{"doi":"10.1016/j.jbc.2022.101763","title":"Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms","abstract":"Voltage-gated sodium channels, Na V s, are responsible for the rapid rise of action potentials in excitable tissues. Na V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-Na V nanobodies (Nbs), Nb17 and Nb82, that recognize the Na V 1.4 (skeletal muscle) and Na V 1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of Na V 1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na V isoform has been challenging because they usually show undesired crossreactivity for different Na V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa V 1.4 or CTNa V 1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa V 1.4 and CTNa V 1.5 with high affinity (K D 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect Na V 1.4 and Na V 1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na V 1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na V reagents to capture Na V s from cell lysates and as molecular visualization agents for Na V s.","journal":"Journal of Biological Chemistry","year":2022,"id":270611,"datarank":0.3958585994422889,"base_score":2.639057329615259,"endowment":2.639057329615259,"self_citation_contribution":0.3958585994422889,"citation_network_contribution":0.0,"self_endowment_contribution":0.3958585994422889,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":13,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9625,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":935369,"name":"Vanina Alzogaray","orcid":null,"position":1,"is_corresponding":false},{"id":935370,"name":"Dakshnamurthy Selvakumar","orcid":null,"position":2,"is_corresponding":false},{"id":286187,"name":"Sara Nathan","orcid":"0000-0002-0090-0303","position":3,"is_corresponding":false},{"id":495686,"name":"Jesse B. Yoder","orcid":"0000-0002-3502-004X","position":4,"is_corresponding":false},{"id":618196,"name":"Katharine M. Wright","orcid":"0000-0001-9060-3849","position":5,"is_corresponding":false},{"id":746387,"name":"Sebastián Klinke","orcid":"0000-0002-8777-0870","position":6,"is_corresponding":false},{"id":934876,"name":"Justin N. Nwafor","orcid":"0000-0001-5348-6196","position":7,"is_corresponding":false},{"id":934877,"name":"María S. Labanda","orcid":"0000-0002-4414-9750","position":8,"is_corresponding":false},{"id":746388,"name":"Fernando A. Goldbaum","orcid":"0000-0001-6235-3002","position":9,"is_corresponding":false},{"id":229152,"name":"Arne Schön","orcid":"0000-0002-2015-2090","position":10,"is_corresponding":false},{"id":934878,"name":"Ernesto Freire","orcid":"0000-0002-2335-6419","position":11,"is_corresponding":false},{"id":144191,"name":"Gordon F. Tomaselli","orcid":null,"position":12,"is_corresponding":false},{"id":495689,"name":"L. Mario Amzel","orcid":"0000-0002-0129-9572","position":13,"is_corresponding":false},{"id":484278,"name":"Manu Ben‐Johny","orcid":"0000-0002-5645-0815","position":14,"is_corresponding":false},{"id":434249,"name":"Sandra B. Gabelli","orcid":"0000-0003-1205-5204","position":15,"is_corresponding":false},{"id":377368,"name":"Lakshmi Srinivasan","orcid":"0000-0002-6786-9523","position":0,"is_corresponding":true}],"reference_count":49,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T00:27:31.019242Z","pmid":"35202650","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":[]}