{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,18]],"date-time":"2026-03-18T03:01:12Z","timestamp":1773802872636,"version":"3.50.1"},"reference-count":49,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2022,6,21]],"date-time":"2022-06-21T00:00:00Z","timestamp":1655769600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institutes of Health National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["GM108580"],"award-info":[{"award-number":["GM108580"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000057","name":"National Institutes of Health National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["GM140947"],"award-info":[{"award-number":["GM140947"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Taylor Family Institute for Innovative Psychiatric Research","award":["GM108580"],"award-info":[{"award-number":["GM108580"]}]},{"name":"Taylor Family Institute for Innovative Psychiatric Research","award":["GM140947"],"award-info":[{"award-number":["GM140947"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>GABAA receptors are a major contributor to fast inhibitory neurotransmission in the brain. The receptors are activated upon binding the transmitter GABA or allosteric agonists including a number of GABAergic anesthetics and neurosteroids. Functional receptors can be formed by various combinations of the nineteen GABAA subunits cloned to date. GABAA receptors containing the \u03b5 subunit exhibit a significant degree of constitutive activity and have been suggested to be unresponsive to allosteric agents. In this study, we have characterized the functional properties of the rat \u03b11\u03b22\u03b5 GABAA receptor. We confirm that the \u03b11\u03b22\u03b5 receptor exhibits a higher level of constitutive activity than typical of GABAA receptors and show that it is inefficaciously activated by the transmitter and the allosteric agonists propofol, pentobarbital, and allopregnanolone. Manipulations intended to alter \u03b5 subunit expression and receptor stoichiometry were largely without effect on receptor properties including sensitivity to GABA and allosteric agonists. Surprisingly, amino acid substitutions at the conserved 9\u2019 and 6\u2019 positions in the second transmembrane (TM2) domain in the \u03b5 subunit did not elicit the expected functional effects of increased constitutive activity and resistance to the channel blocker picrotoxin, respectively. We tested the accessibility of TM2 residues mutated to cysteine using the cysteine-modifying reagent 4-(hydroxymercuri)benzoic acid and found a unique pattern of water-accessible residues in the \u03b5 subunit.<\/jats:p>","DOI":"10.3390\/biom12070868","type":"journal-article","created":{"date-parts":[[2022,6,21]],"date-time":"2022-06-21T21:55:19Z","timestamp":1655848519000},"page":"868","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Activation of the Rat \u03b11\u03b22\u03b5 GABAA Receptor by Orthosteric and Allosteric Agonists"],"prefix":"10.3390","volume":"12","author":[{"given":"Allison L.","family":"Germann","sequence":"first","affiliation":[{"name":"Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ariel B.","family":"Burbridge","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Spencer R.","family":"Pierce","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gustav","family":"Akk","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/bs.apha.2017.03.003","article-title":"GABAA Receptors and the Diversity in their Structure and Pharmacology","volume":"79","author":"Chua","year":"2017","journal-title":"Adv. Pharmacol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"820","DOI":"10.1038\/385820a0","article-title":"Insensitivity to anaesthetic agents conferred by a class of GABAA receptor subunit","volume":"385","author":"Davies","year":"1997","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"5027","DOI":"10.1523\/JNEUROSCI.17-13-05027.1997","article-title":"Neuronally restricted RNA splicing regulates the expression of a novel GABAA receptor subunit conferring atypical functional properties [corrected; erratum to be published]","volume":"17","author":"Whiting","year":"1997","journal-title":"J. Neurosci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"3588","DOI":"10.1523\/JNEUROSCI.20-10-03588.2000","article-title":"GABAA receptor \u03b5 and \u03b8 subunits display unusual structural variation between species and are enriched in the rat locus ceruleus","volume":"20","author":"Sinkkonen","year":"2000","journal-title":"J. Neurosci."},{"key":"ref_5","first-page":"4318","article-title":"cDNA cloning and expression of a \u03b3-aminobutyric acid A receptor \u03b5-subunit in rat brain","volume":"12","author":"Moragues","year":"2000","journal-title":"Eur. J. Neurosci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e1388","DOI":"10.1002\/mgg3.1388","article-title":"Rare variants in the GABAA receptor subunit \u03b5 identified in patients with a wide spectrum of epileptic phenotypes","volume":"8","author":"Markus","year":"2020","journal-title":"Mol. Genet. Genomic Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1124\/mol.55.1.168","article-title":"Spontaneous and \u03b3-aminobutyric acid (GABA)-activated GABAA receptor channels formed by \u03b5 subunit-containing isoforms","volume":"55","author":"Neelands","year":"1999","journal-title":"Mol. Pharmacol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1258","DOI":"10.1111\/j.1471-4159.2007.04833.x","article-title":"Trafficking and potential assembly patterns of \u03b5-containing GABAA receptors","volume":"103","author":"Jones","year":"2007","journal-title":"J. Neurochem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"17","DOI":"10.1016\/S0014-2999(03)02125-3","article-title":"Pharmacology of GABAA receptors exhibiting different levels of spontaneous activity","volume":"476","author":"Mortensen","year":"2003","journal-title":"Eur. J. Pharmacol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Ranna, M., Sinkkonen, S.T., Moykkynen, T., Uusi-Oukari, M., and Korpi, E.R. (2006). Impact of \u03b5 and \u03b8 subunits on pharmacological properties of \u03b13\u03b21 GABAA receptors expressed in Xenopus oocytes. BMC Pharmacol., 6.","DOI":"10.1186\/1471-2210-6-1"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"10462","DOI":"10.1523\/JNEUROSCI.1658-05.2005","article-title":"Kinetics and spontaneous open probability conferred by the \u03b5 subunit of the GABAA receptor","volume":"25","author":"Wagner","year":"2005","journal-title":"J. Neurosci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1097\/00000542-200209000-00027","article-title":"Pentobarbital enhances GABAergic neurotransmission to cardiac parasympathetic neurons, which is prevented by expression of GABAA \u03b5 subunit","volume":"97","author":"Irnaten","year":"2002","journal-title":"Anesthesiology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"662","DOI":"10.1016\/S0028-3908(02)00162-4","article-title":"Overexpression of the GABAA receptor \u03b5 subunit results in insensitivity to anaesthetics","volume":"43","author":"Thompson","year":"2002","journal-title":"Neuropharmacology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1016\/S0028-3908(02)00153-3","article-title":"Alternative transcripts of the GABAA receptor \u03b5 subunit in human and rat","volume":"43","author":"Davies","year":"2002","journal-title":"Neuropharmacology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1124\/mol.118.113787","article-title":"Applying the Monod-Wyman-Changeux Allosteric Activation Model to Pseudo-Steady-State Responses from GABAA Receptors","volume":"95","author":"Steinbach","year":"2019","journal-title":"Mol. Pharmacol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"411","DOI":"10.1097\/ACO.0b013e328354feea","article-title":"Monod-Wyman-Changeux allosteric mechanisms of action and the pharmacology of etomidate","volume":"25","author":"Forman","year":"2012","journal-title":"Curr. Opin. Anaesthesiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"772","DOI":"10.2174\/1570159X14666160202121319","article-title":"Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol","volume":"14","author":"Eaton","year":"2016","journal-title":"Curr. Neuropharmacol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1126\/science.1384130","article-title":"Acetylcholine receptor channel structure probed in cysteine-substitution mutants","volume":"258","author":"Akabas","year":"1992","journal-title":"Science"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1085\/jgp.107.2.195","article-title":"Identification of channel-lining residues in the M2 membrane-spanning segment of the GABAA receptor \u03b11 subunit","volume":"107","author":"Xu","year":"1996","journal-title":"J. Gen. Physiol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3110","DOI":"10.1111\/bph.13566","article-title":"Activation and modulation of recombinant glycine and GABAA receptors by 4-halogenated analogues of propofol","volume":"173","author":"Germann","year":"2016","journal-title":"Br. J. Pharmacol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e14230","DOI":"10.14814\/phy2.14230","article-title":"Steady-state activation and modulation of the synaptic-type \u03b11\u03b22\u03b32L GABAA receptor by combinations of physiological and clinical ligands","volume":"7","author":"Germann","year":"2019","journal-title":"Physiol. Rep."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1124\/mol.117.110569","article-title":"GABA Type A Receptor Activation in the Allosteric Coagonist Model Framework: Relationship between EC50 and Basal Activity","volume":"93","author":"Akk","year":"2018","journal-title":"Mol. Pharmacol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"20982","DOI":"10.1074\/jbc.M400472200","article-title":"Gating allosterism at a single class of etomidate sites on \u03b11\u03b22\u03b32L GABAA receptors accounts for both direct activation and agonist modulation","volume":"279","author":"Rusch","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1016\/0014-5793(89)80271-6","article-title":"Cloning and expression of a novel rat GABAA receptor","volume":"246","author":"Lolait","year":"1989","journal-title":"FEBS Lett."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1837","DOI":"10.1111\/j.1476-5381.1991.tb12337.x","article-title":"GABAA receptors are differentially sensitive to zinc: Dependence on subunit composition","volume":"103","author":"Smart","year":"1991","journal-title":"Br. J. Pharmacol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1124\/mol.117.110403","article-title":"Propofol is an allosteric agonist with multiple binding sites on concatemeric ternary GABAA receptors","volume":"93","author":"Shin","year":"2018","journal-title":"Mol. Pharmacol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1097\/ALN.0b013e31823d0c36","article-title":"An allosteric coagonist model for propofol effects on \u03b11\u03b22\u03b32L \u03b3-aminobutyric acid type A receptors","volume":"116","author":"Ruesch","year":"2012","journal-title":"Anesthesiology"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"468","DOI":"10.1124\/mol.117.111435","article-title":"High Constitutive Activity Accounts for the Combination of Enhanced Direct Activation and Reduced Potentiation in Mutated GABAA Receptors","volume":"93","author":"Germann","year":"2018","journal-title":"Mol. Pharmacol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1038\/nature05324","article-title":"Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites","volume":"444","author":"Hosie","year":"2006","journal-title":"Nature"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1124\/mol.111.071662","article-title":"Occupation of Either Site for the Neurosteroid Allopregnanolone Potentiates the Opening of the GABAA Receptor Induced from Either Transmitter Binding Site","volume":"80","author":"Bracamontes","year":"2011","journal-title":"Mol. Pharmacol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1124\/mol.118.113464","article-title":"Analysis of GABAA receptor activation by combinations of agonists acting at the same or distinct binding sites","volume":"95","author":"Shin","year":"2019","journal-title":"Mol. Pharmacol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1124\/molpharm.121.000268","article-title":"Intrasubunit and Intersubunit Steroid Binding Sites Independently and Additively Mediate \u03b11\u03b22\u03b32L GABAA Receptor Potentiation by the Endogenous Neurosteroid Allopregnanolone","volume":"100","author":"Germann","year":"2021","journal-title":"Mol. Pharmacol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Ziemba, A.M., and Forman, S.A. (2016). Correction for Inhibition Leads to an Allosteric Co-Agonist Model for Pentobarbital Modulation and Activation of \u03b11\u03b23\u03b32L GABAA Receptors. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0154031"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"5415","DOI":"10.1523\/JNEUROSCI.16-17-05415.1996","article-title":"Stoichiometry of a recombinant GABAA receptor","volume":"16","author":"Chang","year":"1996","journal-title":"J. Neurosci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1111\/bph.12514","article-title":"Stoichiometry of \u03b4 subunit containing GABAA receptors","volume":"171","author":"Patel","year":"2014","journal-title":"Br. J. Pharmacol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2542","DOI":"10.1016\/S0006-3495(99)77089-X","article-title":"Allosteric activation mechanism of the \u03b11\u03b22\u03b32 \u03b3-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine","volume":"77","author":"Chang","year":"1999","journal-title":"Biophys. J."},{"key":"ref_37","first-page":"13","article-title":"Point mutations in the M2 region of the \u03b1, \u03b2, or \u03b3 subunit of the GABAA channel that abolish block by picrotoxin","volume":"3","author":"Gurley","year":"1995","journal-title":"Recept. Channels"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1113\/jphysiol.2006.120287","article-title":"Stoichiometry of a pore mutation that abolishes picrotoxin-mediated antagonism of the GABAA receptor","volume":"577","author":"Sedelnikova","year":"2006","journal-title":"J. Physiol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Akk, G., Eaton, M., Li, P., Zheng, S., Lo, J., and Steinbach, J.H. (2013). Energetic contributions to channel gating of residues in the muscle nicotinic receptor \u03b21 subunit. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0078539"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"42","DOI":"10.1196\/annals.1286.005","article-title":"Pathogenesis in menstrual cycle-linked CNS disorders","volume":"1007","author":"Backstrom","year":"2003","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Hengen, K.B., Nelson, N.R., Stang, K.M., Johnson, S.M., Crader, S.M., Watters, J.J., Mitchell, G.S., and Behan, M. (2012). Increased GABAA receptor \u03b5-subunit expression on ventral respiratory column neurons protects breathing during pregnancy. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0030608"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2312","DOI":"10.1152\/jn.00227.2009","article-title":"Inhibitory transmission in locus coeruleus neurons expressing GABAA receptor \u03b5 subunit has a number of unique properties","volume":"102","author":"Belujon","year":"2009","journal-title":"J. Neurophysiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1523\/JNEUROSCI.3209-04.2005","article-title":"Pharmacological properties of GABAA receptors in rat hypothalamic neurons expressing the \u03b5-subunit","volume":"25","author":"Sergeeva","year":"2005","journal-title":"J. Neurosci."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"R1028","DOI":"10.1152\/ajpregu.00239.2009","article-title":"Hibernation induces pentobarbital insensitivity in medulla but not cortex","volume":"297","author":"Hengen","year":"2009","journal-title":"Am. J. Physiol. Regul. Integr. Comp. Physiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"R272","DOI":"10.1152\/ajpregu.00607.2010","article-title":"Changes in ventral respiratory column GABAaR \u03b5- and \u03b4-subunits during hibernation mediate resistance to depression by EtOH and pentobarbital","volume":"300","author":"Hengen","year":"2011","journal-title":"Am. J. Physiol. Regul. Integr. Comp. Physiol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.neuroscience.2009.02.043","article-title":"Expression of GABAA receptor \u03b13-, \u03b8-, and \u03b5-subunit mRNAs during rat CNS development and immunolocalization of the \u03b5 subunit in developing postnatal spinal cord","volume":"160","author":"Pape","year":"2009","journal-title":"Neuroscience"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"657","DOI":"10.1016\/S0306-4522(02)00033-7","article-title":"Localisation of GABAA receptor \u03b5-subunit in cholinergic and aminergic neurones and evidence for co-distribution with the \u03b8-subunit in rat brain","volume":"111","author":"Moragues","year":"2002","journal-title":"Neuroscience"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1111\/j.1469-7793.2001.0101k.x","article-title":"Evidence for the formation of functionally distinct \u03b1\u03b2\u03b3\u03b5 GABAA receptors","volume":"537","author":"Davies","year":"2001","journal-title":"J. Physiol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"4895","DOI":"10.1523\/JNEUROSCI.5039-09.2010","article-title":"The short splice variant of the \u03b32 subunit acts as an external modulator of GABAA receptor function","volume":"30","author":"Boileau","year":"2010","journal-title":"J. Neurosci"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/12\/7\/868\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:36:47Z","timestamp":1760139407000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/12\/7\/868"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,21]]},"references-count":49,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2022,7]]}},"alternative-id":["biom12070868"],"URL":"https:\/\/doi.org\/10.3390\/biom12070868","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,21]]}}}