Anhydrous grade solvents

4-Bromo-2,6-diisopropylaniline and anhydrous grade solvents used in synthesis were from Aldrich, and were not further dried or purified. Common chemicals, etomidate, asolectin, FLAG peptide and polyethyleneimine were from Sigma. Buffer chemicals, CHAPS and DDM were from Fisher–Anatrace. pTFD-BnOH was obtained from TCI America. R–mTFD-MPAB, [³H]R–mTFD-MPAB (38 Ci/mmol, 26 μM in ethanol, and [³H]azietomidate (19 Ci/mmol, 53 μM in ethanol) were synthesized and tritiated previously [22 (link), 32 (link)]. [³H]Muscimol and [³H]flunitrazepam were from Perkin Elmer (Cat. # NET 574 250UC and NET 567250UC respectively).
The human GABA_ARs used for the biochemical and photolabeling studies described herein and designated as α1β3γ2L or α1β3 had the composition N-FLAG–α1β3γ2L–C–(GGS)3GK–1D4 or N-FLAG–α1β3 respectively and were expressed in tetracycline-inducible HEK293 cells as previously described [33 (link), 34 (link)]. They were used as native membranes or after solubilization and purification on a FLAG antibody column, from which they were eluted in micelles of 200 μM asolectin and 5 mM CHAPS with 100 μg/mL (~100 μM) FLAG peptide. Membranes and reconstituted receptors were stored at (–80°) until needed. In electrophysiological studies, the human subunits lacked the purification tags (see below).

11α-Hydroxyprogesterone was from Santa Cruz Biotechnology, Inc. Anhydrous grade solvents were from Aldrich, and were not further dried or purified. [³H]F4N3Bzox-AP (45 Ci/mmol) and [³H]-11-aziAP (40 Ci/mmol) were synthesized by ViTrax Company (Placentia, CA) by tritiation of compounds 15 and 25, respectively, using the procedure described below. [³H]Azietomidate (12 Ci/mmol) and [³H]R-mTFD-MPAB (38 Ci/mmol) were prepared as described.^{30 (link),40} Human α1β3γ2 GABA_ARs with a FLAG epitope on the N-terminus of the α1 subunit was purified as described^{41 (link)} on an anti-FLAG antibody column from detergent extracts of membrane fractions from a tetracycline-inducible HEK293S cell line. GABA_AR was eluted from the column with 0.1 mM FLAG peptide in elution buffer containing 5 mM CHAPS, 0.2 mM asolectin and then stored at −80°C until use.

11α-Hydroxy-progesterone 16 was from Santa Cruz Biotechnology, Inc. Allopregnanolone [5α-pregnan-3α-ol-20-one (3α,5α-P)], alphaxalone (5α-pregnan-3α-ol-11,20-dione), and etomidate were from Tocris Bioscience, and 5α-pregnan-3α, 21-diol-20-one (3α,5α-THDOC) was from Steraloids, Inc. Pregnanolone [5β-pregnan-3α-ol-20-one (3α,5β-P)] was from Research Plus and pregnenolone sulfate (PS) was from Santa Cruz Biotechnology. Bicuculline methochloride was from Abcam. Human α1β3 and 3α,21-Dihydroxy-5α-pregnan-20-one 3, TFD-benzoic acids 21 and 22 were prepared according to the known methods.^{25 ,26 (link)} Anhydrous grade solvents were from Aldrich, and were not further dried or purified. R-mTFD-MPAB was synthesized as described previously.^{29 (link)} α1β3γ2 GABA_ARs with a FLAG epitope on the N-terminus of α1 subunit were expressed in tetracycline-inducible HEK293S cells and purified from detergent extracts as described^{18 (link),19 (link),33 (link),34 (link)} by use of an anti-FLAG antibody column. After elution from columns in the presence of 0.1 mM FLAG peptide, purified GABA_ARs were stored at −80^○C until use in elution buffer containing 5 mM CHAPS and 0.2 mM asolectin.