIM1 recruitment to the TIRF plane by thapsigargin and disassembled RFP-STIM1 clusters forming after store depletion, preventing and acutely reversing SOCE. These effects persisted in STIM1 mutants truncated after the SOAR and were prevented by genetic or pharmacological invalidation of TPC2, Calmodulin (CaM) inhibition, and cytosolic Ca\u003csup\u003e2+\u003c/sup\u003e chelation. We conclude that Ca\u003csup\u003e2+\u003c/sup\u003e ions released by TPC2 channels on lysosomes regulate CaM-dependent STIM1 inactivation.","manuscriptTitle":"Two-pore channel-2 controls calmodulin-dependent STIM1 inactivation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-03 05:30:19","doi":"10.21203/rs.3.rs-8701585/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"86581ddf-a7ac-4307-ae3f-c7cd715ccc64","owner":[],"postedDate":"February 3rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62094889,"name":"Biological sciences/Cell biology/Cell signalling/Calcium signalling"},{"id":62094890,"name":"Biological sciences/Cell biology/Organelles/Lysosomes"}],"tags":[],"updatedAt":"2026-04-24T13:41:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-03 05:30:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8701585","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8701585","identity":"rs-8701585","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}