{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T20:05:26Z","timestamp":1768507526850,"version":"3.49.0"},"posted":{"date-parts":[[2023,7,9]]},"group-title":"Biophysics","reference-count":38,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,7,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Ca\n                  <jats:sup>2+<\/jats:sup>\n                  leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide, (DPc10) detects leaky RyR2 in cardiomyocytes. Conversely, calmodulin (CaM) inhibits RyR2 leak. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. Here we used FRET to understand the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca\n                  <jats:sup>2+<\/jats:sup>\n                  , DPc10 decreased both FRET\n                  <jats:sub>max<\/jats:sub>\n                  (under saturating [A-CaM]) and the CaM\/RyR2 binding affinity. In micromolar Ca\n                  <jats:sup>2+<\/jats:sup>\n                  , DPc10 decreased FRET\n                  <jats:sub>max<\/jats:sub>\n                  without affecting CaM\/RyR2 binding affinity. This correlates with analysis of fluorescence-lifetime-detected FRET indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca\n                  <jats:sup>2+<\/jats:sup>\n                  and lengthens D-FKBP\/A-CaM distances independent of [Ca\n                  <jats:sup>2+<\/jats:sup>\n                  ]. To observe DPc10\/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10\/RyR2 binding, this effect being larger in micromolar vs. nanomolar Ca\n                  <jats:sup>2+<\/jats:sup>\n                  . Moreover, A-DPc10\/RyR2 binding is cooperative in CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.\n                <\/jats:p>","DOI":"10.1101\/2023.07.07.548138","type":"posted-content","created":{"date-parts":[[2023,7,9]],"date-time":"2023-07-09T04:20:12Z","timestamp":1688876412000},"source":"Crossref","is-referenced-by-count":1,"title":["Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3932-2376","authenticated-orcid":false,"given":"Bengt","family":"Svensson","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9768-6333","authenticated-orcid":false,"given":"Florentin R.","family":"Nitu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3840-2286","authenticated-orcid":false,"given":"Robyn T.","family":"Rebbeck","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lindsey M.","family":"McGurran","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tetsuro","family":"Oda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8822-2040","authenticated-orcid":false,"given":"David D.","family":"Thomas","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2237-9483","authenticated-orcid":false,"given":"Donald M.","family":"Bers","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5739-0992","authenticated-orcid":false,"given":"Razvan 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