{"doi":"10.1101/2023.07.07.548138","title":"Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State","abstract":"Abstract Ca 2+ 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 2+ , DPc10 decreased both FRET max (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca 2+ , DPc10 decreased FRET max 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 2+ and lengthens D-FKBP/A-CaM distances independent of [Ca 2+ ]. 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 2+ . 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":400057,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9498,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1137512,"name":"Florentin R. Nitu","orcid":"0000-0001-9768-6333","position":1,"is_corresponding":false},{"id":417546,"name":"Robyn T. Rebbeck","orcid":"0000-0003-3840-2286","position":2,"is_corresponding":false},{"id":788141,"name":"Lindsey M. McGurran","orcid":"0000-0002-0708-1312","position":3,"is_corresponding":false},{"id":542859,"name":"Tetsuro Oda","orcid":"0000-0003-3307-1000","position":4,"is_corresponding":false},{"id":417547,"name":"David D. Thomas","orcid":"0000-0002-8822-2040","position":5,"is_corresponding":false},{"id":229037,"name":"Donald M. Bers","orcid":"0000-0002-2237-9483","position":6,"is_corresponding":false},{"id":417548,"name":"Rǎzvan L. Cornea","orcid":"0000-0001-5739-0992","position":7,"is_corresponding":false},{"id":430804,"name":"Bengt Svensson","orcid":"0000-0003-3932-2376","position":0,"is_corresponding":true}],"reference_count":39,"raw_metadata":null,"created_at":"2026-07-19T01:20:00.023537Z","pmid":"37461514","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}