{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T19:32:36Z","timestamp":1780515156661,"version":"3.54.1"},"reference-count":26,"publisher":"Public Library of Science (PLoS)","issue":"11","license":[{"start":{"date-parts":[[2025,11,6]],"date-time":"2025-11-06T00:00:00Z","timestamp":1762387200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"US National Institutes of Health","doi-asserted-by":"crossref","award":["R35GM141951"],"award-info":[{"award-number":["R35GM141951"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:sec id=\"sec001\">\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      General anesthesia induced by etomidate, barbiturates and propofol is associated with positive modulation of synaptic \u03b1\u03b2\u03b3 GABA\n                      <jats:sub>A<\/jats:sub>\n                      receptors, inhibitory hetero-pentameric ligand-gated ion channels formed from homologous subunits arranged \u03b2-\u03b1-\u03b2-\u03b1-\u03b3 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 \u03b2+\/\u03b1\u2013 inter-subunit sites per receptor. These approaches also reveal that the potent barbiturate photolabel R-mTFD-MPAB binds selectively in homologous sites formed at \u03b1+\/\u03b2\u2013 and \u03b3+\/\u03b2\u2013 interfaces. The anesthetic photolabel, pTFD-di-iPr-BnOH, was proposed to bind selectively in \u03b1+\/\u03b2\u2013 and \u03b1+\/\u03b3\u2013 homologs of the etomidate sites, based largely on functional analysis of only 5 point mutations in \u03b11\u03b23\u03b32L receptors.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec002\">\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>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 \u03b1+ and \u03b3\u2013 loci. Control SCAMP studies were performed using etomidate and R-mTFD-MPAB.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec003\">\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Incorporation of single cysteine mutations (\u03b11M236C, \u03b11S280C, \u03b11A291C, \u03b23L231C, \u03b23M286C, \u03b32I242C, \u03b32L246C, and \u03b32S301C) 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 \u03b1+\u2009, \u03b2\u2013, and \u03b3\u2013, but not in \u03b1\u2013, \u03b2+\u2009, or \u03b3+ interfacial loci. Protection patterns with etomidate and R-mTFD-MPAB mirrored prior results.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec id=\"sec004\">\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>SCAMP results further support the hypothesis that pTFD-di-iPr-BnOH binds selectively in \u03b1+\/\u03b2\u2013 and \u03b1+\/\u03b3\u2013 interfacial sites that are homologs of the \u03b2+\/\u03b1\u2013 etomidate sites.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1371\/journal.pone.0336606","type":"journal-article","created":{"date-parts":[[2025,11,6]],"date-time":"2025-11-06T18:36:24Z","timestamp":1762454184000},"page":"e0336606","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Substituted cysteine modification and protection indicates selective interactions of the anesthetic photolabel pTFD-di-iPr-BnOH with \u03b1+\/\u03b2\u2013 and \u03b1+\/\u03b3\u2013 transmembrane subunit interfaces of synaptic GABAA receptors"],"prefix":"10.1371","volume":"20","author":[{"given":"Kieran","family":"Bhave","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6340-2478","authenticated-orcid":true,"given":"Stuart A.","family":"Forman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2025,11,6]]},"reference":[{"key":"pone.0336606.ref001","doi-asserted-by":"crossref","DOI":"10.1017\/S0033583524000179","article-title":"Molecular mechanisms of the GABA type A receptor function","volume":"58","author":"MA Micha\u0142owski","year":"2025","journal-title":"Q Rev Biophys"},{"key":"pone.0336606.ref002","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/bs.mie.2018.01.009","article-title":"Identification of General Anesthetic Target Protein-Binding Sites by 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