{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T18:09:08Z","timestamp":1788890948435,"version":"build-2803163510"},"reference-count":44,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01GM148591"],"award-info":[{"award-number":["R01GM148591"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"name":"The University of Texas at Austin"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    GABA\n                    <jats:sub>A<\/jats:sub>\n                    receptors (GABA\n                    <jats:sub>A<\/jats:sub>\n                    Rs) are pentameric ligand-gated ion channels (pLGICs) essential for inhibitory synaptic transmission throughout the central nervous system. Despite progress in understanding their three-dimensional structure, the molecular basis for how neurotransmitter binding is transduced to ion channel gating remains poorly understood. Furthermore, relatively little is known about the contributions of distinct subunits to this coupling within typical heteromeric receptors. A highly conserved proline (site 1) in the M2-M3 linker of pLGIC subunits is involved in channel gating \u2013 e.g., P273 in the GABA\n                    <jats:sub>A<\/jats:sub>\n                    R \u03b22 subunit. In GABA\n                    <jats:sub>A<\/jats:sub>\n                    Rs, only the \u03b2 subunits have an additional proline in the M2-M3 linker (site 2) \u2013 e.g., \u03b22(P276) \u2013 whereas all other subunits have a non-proline at the homologous site 2 position. Here, we investigate the functional contribution of proline at site 2 in distinct subunits of \u03b11\u03b22\u03b32 GABA\n                    <jats:sub>A<\/jats:sub>\n                    Rs. We expressed wild type or mutant \u03b11\u03b22\u03b32 GABA\n                    <jats:sub>A<\/jats:sub>\n                    Rs in\n                    <jats:italic>Xenopus laevis<\/jats:italic>\n                    oocytes and used two-electrode voltage clamp electrophysiology to record channel currents in response to GABA and\/or other ligands. First, we introduced a proline at site 2 in \u03b11 or \u03b32 subunits: \u03b11(A280P) and \u03b32(S291P). Second, we replaced the site 2 proline in the \u03b22 subunit with its homologous non-proline residue from \u03b11 or \u03b32 subunits: \u03b22(P276A) or \u03b22(P276S). We show that \u03b11(A280P) confers enhanced GABA-sensitivity and spontaneous unliganded channel activity, whereas \u03b32(S291P) has minor effects on channel activation. In contrast, \u03b22(P276A) or \u03b22(P276S) either had no effect or enhanced GABA-activation, respectively, indicating complex functional dependence on the side chain at site 2 in the \u03b22 subunit. When in combination with other substitutions, the presence or absence of \u03b11(A280P) was consistently correlated with enhanced GABA-sensitivity and spontaneous unliganded opening. Thus, introduction of a proline at site 2 in the \u03b11 M2-M3 linker biases the channel towards an activated state and prevents it from remaining closed at rest.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0357367","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T17:40:33Z","timestamp":1788889233000},"page":"e0357367","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Introducing a proline in the \u03b11 M2-M3 linker relieves a molecular brake on channel activation in \u03b11\u03b22\u03b32 GABAA receptors"],"prefix":"10.1371","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-5807-1413","authenticated-orcid":true,"given":"Netrang G.","family":"Desai","sequence":"first","affiliation":[{"name":"University of Texas at Austin","place":["Austin, Texas, United States of America"],"department":["Department of 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extracellular domain","volume":"286","author":"M Moroni","year":"2011","journal-title":"J Biol Chem"},{"issue":"9","key":"pone.0357367.ref004","doi-asserted-by":"crossref","first-page":"6714","DOI":"10.1074\/jbc.M111.316836","article-title":"Structural link between \u03b3-aminobutyric acid type A (GABAA) receptor agonist binding site and inner \u03b2-sheet governs channel activation and allosteric drug modulation","volume":"287","author":"SP Venkatachalan","year":"2012","journal-title":"J Biol Chem"},{"issue":"6","key":"pone.0357367.ref005","doi-asserted-by":"crossref","first-page":"4887","DOI":"10.1074\/jbc.M311441200","article-title":"Charged residues in the \u03b22 subunit involved in GABAA receptor activation","volume":"279","author":"TL Kash","year":"2004","journal-title":"J Biol Chem"},{"issue":"14","key":"pone.0357367.ref006","doi-asserted-by":"crossref","first-page":"2085","DOI":"10.1016\/j.bpj.2024.02.016","article-title":"GABAA receptor subunit M2-M3 linkers have asymmetric roles in pore gating and diazepam modulation","volume":"123","author":"JW Nors","year":"2024","journal-title":"Biophys J"},{"issue":"1","key":"pone.0357367.ref007","doi-asserted-by":"crossref","first-page":"6107","DOI":"10.1038\/s41467-025-61447-0","article-title":"A single main-chain hydrogen bond required to keep GABAA receptors closed","volume":"16","author":"CM Borghese","year":"2025","journal-title":"Nature Communications"},{"issue":"3","key":"pone.0357367.ref008","doi-asserted-by":"crossref","first-page":"641","DOI":"10.1124\/mol.109.055111","article-title":"Inverse effects on gating and modulation caused by a mutation in the M2-M3 Linker of the GABA(A) receptor gamma subunit","volume":"76","author":"SM O\u2019Shea","year":"2009","journal-title":"Mol Pharmacol"},{"issue":"30","key":"pone.0357367.ref009","doi-asserted-by":"crossref","first-page":"22764","DOI":"10.1074\/jbc.M001299200","article-title":"Arg-274 and Leu-277 of the \u03b3-aminobutyric acid type A receptor \u03b12 subunit define agonist efficacy and potency","volume":"275","author":"SM O\u2019Shea","year":"2000","journal-title":"J Biol Chem"},{"issue":"25","key":"pone.0357367.ref010","doi-asserted-by":"crossref","first-page":"17034","DOI":"10.1074\/jbc.M603599200","article-title":"An asymmetric contribution to \u03b3-aminobutyric type A receptor function of a conserved lysine within TM2-3 of \u03b11, \u03b22, and \u03b32 subunits","volume":"281","author":"TG Hales","year":"2006","journal-title":"J Biol Chem"},{"issue":"50","key":"pone.0357367.ref011","doi-asserted-by":"crossref","first-page":"41655","DOI":"10.1074\/jbc.M508635200","article-title":"A unified view of the role of electrostatic interactions in modulating the gating of Cys loop receptors","volume":"280","author":"X Xiu","year":"2005","journal-title":"J Biol Chem"},{"issue":"4","key":"pone.0357367.ref012","doi-asserted-by":"crossref","first-page":"683","DOI":"10.1006\/mcne.2002.1143","article-title":"A lysine residue in the beta3 subunit contributes to the regulation of GABA(A) receptor activity by voltage","volume":"20","author":"JL Fisher","year":"2002","journal-title":"Mol Cell Neurosci"},{"key":"pone.0357367.ref013","first-page":"10","article-title":"A critical residue in the \u03b11M2-M3 linker regulating mammalian GABAA receptor pore gating by diazepam","author":"JW Nors","year":"2021","journal-title":"eLife"},{"issue":"1","key":"pone.0357367.ref014","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1002\/pro.3943","article-title":"UCSF ChimeraX: Structure visualization for researchers, educators, and developers","volume":"30","author":"EF Pettersen","year":"2021","journal-title":"Protein Sci"},{"issue":"40","key":"pone.0357367.ref015","doi-asserted-by":"crossref","first-page":"13216","DOI":"10.1021\/ja8015457","article-title":"A stereochemical test of a proposed structural feature of the nicotinic acetylcholine receptor","volume":"130","author":"AP Hanek","year":"2008","journal-title":"J Am Chem Soc"},{"issue":"2","key":"pone.0357367.ref016","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1085\/jgp.200810014","article-title":"Nicotinic receptor interloop proline anchors beta1-beta2 and Cys loops in coupling agonist binding to channel gating","volume":"132","author":"WY Lee","year":"2008","journal-title":"J Gen Physiol"},{"issue":"6943","key":"pone.0357367.ref017","doi-asserted-by":"crossref","first-page":"949","DOI":"10.1038\/nature01748","article-title":"Structure and gating mechanism of the acetylcholine receptor pore","volume":"423","author":"A Miyazawa","year":"2003","journal-title":"Nature"},{"issue":"4","key":"pone.0357367.ref018","doi-asserted-by":"crossref","first-page":"765","DOI":"10.1006\/jmbi.1999.2721","article-title":"Nicotinic acetylcholine receptor at 4.6 A resolution: transverse tunnels in the channel wall","volume":"288","author":"A Miyazawa","year":"1999","journal-title":"J Mol Biol"},{"issue":"5","key":"pone.0357367.ref019","doi-asserted-by":"crossref","first-page":"1165","DOI":"10.1016\/S0022-2836(02)00381-9","article-title":"Activation of the nicotinic acetylcholine receptor involves a switch in conformation of the alpha subunits","volume":"319","author":"N Unwin","year":"2002","journal-title":"J Mol Biol"},{"issue":"4","key":"pone.0357367.ref020","doi-asserted-by":"crossref","first-page":"967","DOI":"10.1016\/j.jmb.2004.12.031","article-title":"Refined structure of the nicotinic acetylcholine receptor at 4A resolution","volume":"346","author":"N Unwin","year":"2005","journal-title":"J Mol Biol"},{"issue":"10","key":"pone.0357367.ref021","article-title":"Binding to gating transduction in nicotinic receptors: Cys-loop energetically couples to pre-M1 and M2-M3 regions","volume":"29","author":"WY Lee","year":"2009","journal-title":"J Neurosci"},{"issue":"7065","key":"pone.0357367.ref022","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1038\/nature04156","article-title":"Principal pathway coupling agonist binding to channel gating in nicotinic receptors","volume":"438","author":"WY Lee","year":"2005","journal-title":"Nature"},{"issue":"7","key":"pone.0357367.ref023","doi-asserted-by":"crossref","first-page":"3327","DOI":"10.1021\/acschemneuro.9b00315","article-title":"Proline Residues in the Transmembrane\/Extracellular Domain Interface Loops Have Different Behaviors in 5-HT3 and nACh Receptors","volume":"10","author":"R Mosesso","year":"2019","journal-title":"ACS Chem Neurosci"},{"key":"pone.0357367.ref024","doi-asserted-by":"crossref","first-page":"120943","DOI":"10.1016\/j.lfs.2022.120943","article-title":"GABAA receptor proline 273 at the interdomain interface of the \u03b22 subunit regulates entry into desensitization and opening\/closing transitions","volume":"308","author":"M Brodzki","year":"2022","journal-title":"Life Sci"},{"issue":"21","key":"pone.0357367.ref025","doi-asserted-by":"crossref","first-page":"3044","DOI":"10.1021\/acschemneuro.2c00401","article-title":"\u03b11 proline 277 residues regulate GABAAR gating through M2-M3 loop interaction in the interface region","volume":"13","author":"PT Kaczor","year":"2022","journal-title":"ACS Chem Neurosci"},{"issue":"7065","key":"pone.0357367.ref026","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1038\/nature04130","article-title":"Cis-trans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel","volume":"438","author":"SCR Lummis","year":"2005","journal-title":"Nature"},{"issue":"4","key":"pone.0357367.ref027","doi-asserted-by":"crossref","first-page":"695","DOI":"10.1016\/S0028-3908(02)00036-9","article-title":"The relative amount of cRNA coding for gamma2 subunits affects stimulation by benzodiazepines in GABA(A) receptors expressed in Xenopus oocytes","volume":"43","author":"AJ Boileau","year":"2002","journal-title":"Neuropharmacology"},{"issue":"14","key":"pone.0357367.ref028","doi-asserted-by":"crossref","first-page":"1954","DOI":"10.1016\/j.bpj.2023.09.014","article-title":"Potentiation of the GABAAR reveals variable energetic contributions by etiocholanolone and propofol","volume":"123","author":"SR Pierce","year":"2024","journal-title":"Biophys J"},{"issue":"1","key":"pone.0357367.ref029","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1085\/jgp.200910317","article-title":"The activation mechanism of \u03b11\u03b22\u03b32S and \u03b13\u03b23\u03b32S GABAA receptors","volume":"135","author":"A Keramidas","year":"2010","journal-title":"J Gen Physiol"},{"issue":"2","key":"pone.0357367.ref030","doi-asserted-by":"crossref","first-page":"386","DOI":"10.1016\/S0026-895X(25)09699-3","article-title":"Thermodynamics of gamma-aminobutyric acid type A receptor binding differentiate agonists from antagonists","volume":"46","author":"G Maksay","year":"1994","journal-title":"Mol Pharmacol"},{"issue":"7","key":"pone.0357367.ref031","doi-asserted-by":"crossref","first-page":"984","DOI":"10.1038\/sj.bjp.0706796","article-title":"Mechanism of action of benzodiazepines on GABAA receptors","volume":"148","author":"C Campo-Soria","year":"2006","journal-title":"Br J Pharmacol"},{"key":"pone.0357367.ref032","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/S0014-2999(03)02125-3","article-title":"Pharmacology of GABA(A) receptors exhibiting different levels of spontaneous activity","volume":"476","author":"M Mortensen","year":"2003","journal-title":"Eur J Pharmacol"},{"issue":"6","key":"pone.0357367.ref033","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1124\/mol.114.094813","article-title":"\u03b3-aminobutyric acid type A \u03b14, \u03b22, and \u03b4 subunits assemble to produce more than one functionally distinct receptor type","volume":"86","author":"MM Eaton","year":"2014","journal-title":"Mol Pharmacol"},{"issue":"4","key":"pone.0357367.ref034","doi-asserted-by":"crossref","first-page":"943","DOI":"10.1097\/00001756-199603220-00023","article-title":"The agonistic action of pentobarbital on GABAA beta-subunit homomeric receptors","volume":"7","author":"IN Cestari","year":"1996","journal-title":"Neuroreport"},{"issue":"11","key":"pone.0357367.ref035","doi-asserted-by":"crossref","first-page":"2733","DOI":"10.1523\/JNEUROSCI.4316-03.2004","article-title":"An arginine involved in GABA binding and unbinding but not gating of the GABAA receptor","volume":"24","author":"DA Wagner","year":"2004","journal-title":"J Neurosci"},{"issue":"4","key":"pone.0357367.ref036","doi-asserted-by":"crossref","first-page":"647","DOI":"10.1124\/mol.111.072033","article-title":"Three arginines in the GABAA receptor binding pocket have distinct roles in the formation and stability of agonist- versus antagonist-bound complexes","volume":"80","author":"MP Goldschen-Ohm","year":"2011","journal-title":"Mol Pharmacol"},{"issue":"7824","key":"pone.0357367.ref037","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1038\/s41586-020-2654-5","article-title":"Shared structural mechanisms of general anaesthetics and benzodiazepines","volume":"585","author":"JJ Kim","year":"2020","journal-title":"Nature"},{"issue":"7740","key":"pone.0357367.ref038","doi-asserted-by":"crossref","first-page":"454","DOI":"10.1038\/s41586-018-0832-5","article-title":"GABAA receptor signalling mechanisms revealed by structural pharmacology","volume":"565","author":"S Masiulis","year":"2019","journal-title":"Nature"},{"issue":"1","key":"pone.0357367.ref039","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1046\/j.1471-4159.2001.00527.x","article-title":"Mutagenesis of the GABA(A) receptor alpha1 subunit reveals a domain that affects sensitivity to GABA and benzodiazepine-site ligands","volume":"79","author":"M Davies","year":"2001","journal-title":"J Neurochem"},{"issue":"17","key":"pone.0357367.ref040","doi-asserted-by":"crossref","DOI":"10.1523\/JNEUROSCI.16-17-05415.1996","article-title":"Stoichiometry of a recombinant GABAA receptor","volume":"16","author":"Y Chang","year":"1996","journal-title":"J Neurosci"},{"issue":"48","key":"pone.0357367.ref041","doi-asserted-by":"crossref","first-page":"40224","DOI":"10.1074\/jbc.R112.386664","article-title":"Structure, function, and modulation of GABA(A) receptors","volume":"287","author":"E Sigel","year":"2012","journal-title":"J Biol Chem"},{"key":"pone.0357367.ref042","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1016\/j.sbi.2019.03.023","article-title":"A structural perspective on GABAA receptor pharmacology","volume":"54","author":"S Scott","year":"2019","journal-title":"Curr Opin Struct Biol"},{"issue":"7740","key":"pone.0357367.ref043","doi-asserted-by":"crossref","first-page":"516","DOI":"10.1038\/s41586-018-0833-4","article-title":"Cryo-EM structure of the human \u03b11\u03b23\u03b32 GABAA receptor in a lipid bilayer","volume":"565","author":"D Laverty","year":"2019","journal-title":"Nature"},{"issue":"1","key":"pone.0357367.ref044","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1007\/s00424-006-0183-1","article-title":"Optimized expression vector for ion channel studies in Xenopus oocytes and mammalian cells using alfalfa mosaic virus","volume":"454","author":"SP Venkatachalan","year":"2007","journal-title":"Pflugers Arch"}],"container-title":["PLOS 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