{"doi":"10.1371/journal.pone.0336606","title":"Substituted cysteine modification and protection indicates selective interactions of the anesthetic photolabel pTFD-di-iPr-BnOH with α+/β– and α+/γ– transmembrane subunit interfaces of synaptic GABAA receptors","abstract":"BACKGROUND: General anesthesia induced by etomidate, barbiturates and propofol is associated with positive modulation of synaptic αβγ GABAA receptors, inhibitory hetero-pentameric ligand-gated ion channels formed from homologous subunits arranged β-α-β-α-γ around a central gated chloride channel. Approaches based on mutations, amino-acid level analysis of photolabel incorporation, and cryo-electron micrography (cryo-EM) all indicate that etomidate binds selectively in two outer transmembrane β+/α- inter-subunit sites per receptor. These approaches also reveal that the potent barbiturate photolabel R-mTFD-MPAB binds selectively in homologous sites formed at α+/β- and γ+/β- interfaces. The anesthetic photolabel, pTFD-di-iPr-BnOH, was proposed to bind selectively in α+/β- and α+/γ- homologs of the etomidate sites, based largely on functional analysis of only 5 point mutations in α1β3γ2L receptors. METHODS: To further test the interactions of receptor-bound pTFD-di-iPr-BnOH with outer transmembrane inter-subunit sites, we used voltage-clamp electrophysiology in substituted cysteine modification and protection (SCAMP) experiments at 8 residues located in the five homologous sites, focusing on α+ and γ- loci. Control SCAMP studies were performed using etomidate and R-mTFD-MPAB. RESULTS: Incorporation of single cysteine mutations (α1M236C, α1S280C, α1A291C, β3L231C, β3M286C, γ2I242C, γ2L246C, and γ2S301C) produced functional GABA-responsive receptors that retained sensitivity to pTFD-di-iPr-BnOH modulation and displayed increased GABA sensitivity following exposure to the covalent sulfhydryl modifier p-chloromercuribenzenesulfonate (pCMBS). In the presence of pTFD-di-iPr-BnOH, pCMBS modification effects were reduced (evidence of steric protection) in receptors with cysteine mutations in α+ , β-, and γ-, but not in α-, β+ , or γ+ interfacial loci. Protection patterns with etomidate and R-mTFD-MPAB mirrored prior results. DISCUSSION: SCAMP results further support the hypothesis that pTFD-di-iPr-BnOH binds selectively in α+/β- and α+/γ- interfacial sites that are homologs of the β+/α- etomidate sites.","journal":"PLoS ONE","year":2025,"id":537084,"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":0.9622,"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":380466,"name":"Stuart A. Forman","orcid":"0000-0001-6340-2478","position":1,"is_corresponding":false},{"id":382608,"name":"Kieran Bhave","orcid":"0000-0002-8023-5525","position":0,"is_corresponding":true}],"reference_count":25,"raw_metadata":null,"created_at":"2026-07-19T02:52:09.056872Z","pmid":"41196917","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":[]}