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.

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