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leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide (DPc10) binding promotes leaky RyR2 in cardiomyocytes and reports on that endogenous state. Conversely, calmodulin (CaM) binding inhibits RyR2 leak and low CaM affinity is diagnostic of leaky RyR2. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. We used FRET to clarify 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 Ca2+, DPc10 decreased both FRETmax (under saturating [A-CaM]) and the CaM\/RyR2 binding affinity. In micromolar Ca2+, DPc10 decreased FRETmax without affecting CaM\/RyR2 binding affinity. This correlates with the analysis of fluorescence-lifetime-detected FRET, indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca2+ and lengthens D-FKBP\/A-CaM distances independent of [Ca2+]. To observe DPc10\/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10\/RyR2 binding, with this effect being larger in micromolar versus nanomolar Ca2+. Moreover, A-DPc10\/RyR2 binding is cooperative in a CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by the analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.<\/jats:p>","DOI":"10.3390\/ijms241612547","type":"journal-article","created":{"date-parts":[[2023,8,8]],"date-time":"2023-08-08T12:26:39Z","timestamp":1691497599000},"page":"12547","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3932-2376","authenticated-orcid":false,"given":"Bengt","family":"Svensson","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9768-6333","authenticated-orcid":false,"given":"Florentin R.","family":"Nitu","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3840-2286","authenticated-orcid":false,"given":"Robyn T.","family":"Rebbeck","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lindsey M.","family":"McGurran","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tetsuro","family":"Oda","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, University of California, Davis, CA 95616, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8822-2040","authenticated-orcid":false,"given":"David D.","family":"Thomas","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2237-9483","authenticated-orcid":false,"given":"Donald M.","family":"Bers","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, University of California, Davis, CA 95616, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5739-0992","authenticated-orcid":false,"given":"Razvan L.","family":"Cornea","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,8,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1146\/annurev-physiol-020911-153308","article-title":"Cardiac sarcoplasmic reticulum calcium leak: Basis and roles in cardiac dysfunction","volume":"76","author":"Bers","year":"2014","journal-title":"Annu. 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