{"doi":"10.1016/j.abb.2025.110432","title":"Calcium-dependent levels of phospholamban pentamer in native heart membranes reflect interactions of monomers with calcium-free SERCA2a","abstract":"In sarcoplasmic reticulum (SR) membranes, phospholamban (PLB) exists in an equilibrium of non-inhibitory homopentamers (PLB 5 ) and inhibitory monomers (PLB 1 ) that bind to SERCA2a. A new approach is needed to determine the full scheme of interactions between PLB and SERCA2a in native cardiac SR membranes, which remains poorly understood. Dog cardiac SR membranes (dSR) were switched between EGTA and Ca 2+ buffers to convert SERCA2a between the Ca 2+ -free, E 2 and the high Ca 2+ -affinity, E 1 conformations. Reactions were stopped by SDS to preserve PLB 5 structures in dSR before immunoblotting. Converting SERCA2a from E 2 to E 1, Ca 2+ addition significantly increased PLB 5 /PLB 1 ratios, suggesting that PLB 1 is dissociated from E 1, and assembled into PLB 5 in dSR. This Ca 2+ -induced increase in PLB 5 /PLB 1 was reversed by the subsequent addition of EGTA, revealing the processes of PLB 1 binding to E 2 and disassembly of PLB 5 . In both cases, PLB 5 /PLB 1 reached new steady states in <2 s. Furthermore, PLB antibody eliminated Ca 2+ -dependent shifts in PLB 5 /PLB 1 . PLB phosphorylation caused similar leftward shifts in the Ca 2+ -dependent curve for PLB 5 /PLB 1 and Ca 2+ -ATPase activity. We have developed a simple, effective method and revealed that the levels of SERCA2a inhibition are controlled by an equilibrium between PLB 1 association with E 2 and its dissociation from E 1, and the formation of PLB 5 in native cardiac SR membranes. With intact regulatory components in their natural phospholipid environment, Ca 2+ -dependent shifts in PLB 5 /PLB 1 can expose PLB-SERCA2a protein-protein interactions in native membranes from normal and diseased hearts, in which proteomes and lipidomes are likely to vary. • μM Ca 2+ increases phospholamban pentamers (PLB 5 ) and reduces PLB 1 in native cardiac sarcoplasmic reticulum membranes (SR). • Phosphorylation, antibody, and thapsigargin alter the Ca 2+ -dependent PLB 5 /PLB 1 . • PLB 5 /PLB 1 reached a new Ca 2+ -dependent steady state in <2 s. • Ca 2+ dissociates PLB 1 bound to the Ca 2+ -free SERCA2a, forming PLB 5 in native SR.","journal":"Archives of Biochemistry and Biophysics","year":2025,"id":543833,"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":0.9548,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1434152,"name":"Kazuhiro Aonuma","orcid":null,"position":1,"is_corresponding":false},{"id":1434153,"name":"Richard Kovacs","orcid":null,"position":2,"is_corresponding":false},{"id":329559,"name":"Zhenhui Chen","orcid":"0000-0002-1781-5332","position":3,"is_corresponding":false},{"id":951270,"name":"Jin Guo","orcid":"0000-0002-0093-1927","position":0,"is_corresponding":true}],"reference_count":53,"raw_metadata":null,"created_at":"2026-07-19T02:53:08.338070Z","pmid":"40262694","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":[]}