{"doi":"10.1101/2021.10.07.463355","title":"Tuning the sensitivity of genetically encoded fluorescent potassium indicators through structure-guided and genome mining strategies","abstract":"Abstract Genetically encoded potassium indicators lack optimal binding affinity for monitoring intracellular dynamics in mammalian cells. Through structure-guided design and genome mining of potassium binding proteins, we developed green fluorescent potassium indicators with a broad range of binding affinities. KRaION1, based on the insertion of a potassium binding protein (Ec-Kbp) into the fluorescent protein mNeonGreen, exhibits an isotonically measured K d of 69±10 (mM; mean ± standard deviation used throughout). We identified Ec-Kbp’s binding site using NMR spectroscopy to detect protein-thallium scalar couplings and refined the structure of Ec-Kbp in its potassium-bound state. Guided by this structure, we modified KRaION1, yielding KRaION2, which exhibits an isotonically measured K d of 96±9 (mM). We identified four Ec-Kbp homologs as potassium binding proteins, which yielded indicators with isotonically measured binding affinities in the 39-112 (mM) range. KRaIONs expressed and functioned in HeLa cells, but exhibited lower K d values, which were mirrored by lower K d values measured in vitro when holding sodium constant. Thus, potassium indicator K d may need to be evaluated in the context of a given experimental goal.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":217863,"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":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9547,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":815727,"name":"Minghan Yang","orcid":null,"position":1,"is_corresponding":false},{"id":11613,"name":"Cuixin Lai","orcid":"0000-0003-4257-4013","position":2,"is_corresponding":false},{"id":815179,"name":"Lina Yang","orcid":"0009-0001-9204-3264","position":3,"is_corresponding":false},{"id":311266,"name":"Fedor V. Subach","orcid":"0000-0003-2720-7821","position":4,"is_corresponding":false},{"id":576883,"name":"Brian O. Smith","orcid":"0000-0003-3363-4168","position":5,"is_corresponding":false},{"id":11616,"name":"Kiryl D. Piatkevich","orcid":"0000-0002-7777-9468","position":6,"is_corresponding":false},{"id":11617,"name":"Edward S. Boyden","orcid":"0000-0002-0419-3351","position":7,"is_corresponding":false},{"id":815726,"name":"Cristina C. Torres Cabán","orcid":null,"position":0,"is_corresponding":true}],"reference_count":46,"raw_metadata":null,"created_at":"2026-07-18T23:53:24.683898Z","pmid":null,"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":[]}