Chen ZW , Bracamontes JR , Budelier MM , Germann AL , Shin DJ , Kathiresan K , Qian MX , Manion B , Cheng WWL , Reichert DE , Akk G , Covey DF , Evers AS .

K08 GM126336 NIGMS NIH HHS , R01 GM108580 NIGMS NIH HHS , R01 GM108799 NIGMS NIH HHS , T32 GM108539 NIGMS NIH HHS

	Fig 1. Allopregnanolone-analogue photolabeling reagents.(A) The structure of allopregnanolone, KK123, KK200, and KK202. (B). Allopregnanolone (1 μM) and the photolabeling reagents (10 μM) potentiate GABA-elicited currents of α1β3-GABAA receptors; potentiation is blocked by mutation of α1Q242Lβ3, indicating that these photolabeling reagents mimic the action of allopregnanolone. The numerical data are included in S1 Data. AlloP, allopregnanolone; GABAA, gamma amino-butyric acid Type A; NS, neurosteroid; WT, wild-type.
	Fig 2. KK123 photolabels α1-TM4 and β3-TM4 peptides.(A) A representative MS fragmentation spectrum of a KK123 photolabeled α1-TM4 peptide (m/z = 875.503, z = 4). The y9-y14 ions (red) contain the KK123 adduct. The site-defining ions y8 and y9 indicate that α1-Y415 (red) was photolabeled by KK123. Fragment ions y10− to y14− represent neutral loss of the KK123 adduct. (B). MS1 pair of light and heavy form of FLI-tag-KK123 photolabeled β3 TM4 peptide (m/z = 1,073.246 and m/z = 1,076.580, z = 3). (C) An overlay of light (black) and heavy (red) MS fragmentation spectra of FLI-tag-KK123 photolabeled β3 TM4 peptide. The KK123 adduct-containing b or y ions are labeled with “*”. Site-defining y4 and b17 ions identify β3-Y442 as the photolabeled residue. The photolabeled residues in α1- and β3-TM4 were identified in three replicate experiments. (D) KK123 photolabeled residues are shown in a homology model of the structure of an α1β3 GABAA receptor. In the α1 subunit, the labeled TM4 tyrosine (red) points toward TM1, whereas in the β3 subunit, the labeled tyrosine residue (orange) points toward TM3. The numerical data are included in S2 Data. GABAA, gamma amino-butyric acid Type A; MS, mass spectrometry; TM, transmembrane helix.
	Fig 3. Fragmentation spectra of KK200- and KK202-photolabeled GABAA receptor peptides.(A) An α1-TM4 peptide (m/z = 898.002, z = 4) is photolabeled by KK200 at N408; (B) A β3-TM3 peptide (m/z = 1,188.352, z = 4) is photolabeled by KK200 at [308GR309]. (C and D) β3-TM3 peptides (m/z = 811.453, z = 6) are photolabeled by KK202 at 226VKA228 (panel C) and L294 (panel D). Fragment ions labeled in red contain the neurosteroid adduct. The C* indicates that the cysteine is alkylated by NEM or NEM + DTT. The photolabeled residues shown in panels A–D were all observed in three replicate experiments. The inset schematic of a GABAA receptor subunit in each panel indicates the approximate location of the residues labeled by KK200 (green) and KK202 (blue). The numerical data are included in S3 Data. DTT, 1, 4-dithiothreitol; GABAA, gamma amino-butyric acid Type A; NEM, N-ethylmaleimide; TM, transmebrane helix.
	Fig 4. The α1 subunit is critical for neurosteroid modulation of α1β3 GABAA receptors.(A) Photolabeling with 3 μM KK123, KK200, and KK202 (black) is prevented by labeling in the presence of 30 μM allopregnanolone (red). Each circle represents a replicate experiment, and the bars represent mean ± SD, n = 3. (B) Sample current traces showing modulation of WT α1β3, α1V227Wβ3 and α1N408A/Y411Fβ3 GABAA receptors by allopregnanolone or propofol. (C1) Allopregnanolone potentiation of GABA-elicited currents is reduced in α1V227Wβ3 (orange) and α1N408A/Y411Fβ3 (blue) receptors compared to WT (black). Potentiation is given as the ratio of peak responses to GABA + steroid to GABA alone. Potentiation value of 1 indicates no potentiation by neurosteroids. (C2) Direct activation of α1β3 GABAA receptors by allopregnanolone is reduced in α1V227Wβ3 receptors (orange). Direct activation is given in percentage of the peak response to steroid to peak response to saturating GABA + 100 μM propofol. (C3) Potentiation of GABA-elicited currents by propofol, or (C4) direct activation of the receptor in the presence of propofol is not affected by the α1V227W or α1N408A/Y411F mutations. Each data point represents a replicate experiment. Bars show the mean ± SD (n = 6–8). Data were compared by a one-way analysis of variance followed by a Dunnett’s multiple comparison tests of the means. ***p < 0.001; **p < 0.01; *p < 0.05. The numerical data are included in S4 Data. AlloP, allopregnanolone; GABAA, gamma amino-butyric acid Type A; WT, wild-type.
	Fig 5. Allopregnanolone docking in the three neurosteroid binding sites identified by photolabeling.(A) The six photolabeling sites identified by photolabeling with KK123, KK200, and KK202 grouped into three clusters: β3(+)/α1(−) intersubunit sites (brown circle), β3 intrasubunit sites (blue circle), and α1 intrasubunit sites (red circle); docking of allopregnanolone to the β3 intrasubunit site (B), α1 intrasubunit site (C), and β3(+)/α1(−) intersubunit site (D). Residues photolabeled by KK123, α1-Y415, and β3-Y442 are colored red; Residues photolabeled by KK200 α1-N408 and β3-G308 are colored green; and residues photolabeled by KK202 β3-278VKA280 and L294 are colored blue. Residues previously identified as contributing to an intersubunit neurosteroid binding site—α1-Q242 and β3-F301—are shown in yellow, as is α1-V227, a residue in the α1 intrasubunit site shown to affect neurosteroid action by site-directed mutagenesis.
	Fig 6. Computational docking of neurosteroid photolabeling analogues to their photolabeling sites on α1β3-GABAA receptors.(A) KK200 (light green) and (B) KK123 (pink) in α1 intrasubunit site. (C) KK123 and (D) KK202 (light blue) in β3 intrasubunit site. (E) KK200 and (F) KK202 in β3 (+)/-α1(−) intersubunit site. The KK123 photolabeled residues α1-Y415 and β3-Y442 are colored red, the KK200 photolabeled residues α1-N408 and β3-G308 are colored green, and the KK202 β3-278VKA280 and L294 are colored blue. The canonical neurosteroid binding site residues Q242, F301, and the new mutation site V227 are colored yellow. The photolabeling diazirine group is indicated with red “*”. GABAA, gamma amino-butyric acid Type A.

Agís-Balboa, Characterization of brain neurons that express enzymes mediating neurosteroid biosynthesis. 2006, Pubmed

Agís-Balboa, Characterization of brain neurons that express enzymes mediating neurosteroid biosynthesis. 2006, Pubmed
Akk, The influence of the membrane on neurosteroid actions at GABA(A) receptors. 2009, Pubmed
Akk, Neurosteroid access to the GABAA receptor. 2005, Pubmed
Akk, Mutations of the GABA-A receptor alpha1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids. 2008, Pubmed
Akk, Neuroactive steroids have multiple actions to potentiate GABAA receptors. 2004, Pubmed
Barbaccia, Endogenous gamma-aminobutyric acid (GABA)(A) receptor active neurosteroids and the sedative/hypnotic action of gamma-hydroxybutyric acid (GHB): a study in GHB-S (sensitive) and GHB-R (resistant) rat lines. 2005, Pubmed
Barrantes, From hopanoids to cholesterol: Molecular clocks of pentameric ligand-gated ion channels. 2016, Pubmed
Baulieu, Neurosteroids: a novel function of the brain. 1998, Pubmed
Belelli, The influence of subunit composition on the interaction of neurosteroids with GABA(A) receptors. 2002, Pubmed , Xenbase
Bracamontes, Occupation of either site for the neurosteroid allopregnanolone potentiates the opening of the GABAA receptor induced from either transmitter binding site. 2011, Pubmed , Xenbase
Bracamontes, Multiple modes for conferring surface expression of homomeric beta1 GABAA receptors. 2008, Pubmed
Brunner, New photolabeling and crosslinking methods. 1993, Pubmed
Budelier, Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins. 2017, Pubmed
Carver, Neurosteroid Structure-Activity Relationships for Functional Activation of Extrasynaptic δGABA(A) Receptors. 2016, Pubmed
Chang, A single M1 residue in the beta2 subunit alters channel gating of GABAA receptor in anesthetic modulation and direct activation. 2003, Pubmed , Xenbase
Chen, 11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors. 2014, Pubmed , Xenbase
Chen, Neurosteroid analog photolabeling of a site in the third transmembrane domain of the β3 subunit of the GABA(A) receptor. 2012, Pubmed
Chen, Deep amino acid sequencing of native brain GABAA receptors using high-resolution mass spectrometry. 2012, Pubmed
Cheng, Mapping two neurosteroid-modulatory sites in the prototypic pentameric ligand-gated ion channel GLIC. 2018, Pubmed
Chiara, Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor. 2013, Pubmed
Chiara, Photoaffinity labeling the propofol binding site in GLIC. 2014, Pubmed , Xenbase
Darbandi-Tonkabon, Photoaffinity labeling with a neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-dependent anion channel-1 in rat brain. 2003, Pubmed , Xenbase
Das, Aliphatic diazirines as photoaffinity probes for proteins: recent developments. 2011, Pubmed
Eaton, Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol. 2016, Pubmed
Eaton, Mutational Analysis of the Putative High-Affinity Propofol Binding Site in Human β3 Homomeric GABAA Receptors. 2015, Pubmed , Xenbase
Eaton, γ-aminobutyric acid type A α4, β2, and δ subunits assemble to produce more than one functionally distinct receptor type. 2014, Pubmed , Xenbase
Edgar, MUSCLE: a multiple sequence alignment method with reduced time and space complexity. 2004, Pubmed
Evers, A synthetic 18-norsteroid distinguishes between two neuroactive steroid binding sites on GABAA receptors. 2010, Pubmed , Xenbase
Faroni, The neurosteroid allopregnanolone modulates specific functions in central and peripheral glial cells. 2011, Pubmed
Gurley, Point mutations in the M2 region of the alpha, beta, or gamma subunit of the GABAA channel that abolish block by picrotoxin. 1995, Pubmed , Xenbase
Harrison, Structure-activity relationships for steroid interaction with the gamma-aminobutyric acidA receptor complex. 1987, Pubmed
Hosie, Neurosteroid binding sites on GABA(A) receptors. 2007, Pubmed
Hosie, Conserved site for neurosteroid modulation of GABA A receptors. 2009, Pubmed
Hosie, Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. 2006, Pubmed
Jansen, Modular design of Cys-loop ligand-gated ion channels: functional 5-HT3 and GABA rho1 receptors lacking the large cytoplasmic M3M4 loop. 2008, Pubmed , Xenbase
Jayakar, Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog. 2014, Pubmed
Jiang, A clickable neurosteroid photolabel reveals selective Golgi compartmentalization with preferential impact on proximal inhibition. 2016, Pubmed , Xenbase
Jurd, General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. 2003, Pubmed
Kanes, Brexanolone (SAGE-547 injection) in post-partum depression: a randomised controlled trial. 2017, Pubmed
Krasowski, Methionine 286 in transmembrane domain 3 of the GABAA receptor beta subunit controls a binding cavity for propofol and other alkylphenol general anesthetics. 2001, Pubmed
Krasowski, Propofol and other intravenous anesthetics have sites of action on the gamma-aminobutyric acid type A receptor distinct from that for isoflurane. 1998, Pubmed
Laverty, Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites. 2017, Pubmed
Li, Hydrogen bonding between the 17beta-substituent of a neurosteroid and the GABA(A) receptor is not obligatory for channel potentiation. 2009, Pubmed
Li, Identification of a GABAA receptor anesthetic binding site at subunit interfaces by photolabeling with an etomidate analog. 2006, Pubmed
Li, Natural and enantiomeric etiocholanolone interact with distinct sites on the rat alpha1beta2gamma2L GABAA receptor. 2007, Pubmed
McGibbon, MDTraj: A Modern Open Library for the Analysis of Molecular Dynamics Trajectories. 2015, Pubmed
Mennerick, Selective antagonism of 5alpha-reduced neurosteroid effects at GABA(A) receptors. 2004, Pubmed , Xenbase
Meslamani, Assessing the geometric diversity of cytochrome P450 ligand conformers by hierarchical clustering with a stop criterion. 2009, Pubmed
Middendorp, Accelerated discovery of novel benzodiazepine ligands by experiment-guided virtual screening. 2014, Pubmed
Miller, Structural basis for GABAA receptor potentiation by neurosteroids. 2017, Pubmed
Miller, Crystal structure of a human GABAA receptor. 2014, Pubmed
Nakamura, Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse. 2016, Pubmed
Nury, X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel. 2011, Pubmed
Olsen, GABA A receptors: subtypes provide diversity of function and pharmacology. 2009, Pubmed
Phulera, Cryo-EM structure of the benzodiazepine-sensitive α1β1γ2S tri-heteromeric GABAA receptor in complex with GABA. 2018, Pubmed
Reddy, Enhanced anticonvulsant activity of ganaxolone after neurosteroid withdrawal in a rat model of catamenial epilepsy. 2000, Pubmed
Richardson, A conserved tyrosine in the beta2 subunit M4 segment is a determinant of gamma-aminobutyric acid type A receptor sensitivity to propofol. 2007, Pubmed
Sali, Comparative protein modelling by satisfaction of spatial restraints. 1993, Pubmed
Shen, Statistical potential for assessment and prediction of protein structures. 2006, Pubmed
Shu, Slow actions of neuroactive steroids at GABAA receptors. 2004, Pubmed , Xenbase
Tretter, Stoichiometry and assembly of a recombinant GABAA receptor subtype. 1997, Pubmed
Trott, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. 2010, Pubmed
Unwin, Refined structure of the nicotinic acetylcholine receptor at 4A resolution. 2005, Pubmed
Willenbring, Isoflurane alters the structure and dynamics of GLIC. 2011, Pubmed
Wohlfarth, Enhanced neurosteroid potentiation of ternary GABA(A) receptors containing the delta subunit. 2002, Pubmed
Yip, A propofol binding site on mammalian GABAA receptors identified by photolabeling. 2013, Pubmed
Zhu, Structure of a human synaptic GABAA receptor. 2018, Pubmed
Ziemba, Alphaxalone Binds in Inner Transmembrane β+-α- Interfaces of α1β3γ2 γ-Aminobutyric Acid Type A Receptors. 2018, Pubmed , Xenbase