{"doi":"10.1085/jgp.202113039","title":"Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump","abstract":"The essential transmembrane Na+ and K+ gradients in animal cells are established by the Na+/K+ pump, a P-type ATPase that exports three Na+ and imports two K+ per ATP hydrolyzed. The mechanism by which the Na+/K+ pump distinguishes between Na+ and K+ at the two membrane sides is poorly understood. Crystal structures identify two sites (sites I and II) that bind Na+ or K+ and a third (site III) specific for Na+. The side chain of a conserved tyrosine at site III of the catalytic α-subunit (Xenopus-α1 Y780) has been proposed to contribute to Na+ binding by cation-π interaction. We substituted Y780 with natural and unnatural amino acids, expressed the mutants in Xenopus oocytes and COS-1 cells, and used electrophysiology and biochemistry to evaluate their function. Substitutions disrupting H-bonds impaired Na+ interaction, while Y780Q strengthened it, likely by H-bond formation. Utilizing the non-sense suppression method previously used to incorporate unnatural derivatives in ion channels, we were able to analyze Na+/K+ pumps with fluorinated tyrosine or phenylalanine derivatives inserted at position 780 to diminish cation-π interaction strength. In line with the results of the analysis of mutants with natural amino acid substitutions, the results with the fluorinated derivatives indicate that Na+-π interaction with the phenol ring at position 780 contributes minimally, if at all, to the binding of Na+. All Y780 substitutions decreased K+ apparent affinity, highlighting that a state-dependent H-bond network is essential for the selectivity switch at sites I and II when the pump changes conformational state.","journal":"The Journal of General Physiology","year":2022,"id":280439,"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":10,"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":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":458160,"name":"Daniel T. Infield","orcid":null,"position":1,"is_corresponding":false},{"id":955367,"name":"Hang N. Nielsen","orcid":null,"position":2,"is_corresponding":false},{"id":943781,"name":"Rikke Holm","orcid":null,"position":3,"is_corresponding":false},{"id":465231,"name":"Victoria C. Young","orcid":"0000-0002-4027-8694","position":4,"is_corresponding":false},{"id":499489,"name":"Jason D. Galpin","orcid":"0000-0003-1923-5312","position":5,"is_corresponding":false},{"id":459188,"name":"Christopher A. Ahern","orcid":"0000-0002-7975-2744","position":6,"is_corresponding":false},{"id":943282,"name":"Bente Vilsen","orcid":"0000-0002-4727-9382","position":7,"is_corresponding":false},{"id":465232,"name":"Pablo Artigas","orcid":"0000-0002-6893-9765","position":8,"is_corresponding":false},{"id":465230,"name":"Kerri Spontarelli","orcid":"0000-0002-5988-028X","position":0,"is_corresponding":true}],"reference_count":56,"raw_metadata":null,"created_at":"2026-07-19T00:29:03.247347Z","pmid":"35657726","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":[]}