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Photoaffinity labeling identifies an intersubunit steroid-binding site in heteromeric GABA type A (GABAA) receptors

Open AccessPublished:June 15, 2020DOI:https://doi.org/10.1074/jbc.RA120.013452
      Allopregnanolone (3α5α-P), pregnanolone, and their synthetic derivatives are potent positive allosteric modulators (PAMs) of GABAA receptors (GABAARs) with in vivo anesthetic, anxiolytic, and anti-convulsant effects. Mutational analysis, photoaffinity labeling, and structural studies have provided evidence for intersubunit and intrasubunit steroid-binding sites in the GABAAR transmembrane domain, but revealed only little definition of their binding properties. Here, we identified steroid-binding sites in purified human α1β3 and α1β3γ2 GABAARs by photoaffinity labeling with [3H]21-[4-(3-(trifluoromethyl)-3H-diazirine-3-yl)benzoxy]allopregnanolone ([3H]21-pTFDBzox-AP), a potent GABAAR PAM. Protein microsequencing established 3α5α-P inhibitable photolabeling of amino acids near the cytoplasmic end of the β subunit M4 (β3Pro-415, β3Leu-417, and β3Thr-418) and M3 (β3Arg-309) helices located at the base of a pocket in the β+–α subunit interface that extends to the level of αGln-242, a steroid sensitivity determinant in the αM1 helix. Competition photolabeling established that this site binds with high affinity a structurally diverse group of 3α-OH steroids that act as anesthetics, anti-epileptics, and anti-depressants. The presence of a 3α-OH was crucial: 3-acetylated, 3-deoxy, and 3-oxo analogs of 3α5α-P, as well as 3β-OH analogs that are GABAAR antagonists, bound with at least 1000-fold lower affinity than 3α5α-P. Similarly, for GABAAR PAMs with the C-20 carbonyl of 3α5α-P or pregnanolone reduced to a hydroxyl, binding affinity is reduced by 1,000-fold, whereas binding is retained after deoxygenation at the C-20 position. These results provide a first insight into the structure-activity relationship at the GABAAR β+–α subunit interface steroid-binding site and identify several steroid PAMs that act via other sites.
      Endogenous neurosteroids, including allopregnanolone (3α5α-P) and pregnanolone (3α5β-P), can produce anxiolytic, sedative, and anti-convulsive effects (
      • Belelli D.
      • Lambert J.J.
      Neurosteroids: Endogenous regulators of the GABA(A) receptor.
      ,
      • Belelli D.
      • Hogenkamp D.
      • Gee K.W.
      • Lambert J.J.
      Realising the therapeutic potential of neuroactive steroid modulators of the GABAA receptor.
      ), and their synthetic analogs are in development as general anesthetics and for treatment of epilepsy, anxiety, depression, and other mood disorders (
      • Reddy D.S.
      • Estes W.A.
      Clinical potential of neurosteroids for CNS disorders.
      ,
      • Blanco M.-J.
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      Breakthroughs in neuroactive steroid drug discovery.
      ). These neuroactive steroids act at submicromolar concentrations as potent positive allosteric modulators (PAMs) of γ-aminobutyric acid type A receptors (GABAAR), and at higher concentrations as direct activators in the absence of GABA (
      • Majewska M.D.
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      • Schwartz R.D.
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      Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor.
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      • Puia G.
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      Mechanisms of neurosteroid interactions with GABA(A) receptors.
      ,
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      • Smart T.G.
      Neurosteroid binding sites on GABA(A) receptors.
      ). GABAAR potentiation by steroids demonstrates structural specificity in that the orientation of a hydroxyl group at the C-3 position (Fig. 1) determines activity. Steroids with a 3α-OH, including 3α5α-P and the anesthetic alphaxalone, act as PAMs, whereas their 3β-OH epimers (3β5α-P and betaxalone) at higher concentrations inhibit GABA responses (
      • Harrison N.L.
      • Majewska M.D.
      • Harrington J.W.
      • Barker J.L.
      Structure-activity relationships for steroid interaction with the γ-aminobutyric acidA receptor complex.
      ,
      • Cottrell G.A.
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      • Peters J.A.
      Modulation of GABAA receptor activity by alphaxalone.
      ,
      • Purdy R.H.
      • Morrow A.L.
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      • Paul S.M.
      Synthesis, metabolism, and pharmacological activity of 3α-hydroxy steroids which potentiate GABA-receptor-mediated chloride ion uptake in rat cerebral cortical synaptoneurosomes.
      ,
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      • Fields C.
      • Zeng C.M.
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      • Benz A.
      • Fu T.
      • Zorumski E.
      • Steinbach J.H.
      • Covey D.F.
      • Zorumski C.F.
      • Mennerick S.
      3β-Hydroxypregnane steroids are pregnenolone sulfate-like GABA(A) receptor antagonists.
      ). This structural specificity provided early evidence that steroids might interact with specific binding sites in GABAARs, identification and characterization of which would prove important for the development of novel steroid-based therapeutic agents.
      Figure thumbnail gr1
      Figure 1Locations of general anesthetic binding sites in the TMD of an α1β3γ2 GABAAR and structures of representative neuroactive steroids. Depicted are the four transmembrane helices in each subunit (M1–M4), the homologous binding sites for etomidate and R-mTFD-MPAB, an analog of mephobarbital, in the extracellular third of the β+−α and α++−β subunit TMD interface(s), respectively, and a binding site for neuroactive steroids in the intracellular third of the β+−α interface. The binding sites for GABA are located in the extracellular domain in the β+−α subunit interfaces, and benzodiazepines bind at the homologous site in the α+–γ interface. B, steroid ring structure, with numbering of the carbons, and structures of representative neuroactive steroids that act as positive or negative GABAAR allosteric modulators.
      Functional, structural, and photolabeling studies provide evidence for the existence of multiple steroid-binding sites in αβγ GABAARs. Steroids do not bind to the GABA and benzodiazepine-binding sites at subunit interfaces in the extracellular domain or to the homologous binding sites for intravenous general anesthetics such as propofol, etomidate, and barbiturates that are located at subunit interfaces in the extracellular third of the transmembrane domain (TMD) (Fig. 1) (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ,
      • Forman S.A.
      • Miller K.W.
      Mapping general anesthetic sites in heteromeric γ-aminobutyric acid type a receptors reveals a potential for targeting receptor subtypes.
      ). Binding assays using channel blockers as well as electrophysiological assays identify multiple effects of steroids potentially mediated by distinct sites (
      • Akk G.
      • Bracamontes J.
      • Covey D.F.
      • Evers A.R.
      • Dao T.
      • Steinbach J.H.
      Neuroactive steroids have multiple actions to potentiate GABAA receptors.
      ,
      • Evers A.S.
      • Chen Z.-W.
      • Manion B.D.
      • Han M.
      • Jiang X.
      • Darbandi-Tonkabon R.
      • Kable T.
      • Bracamontes J.
      • Zorumski C.F.
      • Mennerick S.
      • Steinbach J.H.
      • Covey D.F.
      A synthetic 18-norsteroid distinguishes between two neuroactive steroid binding sites on GABAA receptors.
      ). Intersubunit and intrasubunit steroid-binding sites near the extracellular and cytoplasmic surfaces of the TMD are predicted based upon the recently determined α1β3γ2 GABAAR structures (
      • Masiulis S.
      • Desai R.
      • Uchański T.
      • Serna Martin I.
      • Laverty D.
      • Karia D.
      • Malinauskas T.
      • Zivanov J.
      • Pardon E.
      • Kotecha A.
      • Steyaert J.
      • Miller K.W.
      • Aricescu A.R.
      GABAA receptor signalling mechanisms revealed by structural pharmacology.
      ,
      • Laverty D.
      • Desai R.
      • Uchański T.
      • Masiulis S.
      • Stec W.J.
      • Malinauskas T.
      • Zivanov J.
      • Pardon E.
      • Steyaert J.
      • Miller K.W.
      • Aricescu A.R.
      Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer.
      ) and the locations of amino acids identified by mutational analysis as determinants for GABAAR enhancement or direct activation. A site near the cytoplasmic end of the β+–α subunit TMD interface was predicted based upon the identification of α1Gln-242 (human α1 numbering) as a position critical for enhancement by steroids (
      • Hosie A.M.
      • Wilkins M.E.
      • Da Silva H.M.A.
      • Smart T.G.
      Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites.
      ,
      • Akk G.
      • Li P.
      • Bracamontes J.
      • Reichert D.E.
      • Covey D.F.
      • Steinbach J.H.
      Mutations of the GABA-A receptor α1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids.
      ). Consistent with this location, alphaxalone protected against the modification of cysteines substituted in the β3 M3 helix at positions contributing to this interface (
      • Ziemba A.M.
      • Szabo A.
      • Pierce D.W.
      • Haburcak M.
      • Stern A.T.
      • Nourmahnad A.
      • Halpin E.S.
      • Forman S.A.
      Alphaxalone binds in inner transmembrane β+- interfaces of α1β3γ2 γ-aminobutyric acid type A receptors.
      ), and 3α5β-P, tetrahydrodeoxycorticosterone (3α5α-THDOC), and alphaxalone bind to a homologous pocket in crystallographic structures of homopentameric, chimeric receptors with GABAAR α subunit TMDs (
      • Laverty D.
      • Thomas P.
      • Field M.
      • Andersen O.J.
      • Gold M.G.
      • Biggin P.C.
      • Gielen M.
      • Smart T.G.
      Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.
      ,
      • Miller P.S.
      • Scott S.
      • Masiulis S.
      • De Colibus L.
      • Pardon E.
      • Steyaert J.
      • Aricescu A.R.
      Structural basis for GABAA receptor potentiation by neurosteroids.
      ,
      • Chen Q.
      • Wells M.M.
      • Arjunan P.
      • Tillman T.S.
      • Cohen A.E.
      • Xu Y.
      • Tang P.
      Structural basis of neurosteroid anesthetic action on GABA(A) receptors.
      ). In α1β3 GABAARs, there is 3α5α-P inhibitable steroid photolabeling of a residue at the cytoplasmic end of βM3 in proximity to this pocket, with additional residues identified near the extracellular end of the TMD within the α1 and β3 subunits (
      • Chen Z.W.
      • Bracamontes J.R.
      • Budelier M.M.
      • Germann A.L.
      • Shin D.J.
      • Kathiresan K.
      • Qian M.X.
      • Manion B.
      • Cheng W.W.L.
      • Reichert D.E.
      • Akk G.
      • Covey D.F.
      • Evers A.S.
      Multiple functional neurosteroid binding sites on GABAA receptors.
      ).
      Photoaffinity labeling with radiolabeled, photoreactive intravenous general anesthetics has allowed the identification of photolabeled amino acids for site identification and the determination of the pharmacological specificity of these sites by inhibition of photolabeling with nonradioactive anesthetics. Photolabeling with [3H]azietomidate and a mephobarbital analog, [3H]R-mTFD-MPAB, identified homologous binding sites in the α1β3γ2 GABAAR TMD at the β+–α and α++–β subunit interfaces, respectively (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ,
      • Jayakar S.S.
      • Zhou X.J.
      • Chiara D.C.
      • Jarava-Barrera C.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Tortosa M.
      • Miller K.W.
      • Cohen J.B.
      Identifying drugs that bind selectively to intersubunit general anesthetic sites in the α1β3γ2 GABAAR transmembrane domain.
      ). Etomidate and azietomidate bind with 100-fold selectivity to the β+ sites, R-mTFD-MPAB with 50-fold selectivity to the β sites, and other barbiturates and propofol derivatives bind with variable selectivity to the two classes of sites.
      Here we characterize a GABAAR steroid-binding site by use of 21-pTFDBzox-AP (21-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoxy]allopregnanolone), a photoreactive steroid that acts as a potent α1β3 and α1β3γ2 GABAAR PAM (
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      ). Previously, we reported that [3H]21-pTFDBzox-AP primarily photoincorporated into the β3 subunit with ∼80% of the subunit photolabeling inhibitable by 3α5α-P or by alphaxalone, but not by pregnenolone sulfate (PS), an inhibitory neurosteroid, or by etomidate or R-mTFD-MPAB (
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      ). We now identify the amino acids photolabeled by [3H]21-pTFDBzox-AP, which are located at the cytoplasmic ends of the βM3 and βM4 helices and form the base of a pocket at β+–α intersubunit interface that extends up to the level of α1Gln-242 in αM1. By use of competition photolabeling with a panel of steroid GABAAR PAMs and inhibitors, we provide a first definition of the structural determinants important for high affinity binding to this site.

      Results

      Positive and negative steroid GABAAR allosteric modulators enhance [3H]muscimol binding

      In equilibrium binding assays with the agonist [3H]muscimol, GABAAR PAMs, including steroids and other general anesthetics, enhance binding by increasing the fraction of GABAARs in a desensitized state that binds [3H]muscimol with high affinity (
      • Peters J.A.
      • Kirkness E.F.
      • Callachan H.
      • Lambert J.J.
      • Turner A.J.
      Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids.
      ). 21-pTFDBzox-AP was shown previously to enhance [3H]muscimol binding to expressed α1β3 and α1β3γ2 GABAARs in membranes, and after purification in detergent/lipid micelles, with concentrations producing half-maximal enhancement (EC50, 0.2-0.5 μm) similar to those for 3α5α-P, 3α5β-P, and alphaxalone (
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      ). We extended these studies by characterizing [3H]muscimol binding to α1β3 GABAARs in the presence of steroids that act as GABAAR negative allosteric modulators, inhibiting GABA responses noncompetitively: the 3β-epimers of 3α5α-P, 3α5β-P, and alphaxalone, and two 3β-sulfated steroids (PS and dehydroepiandrosterone sulfate (DHEAS)) (
      • Cottrell G.A.
      • Lambert J.J.
      • Peters J.A.
      Modulation of GABAA receptor activity by alphaxalone.
      ,
      • Wang M.D.
      • He Y.J.
      • Eisenman L.N.
      • Fields C.
      • Zeng C.M.
      • Mathews J.
      • Benz A.
      • Fu T.
      • Zorumski E.
      • Steinbach J.H.
      • Covey D.F.
      • Zorumski C.F.
      • Mennerick S.
      3β-Hydroxypregnane steroids are pregnenolone sulfate-like GABA(A) receptor antagonists.
      ,
      • Demirgören S.
      • Majewska M.D.
      • Spivak C.E.
      • London E.D.
      Receptor binding and electrophysiological effects of dehydroepiandrosterone sulfate, an antagonist of the GABAA receptor.
      ,
      • Park-Chung M.
      • Malayev A.
      • Purdy R.H.
      • Gibbs T.T.
      • Farb D.H.
      Sulfated and unsulfated steroids modulate γ-aminobutyric acidA receptor function through distinct sites.
      ) (Fig. 2 and Table 1). The 3β-OH epimers of pregnanolone (3β5β-P) and alphaxalone (betaxalone) enhanced [3H]muscimol binding with EC50 values of 25 and 45 μm, respectively, whereas 3β5α-P at concentrations up to 100 μm did not. PS at concentrations up to 500 μm had no effect on [3H]muscimol binding, whereas DHEAS reduced specific binding maximally by 50% (IC50 = 10 μm). In addition, we found that (3α5α)-17-phenylandrost-16-en-3-ol (17-PA), which antagonizes steroid enhancement of GABA responses but not GABA responses (
      • Mennerick S.
      • He Y.J.
      • Jiang X.
      • Manion B.D.
      • Wang M.D.
      • Shute A.
      • Benz A.
      • Evers A.S.
      • Covey D.F.
      • Zorumski C.F.
      Selective antagonism of 5α-reduced neurosteroid effects at GABAA receptors.
      ), enhanced [3H]muscimol binding with an EC50 of 30 μm.
      Figure thumbnail gr2
      Figure 2Modulation of GABAAR agonist binding by steroid antagonists. The 3β-OH steroid antagonists 3β5β-P and betaxalone and the 3α-OH antagonist 17-PA enhance equilibrium binding of subsaturating concentrations of [3H]muscimol (2 nm) with efficacies similar to that seen for the PAM 3α5α-P but with lower potencies, whereas no enhancement is seen for 3β5α-P. The 3β-sulfate antagonist PS did not enhance binding, whereas DHEAS reduced specific binding maximally by 50%. The data from n independent experiments were combined and fit to determine values of EC50 (in µm), Hill coefficients (nH), and maximal enhancements (Bmax, as % control), that were: 3β5β-P (26 ± 7, 1.0 ± 0.3, 193 ± 8, n =2); betaxalone (45 ± 8, 1.2 ± 0.2, 233 ± 8, n = 2); 17-PA (29 ± 5, 1.6 ± 0.4, 352 ± 22, n = 3); 3α5α-P (EC50 = 0.58 ± 0.22 μm, data from Ref.
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      ). DHEAS (n = 4) inhibited specific binding maximally by 51 ± 2% with IC50 = 10.3 ± 1.6 μm and nH = 1.6 ± 0.3.
      Table 1Comparison of potency of neuroactive steroids as modulators of [3H]muscimol binding and as inhibitors α1β3 and α1β3γ2 GABAAR photolabeling by [3H]21-pTFDBzoxAP
      Steroid
      Catalog numbers are indicated for steroids from Research Plus (xxxx-16) and Steraloids (P-xxxx).
      R1 (C-3)C5R2 (C-17)[3H]Muscimol Binding
      EC50 (±S.E.) values for steroid modulation of 2 nm [3H]muscimol binding to α1β3 GABAAR in membranes, from Fig. 2 and Ref. 27.
      EC50
      [3H]21-pTFDBzox-AP
      IC50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of photolabeling of GABAAR purified in detergent/lipid, were determined as described under “Experimental procedures,” from Fig. 4 and Ref. 27. n, number of experiments.
      IC50 (n)
      α1β3α1β3γ2
      μmμmμm
      21-pTFDBzox-AP-OH, αα-COCH2OCOBzTFD0.36 ± 0.060.21 ± 0.02 (4)0.65 ± 0.08 (4)
      3α5β-P pregnanolone-OH, αβ-COCH30.62 ± 0.080.65 ± 0.13 (4)0.30 ± 0.05 (4)
      3α5α-P allopregnanolone-OH,αα-COCH30.58 ± 0.220.27 ± 0.03 (6)0.40 ± 0.06 (6)
      3β5α-P (3101-16, P3830)-OH, βα-COCH3>100>1000 (6)470 ± 70 (2)
      3β5β-P (3198-16)-OH, ββ-COCH326 ± 7>500 (4)>300 (6)
      3α-Acetyl-5α-P (P3801)CH3CO2-, αα-COCH3>10
      EC50 values for enhancement of [3H]flunitrazepam binding to rat brain membranes (51).
      >300 (4)>300 (6)
      3-Oxo,5α-P (P2970)=Oα-COCH3>10
      EC50 values for enhancement of [3H]flunitrazepam binding to rat brain membranes (51).
      >300 (4)>300 (2)
      3-Deoxy-5α-P (P4230)-Hα-COCH31
      EC50 values for enhancement of GABA responses of expressed α1β2γ2 GABAAR (12, 20).
      >300 (4)>300 (2)
      Alphaxalone (11-oxo)-OH, αα-COCH30.71 ± 0.154.6 ± 0.7 (4)2.4 ± 0.5 (4)
      Betaxalone (11-oxo) (3093-16)-OH, βα-COCH345 ± 8175 ± 25 nH = 0.5 ± 0.1 (6)200 ± 50 (2)
      3α5α-THDOC (P2560)-OH, αα-COCH2OH1.0 ± 0.152.1 ± 0.3 (4)ND
      3α5β-THDOC (3167-16)-OH, αβ-COCH2OH3
      EC50 values for enhancement of GABA responses of expressed α1β2γ2 GABAAR (12, 20).
      3.3 ± 0.5 (4)2.9 ± 0.5 (4)
      DHEAS (5-ene)SO4-, β=OIC50 = 10 ± 2>300 (4)>300 (4)
      PS (5-ene)SO4-, β-COCH3>300>300 (4)>300 (6)
      a Catalog numbers are indicated for steroids from Research Plus (xxxx-16) and Steraloids (P-xxxx).
      b EC50 (±S.E.) values for steroid modulation of 2 nm [3H]muscimol binding to α1β3 GABAAR in membranes, from Fig. 2 and Ref.
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      .
      c IC50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of photolabeling of GABAAR purified in detergent/lipid, were determined as described under “Experimental procedures,” from Fig. 4 and Ref.
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      . n, number of experiments.
      d EC50 values for enhancement of [3H]flunitrazepam binding to rat brain membranes (
      • Hawkinson J.E.
      • Kimbrough C.L.
      • Belelli D.
      • Lambert J.J.
      • Purdy R.H.
      • Lan N.C.
      Correlation of neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding and γ-aminobutyric acidA receptor function.
      ).
      e EC50 values for enhancement of GABA responses of expressed α1β2γ2 GABAAR (
      • Wang M.D.
      • He Y.J.
      • Eisenman L.N.
      • Fields C.
      • Zeng C.M.
      • Mathews J.
      • Benz A.
      • Fu T.
      • Zorumski E.
      • Steinbach J.H.
      • Covey D.F.
      • Zorumski C.F.
      • Mennerick S.
      3β-Hydroxypregnane steroids are pregnenolone sulfate-like GABA(A) receptor antagonists.
      ,
      • Akk G.
      • Li P.
      • Bracamontes J.
      • Reichert D.E.
      • Covey D.F.
      • Steinbach J.H.
      Mutations of the GABA-A receptor α1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids.
      ).

      Pharmacologically specific photolabeling by [3H]21-pTFDBzox-AP in the β3 subunit of α1β3 and α1β3γ2 GABAARs

      In initial photolabeling studies, we compared [3H]21-pTFDBzox-AP photolabeling of α1β3 and α1β3γ2 GABAARs. After photolabeling, GABAAR subunits were resolved by SDS-PAGE, and 3H incorporation into the subunits was characterized by fluorography (Fig. 3A). As reported previously (
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      ), for both receptor subtypes photolabeling was most prominent in the gel bands of 59 and 61 kDa that contain differentially glycosylated β3 subunits (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ,
      • Chiara D.C.
      • Dostalova Z.
      • Jayakar S.S.
      • Zhou X.
      • Miller K.W.
      • Cohen J.B.
      Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-Etomidate, a photoreactive etomidate analogue.
      ), and at a lower level in the 56-kDa gel band containing the α1 and γ2 subunits. Photolabeling of the β3 subunit was inhibited by 30 μm 3α5α-P, but not by PS, etomidate, or R-mTFD-MPAB. To quantify the concentration dependence of inhibition of photolabeling by nonradioactive drugs, receptor aliquots were photolabeled with [3H]21-pTFDBzox-AP in the presence of a range of drug concentrations, with receptor subunits excised from the stained gel after SDS-PAGE and 3H incorporation into the β subunit determined by liquid scintillation counting. In a representative experiment (Fig. 3B), nonradioactive 21-pTFDBzox-AP maximally inhibited [3H]21-pTFDBzox-AP photolabeling of α1β3 and α1β3γ2 GABAARs to the same extent as 30 μm 3α5α-P, with IC50 values of 0.7 and 0.9 μm, respectively. As described under “Experimental procedures,” IC50 values for drugs were determined by combining results from at least four independent experiments using two or more GABAAR purifications, with data from individual experiments combined after normalization to the total specific (i.e. 3α5α-P inhibitable) binding in the absence of competitor. The pooled data for inhibition by 21-pTFDBzox-AP are shown in Fig. 4.
      Figure thumbnail gr3
      Figure 33α5α-P and 21-pTFDBzox-AP, but not PS, inhibit [3H]21-pTFDBzox-AP (0.5 μm) photolabeling of α1β3 and α1β3γ2 GABAARs. GABAAR aliquots were equilibrated in the presence of 300 μm GABA, in the absence or presence of 3α5α-P, PS, etomidate, or R-mTFD-MPAB. After photolabeling, receptor subunits were resolved by SDS-PAGE and 3H incorporation was determined by fluorography (A) or by liquid scintillation counting of excised subunits (B). A, lanes 1 and 2 are representative Coomassie Blue-stained gel lanes from the gel used for fluorography. The fluorogram (lanes 3–12) compares 3H incorporation into α1β3 (lanes 3–7) and α1β3γ (lanes 8–12) GABAARs photolabeled in the presence of GABA (lanes 3 and 8), or in the presence of GABA and 30 μm 3α5α-P (lanes 4 and 9), 100 μm PS (lanes 5 and 10), 300 μm etomidate (lanes 6 and 11), or 60 μm R-mTFD-MPAB (lanes 7 and 12). Indicated on the left of lane 1 are the mobilities of the molecular mass markers (98, 64, and 58 kDa) and between lanes 2 and 3 the calculated mobilities of the GABAAR subunit bands (α1, 56 kDa; β3, 59/61 kDa; with the γ2 subunit distributed diffusely in this region). B, in separate experiments, α1β3 and α1β3γ2 GABAARs were equilibrated with [3H]21-pTFDBxoz-AP, GABA, and increasing concentrations of nonradioactive 21-pTFDBzox-AP or 30 μm 3α5α-P. After photolabeling, receptor subunits were separated by SDS-PAGE, and 3H incorporation was determined in the excised gel bands. For each receptor, two experiments were run in parallel, and the plotted data are the 3H incorporation in the β subunit (59/61 kDa) gel bands (mean ± ½ range). The curves are the fits of the data to a single site model, with Bns fixed at the observed photoincorporation in the presence of 30 μm 3α5α-P (α1β3 (dotted line), 310 ± 30 cpm; α1β3γ2 (dashed line), 460 ± 16 cpm). For α1β3 and α1β3γ2 GABAARs, the IC50 values were 0.65 ± 0.1 μm (R2 = 0.95) and 0.92 ± 0.16 μm (R2 = 0.94), respectively. In the α1 (56 kDa) gel bands, control and nonspecific 3H incorporation (in cpm) were 370 ± 40/140 ± 10 (α1β3) and 430 ± 60/240 ± 13 (α1β3γ2).
      Figure thumbnail gr4
      Figure 4A free 3α-OH is a major determinant of high affinity binding of pregnane steroids to the [3H]21-pTFDBzox-AP GABAAR site. α1β3 GABAARs (A) or α1β3γ2 GABAARs (B) were photolabeled in the presence of GABA and varying concentrations of pregnane steroids containing a 3α-OH (3α5α-P, 3α5β-THDOC, alphaxalone, 21-pTFDBzox-AP), a 3β-OH (3β5α-P, betaxalone), or lacking a free 3-OH (3α-acetyl-5α-P, 3-deoxy-5α-P, 3-oxo-5α-P). After SDS-PAGE, covalent incorporation of 3H in the β subunit was determined by liquid scintillation counting. For each independent experiment, nonspecific photolabeling (Bns) was determined in the presence of 30 μm 3α5α-P, and specific binding was normalized to the 3H cpm incorporated specifically in the control condition (B0Bns). The plotted data are the averages (± S.D.) from the independent experiments. As described under “Experimental procedures,” the pooled data from the independent experiments were fit to . Drug structures, parameters for the fits, and the number of independent experiments are tabulated in . The curves are plotted for fits to nH =1, which were favored by F-test comparison over fits with variable nH, with the exception of betaxalone (α1β3, nH = 0.5 ± 0.1). Based upon an F-test comparison of fits of the data for α1β3 and α1β3γ2 GABAARs to the same (null hypothesis) or separate IC50 values, a common fit was favored for 3α5α-P (p = 0.7, F(DFn,DFd) = 0.22 (1,112)) and 3α5β-THDOC (p = 0.6, F(DFn,DFd) = 0.24(1,63)). Separate fits were favored for 21-pTFDBzox-AP (p < 0.0001, F(DFn,DFd) = 35.9(1,94)), 3α5β-P (p = 0.002, F(DFn,DFd) = 10.4(1,62)), and alphaxalone (p = 0.01, F(DFn,DFd) = 6.7(1,62)).

      In α1β3 and α1β3γ2 GABAARs, a 3α-OH substituent is a major determinant of pregnanolone affinity for this site

      As a test of the pharmacological specificity of the sites identified in α1β3 and α1β3γ2 GABAARs by [3H]21-pTFDBzox-AP photolabeling, we compared inhibition by 3α5α-P with its antagonist 3β-OH isomer (3β5α-P) and with analogs modified at the 3-position by acetylation (3α-acetyl-5α-P), removal of the –OH (3-deoxy-5α-P), or oxidation into a ketone (3-oxo-5α-P) (Fig. 4 and Table 1). 3α5α-P inhibited photolabeling of both receptor subtypes with an IC50 of 0.4 μm, whereas 3β5α-P at 300 μm inhibited photolabeling of α1β3 and α1β3γ2 GABAARs by <10% and ∼40%, respectively. At the highest concentration tested (100 μm), 3-deoxy-5α-P, which is a GABAAR PAM (
      • Akk G.
      • Li P.
      • Bracamontes J.
      • Reichert D.E.
      • Covey D.F.
      • Steinbach J.H.
      Mutations of the GABA-A receptor α1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids.
      ), as well as 3α-acetyl-5α-P and 3-oxo-5α-P each inhibited photolabeling by <10%. We also determined that alphaxalone inhibited α1β3 and α1β3γ2 GABAAR photolabeling with IC50 values of 5 and 2 μm, respectively, whereas for betaxalone, 50% inhibition was seen at ∼200 μm (Fig. 4). Consistent with the importance of a 3α-OH for binding to this site, the sulfated 3β-OH antagonists PS and DHEAS at 100 μm each inhibited photolabeling by <10% (Table 1 and Ref.
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      ). In contrast to the importance of the 3α-OH, the configuration at the 5-position was not important. 3α5β-P inhibited photolabeling with an IC50 of 0.7 μm, similar to that for 3α5α-P, whereas 3α5α-THDOC and 3α5β-THDOC inhibited GABAAR photolabeling with IC50 values of 2-3 μm (Table 1).

      3α5α-P inhibits [3H]21-pTFDBzox-AP photolabeling of amino acids located at the cytoplasmic ends of the βM3 and βM4 helices that contribute to a pocket at the β+–α subunit interface

      Based upon the similar pharmacological properties of the steroid-binding sites in α1β3 and α1β3γ2 GABAARs defined by [3H]21-pTFDBzox-AP photolabeling, we identified the photolabeled amino acids in α1β3 GABAARs, which can be expressed and purified at higher levels than α1β3γ2 GABAARs. β3 subunits were isolated from α1β3 GABAARs photolabeled on a preparative scale with [3H]21-pTFDBzox-AP (0.7 μm) in the presence of 300 μm GABA and in the absence or presence of 30 μm 3α5α-P. In five preparative photolabelings, the specific β subunit photolabeling (i.e. 3α5α-P inhibitable) was 320 ± 70 3H cpm/pmol, which indicated photolabeling of 1.2 ± 0.2% of β subunits based upon the radiochemical specific activity of [3H]21-pTFDBzox-AP (21.8 Ci/mmol) and the amount of GABAAR photolabeled. This efficiency of photolabeling was similar to that seen for GABAAR photolabeling by a photoreactive etomidate analog (
      • Chiara D.C.
      • Dostalova Z.
      • Jayakar S.S.
      • Zhou X.
      • Miller K.W.
      • Cohen J.B.
      Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-Etomidate, a photoreactive etomidate analogue.
      ), but ∼15% the efficiency seen for [3H]R-mTFD-MPAB (
      • Savechenkov P.Y.
      • Zhang X.
      • Chiara D.C.
      • Stewart D.S.
      • Ge R.
      • Zhou X.
      • Raines D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Allyl m-trifluoromethyldiazirine mephobarbital: an unusually potent enantioselective and photoreactive barbiturate general anesthetic.
      ).
      The photolabeled amino acids were identified by protein microsequencing of fragments beginning near the N termini of the β3M4, β3M3, and β3M1 helices that can be produced by digestion with endoproteinase Lys-C (Endo Lys-C) and resolved by reversed-phase HPLC (rpHPLC) (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ,
      • Chiara D.C.
      • Dostalova Z.
      • Jayakar S.S.
      • Zhou X.
      • Miller K.W.
      • Cohen J.B.
      Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-Etomidate, a photoreactive etomidate analogue.
      ,
      • Jayakar S.S.
      • Zhou X.
      • Savechenkov P.Y.
      • Chiara D.C.
      • Desai R.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Positive and negative allosteric modulation of an α1β3γ2 γ-aminobutyric acid type A (GABAA) receptor by binding to a site in the transmembrane domain at the γ+ interface.
      ). When aliquots of the β subunit Endo Lys-C digests were sequenced, peaks of 3H release were seen in cycles 3/4 and 6/7 that were inhibitable by 3α5α-P (Fig. 5A). When the digests were fractionated by rpHPLC (Fig. 5B), the peak of 3H was recovered in a fraction that contained an unlabeled fragment beginning at β3Ala-280 near the N terminus of βM3, with the unlabeled fragment beginning at β3Ile-412 before the N terminus of βM4 eluting one fraction earlier. Additional 3H-containing adducts eluted in the more hydrophobic fractions that contain the unlabeled fragment beginning at β3Arg-216 at the N terminus of βM1 that extends through βM2.
      Figure thumbnail gr5
      Figure 53α5α-P inhibits [3H]21-pTFDBzox-AP photolabeling of β3Ile-414, β3Pro-415, β3Leu-417, and β3Thr-418 near the N terminus of βM4. α1β3 GABAARs were photolabeled on a preparative scale in the presence of 300 μm GABA in the absence or presence of 30 μm 3α5α-P. GABAAR β subunits were isolated by SDS-PAGE and digested with Endo Lys-C. A, when digested, aliquots (10%) were sequenced without further purification, there were peaks of 3H release in cycles 3/4 and 6/7 for the sample photolabeled in the absence (●) but not the presence (○) of 3α5α-P. Shown above are the sequences of the β3 subunit fragments produced by Endo Lys-C digestion that contain transmembrane helices (M1–M2, M3, and M4). B, 3H elution profiles when the Endo Lys-C digests were fractionated by rpHPLC, determined by counting 10% of each fraction. Inset, Edman degradation determination of the masses (I0) of β subunit fragments eluting in rpHPLC fractions 25 (βM4), 26/27 (βM3), and 28-29 (βM1). C, 3H released during sequence analysis of the peak of 3H (rpHPLC fraction 26) from receptors photolabeled in the absence (●, ♦) and presence (○, ⋄) of 3α5α-P and released PTH-derivatives (□, ▵) in the absence of 3α5α-P. Equal aliquots were sequenced normally (●, ○, □) or with sequencing interrupted at cycle 4 for treatment with OPA (♦, ⋄, ▵) to prevent further sequencing of the βM3 fragment not containing a proline at that cycle. In the absence of OPA, the PTH-derivatives from the β3Ala-280 fragment (□, I0 = 1.6 pmol) were detected for 20 cycles of Edman degradation. OPA treatment (▵) prevented further sequencing of that fragment, but did not alter the pattern of 3H release, with peaks in cycles 3/4 and 6/7 without (●) or with (♦) OPA. The persistence of 3H release in cycles 4, 6, and 7 after OPA treatment was consistent with photolabeling of β3Pro-415, β3Leu-417, and β3Thr-418. This photolabeling was inhibitable by 3α5α-P, because the peaks of 3H release were reduced by 90% (without (○) or with (⋄) OPA) when fraction 26 was sequenced from receptors photolabeled in the presence of 3α5α-P.
      Protein sequencing protocols were designed to allow identification of photolabeled amino acids even if the incorporation of the hydrophobic steroid caused the 3H-labeled fragment to elute in more hydrophobic HPLC gradient fractions than the unlabeled fragment directly identifiable by PTH-derivative analysis. When 50% of the fraction containing the peak of 3H was sequenced, there were peaks of 3H release in cycles 3-4 and 6-7 of Edman degradation that were reduced by 90% by 3α5α-P (Fig. 5C), as seen when the total digest was sequenced. There were no additional peaks of 3H release above background in 30 cycles of Edman degradation (not shown). To determine whether the peaks of 3H release originated from labeling in βM3 or βM4, we took advantage of the presence of β3Pro-415 in cycle 4 of Edman degradation of the βM4 fragment and the lack of a proline at that cycle in the βM3 or βM1 fragment. For the remaining 50% of the fraction, sequencing was interrupted at cycle 4 for treatment with o-pthalaldehyde (OPA) to prevent further sequencing of fragments not containing a proline at that cycle (
      • Brauer A.W.
      • Oman C.L.
      • Margolies M.N.
      Use of o-phthalaldehyde to reduce background during automated Edman degradation.
      ,
      • Middleton R.E.
      • Cohen J.B.
      Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label.
      ). After treatment with OPA in cycle 4, the 3H releases in cycles 4, 6, and 7 were preserved, whereas sequencing of the M3 fragment was reduced by >95% (Fig. 5C). Thus, these 3H releases did not originate from the βM3 fragment. Rather, the results were consistent with 3α5α-P inhibitable photolabeling of β3Pro-415 (cycle 4), β3Leu-417, and β3Thr-418 in the fragment beginning at β3Ile-412 before the N terminus of βM4. The 3H release in cycle 3, although not tested by the use of OPA in cycle 4, indicated likely labeling of β3Ile-414. Based upon sequencing nine samples from five independent photolabeling experiments, β3Ile-414 and β3Leu-417 were photolabeled at 55 ± 26 and 155 ± 55 cpm/pmol, respectively, ∼90% inhibitable by 3α5α-P (Table 2). Because of uncertainties in calculating photolabeling efficiency for the second of two successive photolabeled amino acids, similar calculations were not made for β3Pro-415 and β3Thr-418.
      Table 2Pharmacological specificity of [3H]21-pTFDBzox-AP photoincorporation into β3Ile-414, β3Leu-417, and β3Arg-309 in the α1β3 GABAAR in the presence of GABA
      Controln+3α5α-P inhibition
      cpm/pmol%
      β3Ile41455 ± 26992 ± 7
      β3Leu417155 ± 55988 ± 6
      β3Arg30937 ± 22590 ± 7
      Photolabeling of β3Arg-309 at the C terminus of βM3 was identified by sequencing the broad peak of 3H that co-eluted with the unlabeled βM1 fragment (Fig. 6). A peak of 3α5α-P inhibitable 3H release was seen in cycle 30, in addition to the peaks of 3H release in cycles 3/4 and 6/7 attributable to labeling within the βM4 fragment and a peak in cycle 19 not reproduced in other experiments (Fig. 6A). The 3H release in cycle 30 did not result from labeling in βM1, because for a sample sequenced with OPA treatment at cycle 13, the cycle containing β3Pro-228 in βM1, sequencing of the βM1 fragment persisted after treatment but no release of 3H was seen in cycle 30 (not shown). This suggested that the labeled βM3 fragment, similar to the labeled βM4 fragment, eluted in more hydrophobic rpHPLC fractions than the unlabeled fragment, with the 3H release in cycle 30 resulting from photolabeling of β3Arg-309. To test this, we generated a fragment beginning at β3Gly-287 in βM3 by use of cyanogen bromide to cleave at the C terminus of β3Met-287 (as well as other methionines in the sample on the sequencing filter). When this fragment was sequenced, there was a peak of 3α5α-P inhibitable 3H release in cycle 23 consistent with photolabeling of β3Arg-309 (Fig. 6B). Based upon results from 4 independent photolabeling experiments, β3Arg-309 was photolabeled at ∼25% the efficiency as compared with β3Leu-417 (Table 2).
      Figure thumbnail gr6
      Figure 63α5α-P inhibits [3H]21-pTFDBzox-AP photolabeling of β3Arg-309 near the cytoplasmic end of M3. A, 3H (●, ○) and PTH-derivatives (⋄) released during sequence analysis of rpHPLC fractions 28-30 (from B) from receptors photolabeled in the absence (●, ⋄) and presence (○) of 3α5α-P. Samples were sequenced in duplicate, and the 3H release is plotted as mean cpm (±½ range). The primary sequence began at β3Arg-216 at the N terminus of βM1 (⋄, I0 = 6 pmol) with the β3Ala-280 fragment present at 10% of that level (not shown). Also plotted are the PTH-derivatives released (□) for the total amount of the β3Ala-280 fragment sequenced in fractions 26-30 (I0 = 9 pmol). The peak of 3α5αP-inhibitable 3H release in cycles 30/31 of Edman degradation was not seen when fractions 26 or 27 were sequenced (not shown). If the increased hydrophobicity of the photolabeled β3Ala-280 fragment shifted its elution to more hydrophobic fractions than the unlabeled fragment, which eluted in fractions 26-27, the peak of 3H release in cycle 30 would result from 3α5α-P inhibitable photolabeling of β3Arg-309 near the C terminus of βM3. The following experiment tested this hypothesis. B, 3H (●, ○) and PTH-derivatives (□) released during sequence analysis of a β3 subunit fragment beginning at β3Gly-287 confirms 3α5α-P inhibitable photolabeling of β3Arg-309. From an independent preparative photolabeling of GABAARs in the absence (●, □) and presence (○) of 3α5α-P, rpHPLC fractions 25-29 were pooled for sequencing from Endo Lys-C digests of β subunits. Samples were first sequenced for 20 cycles with OPA treatment at cycle 4 (not shown), then sequencing was interrupted for treatment with cyanogen bromide to cleave at methionines (see “Experimental procedures”). After treatment, the fragment beginning at β3Gly-287 (□, I0 = 1.8 pmol) was sequenced along with fragments beginning at β3Pro-228 in βM1 and β3Thr-262 in βM2. The peak of 3H release in cycle 23 seen for photolabeling in the absence (●) but not in the presence (○) of 3α5α-P was consistent with photolabeling of β3Arg-309 at 30 cpm/pmol. This efficiency was the same as that calculated for β3Arg-309 photolabeling based upon the peak of 3H release seen in cycle 30 when a parallel aliquot of the pool of fraction 25–29 was directly sequenced for 35 cycles (not shown). If photolabeling of β3Asp-245 in βM1 was the source of 3H release in cycle 30 (A), the peak of 3H release would have shifted to cycle 18 when sequencing the β3Pro-228 fragment. The identity of the photolabeled amino acid associated with the peak of 3H release in cycle 4 is uncertain. The absence of peaks of 3H release in cycles 11 and 16 of A rules out labeling of βVal-290 in βM3 or βLeu-232 in βM1; β3Asn-265 may be photolabeled at ∼10% the efficiency of β3Leu-417.

      [3H]21-pTFDBzoxy-AP photolabeling in GABAAR α subunit

      Because β subunit photolabeling dominated over that in α and the gel band containing α subunit also contains β subunit at a low level (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ), it was difficult to use our protocols to determine whether α subunit residues were photolabeled at low efficiency. Nonetheless, we searched in particular for 3α5α-P inhibitable photolabeling in αM4 at αAsn-408, which is an intrasubunit residue near the extracellular end of TMD that is a sensitivity determinant for steroid enhancement (
      • Hosie A.M.
      • Wilkins M.E.
      • Da Silva H.M.A.
      • Smart T.G.
      Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites.
      ) and that was photolabeled by an allopreganolone derivative with a photoreactive group at C-21 (
      • Chen Z.W.
      • Bracamontes J.R.
      • Budelier M.M.
      • Germann A.L.
      • Shin D.J.
      • Kathiresan K.
      • Qian M.X.
      • Manion B.
      • Cheng W.W.L.
      • Reichert D.E.
      • Akk G.
      • Covey D.F.
      • Evers A.S.
      Multiple functional neurosteroid binding sites on GABAA receptors.
      ). The latter also photolabeled β3Gly-308 or β3Arg-309 in the β+–α steroid site. In parallel with the β subunit studies, we fractionated α subunit Endo Lys-C digests by rpHPLC and found a 3H distribution similar to that for β subunit digests shown in Fig. 5B. We sequenced fractions 24-27 that would contain the unlabeled and labeled fragments beginning at α1Ileu-392, with OPA treatment in cycle 10 of Edman degradation (α1Pro-401) to associate 3H release beyond cycle 11 (α1Leu-402) with αM4. When the α1Ile-392 fragment (I0 = 6 pmol) was sequenced for 25 cycles, no peaks of 3H release were detected above background after cycle 11. Any photolabeling of α1Asn-408, or residues nearby in the primary structure, if it occurred, would be at less than 10% the efficiency of photolabeling of β3Leu-417.

      Locations of photolabeled residues in α1β3γ2 GABAAR structure

      In Fig. 7 we highlight the positions of four photolabeled residues (β3Pro-415, β3Leu-417, β3Thr-418, and β3Arg-309) in a structure recently solved by cryo-EM (
      • Laverty D.
      • Desai R.
      • Uchański T.
      • Masiulis S.
      • Stec W.J.
      • Malinauskas T.
      • Zivanov J.
      • Pardon E.
      • Steyaert J.
      • Miller K.W.
      • Aricescu A.R.
      Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer.
      ) of α1β3γ2 GABAARs (Protein Data Base 6I53) purified from the same GABAAR-expressing HEK 293T cell line used for our purifications. Most of the 116 amino acids comprising the β3 cytoplasmic domain between the M3 and M4 helices are not defined in this structure, which resolves amino acids beginning with β3Pro-415 and locates β3Val-420 at the cytoplasmic end of the βM4 helix. In this structure, β3Pro-415–β3Asp-419 form a turn at the cytoplasmic end of βM4, with the photolabeled residues (β3Pro-415, β3Leu-417, β3Thr-418, and β3-Arg-309 at the cytoplasmic end of βM3) contributing to the base of a pocket at the β+–α subunit interface (Fig. 7, B and C) that extends between βM3 and αM1 up to the level of α1Gln-242, the amino acid in αM1 identified by mutational analysis as a major sensitivity determinant for many steroid PAMs (
      • Hosie A.M.
      • Wilkins M.E.
      • Da Silva H.M.A.
      • Smart T.G.
      Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites.
      ), including 21-pTFDBzox-AP (
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      ). This pocket is homologous to the intersubunit cleft identified as a binding site for 3α-OH steroid PAMs in crystal structures of homomeric, chimeric receptors containing GABAAR α subunit TMDs (
      • Laverty D.
      • Thomas P.
      • Field M.
      • Andersen O.J.
      • Gold M.G.
      • Biggin P.C.
      • Gielen M.
      • Smart T.G.
      Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.
      ,
      • Miller P.S.
      • Scott S.
      • Masiulis S.
      • De Colibus L.
      • Pardon E.
      • Steyaert J.
      • Aricescu A.R.
      Structural basis for GABAA receptor potentiation by neurosteroids.
      ,
      • Chen Q.
      • Wells M.M.
      • Arjunan P.
      • Tillman T.S.
      • Cohen A.E.
      • Xu Y.
      • Tang P.
      Structural basis of neurosteroid anesthetic action on GABA(A) receptors.
      ). Based upon computational docking using CDocker, 21-pTFDBzoxy-AP can be readily accommodated within this intersubunit pocket in the α1β3γ2 GABAAR β+–α subunit interface, with the lowest energy solutions adopting an orientation with the 3α-OH in proximity to α1Gln-242 and with the reactive diazirine in proximity to the photolabeled residues (Fig. 7C).
      Figure thumbnail gr7
      Figure 7Location of the [3H]21p-TFDBzox-AP labeled residues in the cytoplasmic domain of the α1β3γ2L GABAAR (PDB ). A, a partial alignment of the human α1, β3, and γ2L GABAAR subunits' M3 and M4 helices (denoted by heavy lines) with cytoplasmic extensions, with amino acid numbering of the subunits after signal sequence cleavage. Asterisks (*) denote conserved residues in the alignments, and dashed lines designate residues resolved in the PDB 6I53 GABAAR structure (
      • Laverty D.
      • Desai R.
      • Uchański T.
      • Masiulis S.
      • Stec W.J.
      • Malinauskas T.
      • Zivanov J.
      • Pardon E.
      • Steyaert J.
      • Miller K.W.
      • Aricescu A.R.
      Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer.
      ). Residues photolabeled by [3H]21p-TFDBzox-AP are color-coded: β3Arg-309 (crimson); β3Ile-414 (red); β3Pro-415 (orange); β3Leu-417 (lime green); and β3Thr-418 (magenta). B and C, images of the PDB 6I53 structure with horizontal lines approximating membrane-aqueous interfaces. In B, α-helices are cylinders and β-sheets are ribbons. Binding sites for GABA (green, overlaid from PDB 6HUJ), etomidate (red, docked), and αTHDOC (blue, docked) are included. C, an expanded view of the TMD at a β+–α interface with the [3H]21p-TFDBzox-AP labeled residues (β3Arg-309, β3Pro-415, β3Leu-417, and β3Thr-418) highlighted. These residues are shown as Connolly surfaces, as are the others that contribute to an intersubunit pocket extending to αGln-242 (purple), a residue identified by mutational analysis as a steroid sensitivity determinant (
      • Hosie A.M.
      • Wilkins M.E.
      • Da Silva H.M.A.
      • Smart T.G.
      Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites.
      ). 21p-TFDBzox-AP is in stick format, docked in this pocket in its lowest energy orientation with a transparent Connolly surface of the 9 lowest energy solutions. In this orientation, the photoreactive diazirine is within 5 Å of β3Arg-309, β3Leu-417, and β3Thr-418, and the 3α-OH is within 5 Å of αGln-242 and 3 Å of αTrp-246, a residue also identified by mutational analysis as a steroid sensitivity determinant (
      • Akk G.
      • Li P.
      • Bracamontes J.
      • Reichert D.E.
      • Covey D.F.
      • Steinbach J.H.
      Mutations of the GABA-A receptor α1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids.
      ). Also shown is the etomidate-binding site in the β+–α interface near the extracellular end of the TMD, defined by the residues photolabeled by etomidate analogs and by mutational analysis (β3Met-286, β3Val-290, α1Leu-232, and α1Met-236 (
      • Chiara D.C.
      • Dostalova Z.
      • Jayakar S.S.
      • Zhou X.
      • Miller K.W.
      • Cohen J.B.
      Mapping general anesthetic binding site(s) in human α1β3 γ-aminobutyric acid type A receptors with [3H]TDBzl-Etomidate, a photoreactive etomidate analogue.
      ,
      • Nourmahnad A.
      • Stern A.T.
      • Hotta M.
      • Stewart D.S.
      • Ziemba A.M.
      • Szabo A.
      • Forman S.A.
      Tryptophan and cysteine mutations in M1 helices of α1β3γ2L γ-aminobutyric acid type A receptors indicate distinct intersubunit sites for four intravenous anesthetics and one orphan site.
      )) and a docked etomidate (in stick figure).

      Anesthetic, anticonvulsant, and anxiolytic 3α-OH steroids bind to this site

      By use of competition photolabeling, we established that this site binds with high affinity a structurally diverse group of 3α-OH pregnane GABAAR PAMs that have a wide range of pharmacological activities in vivo (Fig. 8A and Table 3). Org20599, an amino steroid anesthetic containing a 2β-morpholino-substituent to enhance water solubility (
      • Hill-Venning C.
      • Peters J.A.
      • Callachan H.
      • Lambert J.J.
      • Gemmell D.K.
      • Anderson A.
      • Byford A.
      • Hamilton N.
      • Hill D.R.
      • Marshall R.J.
      • Campbell A.C.
      The anaesthetic action and modulation of GABAA receptor activity by the novel water-soluble aminosteroid Org 20599.
      ), inhibited photolabeling with IC50 = 0.2 μm. Substitutions at the 3β- and 17β-positions that improve bioavailability were well tolerated. Thus, GABAAR PAMs that act in vivo as an anticonvulsant (ganaxolone (
      • Carter R.B.
      • Wood P.L.
      • Wieland S.
      • Hawkinson J.E.
      • Belelli D.
      • Lambert J.J.
      • White H.S.
      • Wolf H.H.
      • Mirsadeghi S.
      • Tahir S.H.
      • Bolger M.B.
      • Lan N.C.
      • Gee K.W.
      Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3α-hydroxy-3β-methyl-5α-pregnan-20-one), a selective, high-affinity, steroid modulator of the γ-aminobutyric acid(A) receptor.
      )), an anti-depressant (SAGE-217 (
      • Martinez Botella G.
      • Salituro F.G.
      • Harrison B.L.
      • Beresis R.T.
      • Bai Z.
      • Blanco M.J.
      • Belfort G.M.
      • Dai J.
      • Loya C.M.
      • Ackley M.A.
      • Althaus A.L.
      • Grossman S.J.
      • Hoffmann E.
      • Doherty J.J.
      • Robichaud A.J.
      Neuroactive steroids. 2. 3a-hydroxy-3b-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5b-pregnan-20-one (SAGE-217): a clinical next generation neuroactive steroid positive allosteric modulator of the (γ-aminobutyric acid)A receptor.
      )), or a sedative/hypnotic (CCD-3693 (
      • Edgar D.M.
      • Seidel W.F.
      • Gee K.W.
      • Lan N.C.
      • Field G.
      • Xia H.
      • Hawkinson J.E.
      • Wieland S.
      • Carter R.B.
      • Wood P.L.
      CCD-3693: an orally bioavailable analog of the endogenous neuroactive steroid, pregnanolone, demonstrates potent sedative hypnotic actions in the rat.
      )) each inhibited photolabeling with IC50 < 1 μm, and for UCI-50027, active orally as an anxiolytic (
      • Hogenkamp D.J.
      • Tran M.B.
      • Yoshimura R.F.
      • Johnstone T.B.
      • Kanner R.
      • Gee K.W.
      Pharmacological profile of a 17β-heteroaryl-substituted neuroactive steroid.
      ), the IC50 was 10 μm. 3β-CH3OCH2-3α,5α-THDOC (
      • Tsai S.E.
      • Lee J.C.
      • Uramaru N.
      • Takayama H.
      • Huang G.J.
      • Wong F.F.
      Synthesis and antiproliferative activity of 3α-hydroxyl-3β-methoxymethyl-5α-pregnan-20-one with a C-21 hydrophilic substituent.
      ) (IC50 = 2 μm) was equipotent with 3α,5α-THDOC as an inhibitor. Each of these compounds inhibited photolabeling maximally to the same extent as 30 μm 3α5α-P, with the exception of ganaxolone, which inhibited maximally by only 71 ± 2%.
      Figure thumbnail gr8
      Figure 8Structural determinants for binding of 3α-OH pregnanes and androstanes to the [3H]21p-TFDBzox-AP site in α1β3 GABAARs. GABAARs were photolabeled in the presence of GABA and the indicated concentrations of a panel of GABAAR PAMs or the androstene antagonist 17-PA. Covalent incorporation of 3H was determined by liquid scintillation counting of β3 subunits isolated by SDS-PAGE, and data from independent experiments were normalized and combined as described under “Experimental procedures” and . The plotted data are the mean ± S.D. from the independent experiments. For each steroid tested, the chemical structure, the parameters (IC50, nH) determined from the concentration dependence of inhibition, and the number of independent experiments are presented in Table 3, Table 4. A, substitutions at the 2β- (Org-20599) and 3β- (ganaxolone, SAGE-217, CCD-3693, UCI-50027) positions are well tolerated, as is the presence at C-19 of an -H (SAGE-217, CCD-3693) rather than –CH3. Pregnanes with a carbonyl at C-20 bind with high affinity, but those with an –OH do not. With the exception of ganaxalone (Bns = 28.6 ± 1.6%), curves were calculated from fits with Bns = 0 and nH = 1. B, substituents at steroid carbon 17 (C-17) are a major determinant of binding affinity. 5α-Androstan-3α-ol (3α5α-A), with hydrogens at C-17, and 3α5β-P-20-deoxy, with a 17β-ethyl substituent, bind to this site, but 3α5α-A17α-ol does not. Inhibition curves were calculated from fits with Bns = 0 and variable nH for 5αA3α-ol,17-one (IC50 = 700 ± 390 μm, nH = 0.32 ± 0.05, R2 = 0.72) and 17-PA (IC50 = 85 ± 13 μm, nH = 0.33 ± 0.02, R2 = 0.95). Inhibition by 3α5α-A-17-one was fit equally well to with variable Bns and nH = 1 (Bns = 63 ± 3%, IC50 = 5 ± 2 μm, R2 = 0.71) but not for inhibition by 17-PA (R2 = 0.47).
      Table 3Inhibition of [3H]21-pTFDBzox-AP photolabeling by substituted 3α-OH pregnan steroid GABAAR PAMs
      R1C-5R2R3[3H]21-pTFDBzoxy-AP
      IC50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of α1β3 GABAAR photolabeling, were determined by fit of the data of Fig. 8A to Equation 2 under “Experimental procedures,” with nH = 1 and Bns = 0, or for ganaxlone with variable Bns. n, number of experiments.
      IC50 (n)
      GABA enhancement
      EC50 values for steroid enhancement of GABA responses of α1βγ GABAARs expressed in oocytes, from the literature: Org20599 (37); ganaxolone (38); SAGE-217 (39); UCI-50027 (41).
      EC50
      μmμm
      Org-20599C-2: -Hα-CH3-COCH2Cl0.2 ± 0.02 (4)1.2
      Ganaxolone-CH3α-CH3-COCH30.26 ± 0.04 Bns = 29 ± 2% (8)0.2
      SAGE-217-CH3β-H0.51 ± 0.07 (4)0.4
      3β-CH3OCH2-THDOC-CH2OCH3α-CH3-COCH2OH2.1 ± 0.3 (6)ND
      ND, not determined.
      CCD-3693-CF3β-H-COCH30.95 ± 0.1 (4)0.2
      CD-3693 enhancement of [3H]flunitrazepam binding (40).
      UCI-50027-CH3α-CH310.6 ± 1.5 (4)1.2
      a IC50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of α1β3 GABAAR photolabeling, were determined by fit of the data of Fig. 8A to Equation 2 under “Experimental procedures,” with nH = 1 and Bns = 0, or for ganaxlone with variable Bns. n, number of experiments.
      b EC50 values for steroid enhancement of GABA responses of α1βγ GABAARs expressed in oocytes, from the literature: Org20599 (
      • Hill-Venning C.
      • Peters J.A.
      • Callachan H.
      • Lambert J.J.
      • Gemmell D.K.
      • Anderson A.
      • Byford A.
      • Hamilton N.
      • Hill D.R.
      • Marshall R.J.
      • Campbell A.C.
      The anaesthetic action and modulation of GABAA receptor activity by the novel water-soluble aminosteroid Org 20599.
      ); ganaxolone (
      • Carter R.B.
      • Wood P.L.
      • Wieland S.
      • Hawkinson J.E.
      • Belelli D.
      • Lambert J.J.
      • White H.S.
      • Wolf H.H.
      • Mirsadeghi S.
      • Tahir S.H.
      • Bolger M.B.
      • Lan N.C.
      • Gee K.W.
      Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3α-hydroxy-3β-methyl-5α-pregnan-20-one), a selective, high-affinity, steroid modulator of the γ-aminobutyric acid(A) receptor.
      ); SAGE-217 (
      • Martinez Botella G.
      • Salituro F.G.
      • Harrison B.L.
      • Beresis R.T.
      • Bai Z.
      • Blanco M.J.
      • Belfort G.M.
      • Dai J.
      • Loya C.M.
      • Ackley M.A.
      • Althaus A.L.
      • Grossman S.J.
      • Hoffmann E.
      • Doherty J.J.
      • Robichaud A.J.
      Neuroactive steroids. 2. 3a-hydroxy-3b-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5b-pregnan-20-one (SAGE-217): a clinical next generation neuroactive steroid positive allosteric modulator of the (γ-aminobutyric acid)A receptor.
      ); UCI-50027 (
      • Hogenkamp D.J.
      • Tran M.B.
      • Yoshimura R.F.
      • Johnstone T.B.
      • Kanner R.
      • Gee K.W.
      Pharmacological profile of a 17β-heteroaryl-substituted neuroactive steroid.
      ).
      c ND, not determined.
      d CD-3693 enhancement of [3H]flunitrazepam binding (
      • Edgar D.M.
      • Seidel W.F.
      • Gee K.W.
      • Lan N.C.
      • Field G.
      • Xia H.
      • Hawkinson J.E.
      • Wieland S.
      • Carter R.B.
      • Wood P.L.
      CCD-3693: an orally bioavailable analog of the endogenous neuroactive steroid, pregnanolone, demonstrates potent sedative hypnotic actions in the rat.
      ).

      Substitutents at C-17 in the steroid D ring are important determinants of binding affinity

      In contrast to the high affinity binding of 3α5α-P and 3α5β-P, the presence of a hydroxyl group at the C-20 position in place of the carbonyl resulted in loss of binding. 5α-Pregnan-3α,20α-diol or 5β-pregnan-3α,20β-diol at 300 μm inhibited photolabeling by <10%, although they act as GABAAR PAMs with potencies similar to 3α5α-P (
      • Park-Chung M.
      • Malayev A.
      • Purdy R.H.
      • Gibbs T.T.
      • Farb D.H.
      Sulfated and unsulfated steroids modulate γ-aminobutyric acidA receptor function through distinct sites.
      ,
      • Belelli D.
      • Lambert J.J.
      • Peters J.A.
      • Gee K.W.
      • Lan N.C.
      Modulation of human recombinant GABAA receptors by pregnanediols.
      ) (Fig. 8A). The presence of the –OH at C-20 caused the loss of binding, because 5β-pregnan-3α-ol (3α5β-P-20-deoxy), with hydrogens at C-20, bound with high affinity (IC50 = 4 μm), as did 5α-androstan-3α-ol (3α5α-A, IC50 = 9 μm)) without any C-17 substituent (Fig. 8B and Table 4). Similar to the loss of binding associated with an –OH at C-20, the presence of an –OH or a carbonyl at C-17 also reduced binding affinity. At 300 μm, androsterone (3α5α-A-17-one), a potent GABAAR PAM (
      • Park-Chung M.
      • Malayev A.
      • Purdy R.H.
      • Gibbs T.T.
      • Farb D.H.
      Sulfated and unsulfated steroids modulate γ-aminobutyric acidA receptor function through distinct sites.
      ,
      • Ziegler E.
      • Bodusch M.
      • Song Y.
      • Jahn K.
      • Wolfes H.
      • Steinlechner S.
      • Dengler R.
      • Bufler J.
      • Krampfl K.
      Interaction of androsterone and progesterone with inhibitory ligand-gated ion channels: a patch clamp study.
      ), inhibited photolabeling by only ∼40%, and 5α-androstan-3α,17α-diol (3α5α-A-17α-ol) inhibited photolabeling by <10%. In contrast to the steroid PAMs that bound with high affinity to this site and inhibited photolabeling with a Hill coefficient (nH) close to 1, 3α5α-A-17-one inhibited photolabeling with nH less than 0.5 (IC50 = 700 ± 390 μm, nH = 0.32 ± 0.05). The 3α-OH androstene antagonist 17-phenyl-(3α,5α)-androsten-16-en-3-ol (17-PA) was more potent than 3α5α-A-17-one as an inhibitor for photolabeling, with IC50 = 85 ± 13 μm, nH = 0.33 ± 0.03.
      Table 4Inhibition of [3H]21-pTFDBzox-AP photolabeling by C-17 substituted 3α-OH steroid GABAAR PAMs
      Steroid
      Catalog numbers are indicated for steroids from Steraloids (A/P-xxxx).
      C-5R[3H]21-pTFDBzoxy-AP
      IC50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of GABAAR photolabeling, were determined as described under “Experimental Procedures” from data of Fig. 8B. n, number of experiments.
      IC50 (n)
      GABA enhancement
      EC50 values for steroid enhancement of GABA responses of α1βγ GABAARs expressed in oocytes, from the literature: 21p-TFD-Bzoxy-AP (27); 3α5α-A (67); 5α-pregnan-3α,20α-diol and 5β-pregnan-3α,20β-diol (43); 3α5αA-17-one (52).
      EC50
      μmμm
      21-pTFDBzox-APα-COCH2OCOBzTFD0.21 ± 0.022.7
      3α5α-A (A2150)α-H8.7 ± 1.0 (6)0.3
      3α5β-P-20-deoxy (P7800)β4.2 ± 0.5 (4)ND
      ND, not determined.
      (0.3)
      EC50 for GABAAR enhancement by 3α5α-P-20-deoxy (67).
      5α-Pregan3α,20α-diol (P1950)α>>300 (3)0.2
      5β-Pregnan3α,20β-diol (P6050)β>>300 (4)2
      3α5α-A-17-one (androsterone, A2420)α=O∼700 ± 400 (nH = 0.35) (6)∼3
      3α5α-A-17α-ol (A1150)α>300 (4)ND
      17-PA (5αA3α-ol,16-ene, 17-C6H5)α-C6H585 ± 13 (nH = 0.3 ± 0.03) (4)29 ± 5
      EC50 for enhancement of [3H]muscimol binding (Fig. 2).
      a Catalog numbers are indicated for steroids from Steraloids (A/P-xxxx).
      b IC50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of GABAAR photolabeling, were determined as described under “Experimental Procedures” from data of Fig. 8B. n, number of experiments.
      c EC50 values for steroid enhancement of GABA responses of α1βγ GABAARs expressed in oocytes, from the literature: 21p-TFD-Bzoxy-AP (
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      ); 3α5α-A (
      • Li P.
      • Bandyopadhyaya A.K.
      • Covey D.F.
      • Steinbach J.H.
      • Akk G.
      Hydrogen bonding between the 17β-substituent of a neurosteroid and the GABAA receptor is not obligatory for channel potentiation.
      ); 5α-pregnan-3α,20α-diol and 5β-pregnan-3α,20β-diol (
      • Belelli D.
      • Lambert J.J.
      • Peters J.A.
      • Gee K.W.
      • Lan N.C.
      Modulation of human recombinant GABAA receptors by pregnanediols.
      ); 3α5αA-17-one (
      • Katona B.W.
      • Krishnan K.
      • Cai Z.Y.
      • Manion B.D.
      • Benz A.
      • Taylor A.
      • Evers A.S.
      • Zorumski C.F.
      • Mennerick S.
      • Covey D.F.
      Neurosteroid analogues: 12. potent enhancement of GABA-mediated chloride currents at GABAA receptors by ent-androgens.
      ).
      d ND, not determined.
      e EC50 for GABAAR enhancement by 3α5α-P-20-deoxy (
      • Li P.
      • Bandyopadhyaya A.K.
      • Covey D.F.
      • Steinbach J.H.
      • Akk G.
      Hydrogen bonding between the 17β-substituent of a neurosteroid and the GABAA receptor is not obligatory for channel potentiation.
      ).
      f EC50 for enhancement of [3H]muscimol binding (Fig. 2).

      The binding of C-11 substituted pregnanolones

      As the carbonyl at C-11 in alphaxalone reduced its IC50 value by 20-fold compared with 3α5α-P (Table 1), we used competition photolabeling to determine the effects of other C-11 substituents on binding to this site (Fig. 9 and Table 5). As seen for the C-11 carbonyl in alphaxalone, the affinity for the 5β analog of alphaxalone (renanolone, IC50 =10 μm) was 20-fold weaker than that for 3α5β-P. Substitution of an 11β-OH further reduced potency by 20-fold (3α5β-P-11β-ol, IC50 ∼ 200 μm). In contrast, high affinity binding was retained in the presence at C-11 of either the small azi-group (11-Azi-AP, IC50 = 0.4 μm) or the bulky azidotetrafluorophenyl-group (11-F4N3Bzoxy-AP, IC50 = 0.1 μm) in two recently introduced photoreactive 3α5α-P derivatives that act as potent GABAAR PAMs and general anesthetics (
      • Savechenkov P.Y.
      • Chiara D.C.
      • Desai R.
      • Stern A.T.
      • Zhou X.N.
      • Ziemba A.M.
      • Szabo A.L.
      • Zhang Y.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.
      ).
      Figure thumbnail gr9
      Figure 9Effects of substituents at C-11 on steroid binding affinity for the [3H]21p-TFDBzox-AP site in α1β3 GABAARs. GABAARs were photolabeled in the presence of GABA and the indicated concentrations of C11-substituted derivatives of 3α5α-P (the photoreactive anesthetics 11-azi-AP and 11-F4N3Bzox-AP) or 3α5β-P (renanolone and 3α5β-P-11β-ol). The chemical structures, IC50 values determined from the concentration dependence of inhibition, and the number of independent experiments are presented in . Covalent incorporation of 3H was determined by liquid scintillation counting of β3 subunits isolated by SDS-PAGE, and data from independent experiments were normalized and combined as described under “Experimental procedures” and in . The plotted data are the mean ± S.D. from the independent experiments.
      Table 5Inhibition of [3H]21-pTFDBzox-AP photolabeling by C-11 substituted pregnanolone GABAAR PAMs
      RC-5[3H]21-pTFDBzoxy-AP
      IC50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of α1β3 GABAAR photolabeling, were determined as described under “Experimental Procedures” from data of Fig. 9 or Ref. 27. n, number of experiments.
      IC50 (n)
      GABA enhancement
      Literature EC50 values for steroid enhancement of GABA responses of α1β3γ2 GABAARs expressed in oocytes (alphaxalone, 11-Azi-AP, and 11-F4N3Bzox-AP (45)) or for enhancement of [3H]flunitrazepam binding to rat brain membranes (renanolone (58); 3α5β-pregnan-11β-ol (68)).
      EC50
      μmμm
      Alphaxalone=Oα4.6 ± 0.7 (4)2.2
      11-Azi-APα0.44 ± 0.06 (4)0.2 ± 0.1
      11-F4N3Bzox-APα0.09 ± 0.01 (4)0.5 ± 0.2
      6-AziOAP=Oα44
      From Ref. 27, EC50 for enhancement of [3H]muscimol binding.
      25
      From Ref. 27, EC50 for enhancement of [3H]muscimol binding.
      Renanolone (Res Plus 3183-16)=Oβ10 ± 1.5 (4)3.6
      3α5β-P-11β-ol (Res Plus 3159-16)-OHβ190 ± 32 (6)>300
      6-AziOP =Oβ>100
      From Ref. 27, EC50 for enhancement of [3H]muscimol binding.
      37
      From Ref. 27, EC50 for enhancement of [3H]muscimol binding.
      a IC50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of α1β3 GABAAR photolabeling, were determined as described under “Experimental Procedures” from data of Fig. 9 or Ref.
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      . n, number of experiments.
      b Literature EC50 values for steroid enhancement of GABA responses of α1β3γ2 GABAARs expressed in oocytes (alphaxalone, 11-Azi-AP, and 11-F4N3Bzox-AP (
      • Savechenkov P.Y.
      • Chiara D.C.
      • Desai R.
      • Stern A.T.
      • Zhou X.N.
      • Ziemba A.M.
      • Szabo A.L.
      • Zhang Y.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.
      )) or for enhancement of [3H]flunitrazepam binding to rat brain membranes (renanolone (
      • Prince R.J.
      • Simmonds M.A.
      Differential antagonism by epipregnanolone of alphaxalone and pregnanolone potentiation of [3H]flunitrazepam binding suggests more than one class of binding site for steroids at GABAA receptors.
      ); 3α5β-pregnan-11β-ol (
      • Slavíková B.
      • Bujons J.
      • Matyás L.
      • Vidal M.
      • Babot Z.
      • Kristofikova Z.
      • Suñol C.
      • Kasal A.
      Allopregnanolone and pregnanolone analogues modified in the C ring: synthesis and activity.
      )).
      c From Ref.
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      , EC50 for enhancement of [3H]muscimol binding.

      Simultaneous binding with nonsteroidal GABAAR PAMs at the etomidate site

      We used competition photolabeling to determine whether PAMs of large size that bind to the etomidate site near the extracellular end of the TMD β+–α subunit interface would inhibit [3H]21-pTFDBzox-AP photolabeling of α1β3 GABAARs, whether by steric overlap or by negative allosteric interaction. Less than 10% inhibition was seen at the highest concentrations tested for propofol (300 μm, molecular volume, 191 Å3), etomidate (300 μm, volume 208 Å3), or TG-41 (10 μm, ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate (
      • Mascia M.P.
      • Asproni B.
      • Busonero F.
      • Talani G.
      • Maciocco E.
      • Pau A.
      • Cerri R.
      • Sanna E.
      • Biggio G.
      Ethyl 2-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-4-imidazolecarboxylate is a novel positive modulator of GABAA receptors.
      ), volume 323 Å3), which were tested at 40-, 150-, and 500-fold higher than their IC50 values for inhibition of [3H]azietomidate photolabeling (
      • Jayakar S.S.
      • Zhou X.J.
      • Chiara D.C.
      • Jarava-Barrera C.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Tortosa M.
      • Miller K.W.
      • Cohen J.B.
      Identifying drugs that bind selectively to intersubunit general anesthetic sites in the α1β3γ2 GABAAR transmembrane domain.
      ).
      We also tested ivermectin (volume 880 Å3), a nonanesthetic activator and PAM of GABAARs and other pentameric ligand-gated ion channels (
      • Lynagh T.
      • Lynch J.W.
      Ivermectin binding sites in human and invertebrate Cys-loop receptors.
      ) that binds in a homomeric, invertebrate glutamate-gated chloride channel (Glu-Cl) to an intersubunit site (
      • Hibbs R.E.
      • Gouaux E.
      Principles of activation and permeation in an anion-selective Cys-loop receptor.
      ) homologous to the etomidate and R-mTFD-MPAB sites in α1β3 or α1β3γ2 GABAARs. In α1β2γ2 GABAARs, however, mutational analysis indicate that ivermectin interacts nonequivalently with these sites, with the γ+–β site most important for activation (
      • Estrada-Mondragon A.
      • Lynch J.
      Functional characterization of ivermectin binding sites in α1β2γ2L GABAA receptors.
      ). We found that 100 μm ivermectin inhibited [3H]21-pTFDBzox-AP photolabeling of α1β3 or α1β3γ2 GABAARs by <10% (Fig. 10). In contrast, ivermectin potently inhibited photolabeling by [3H]R-mTFD-MPAB (IC50 = 0.02 ± 0.005 μm, nH = 0.6 ± 0.1) and inhibited [3H]azietomidate photolabeling at higher concentrations (IC50 = 6.4 ± 1.0 μm) (Fig. 10). Thus, ivermectin at 100 μm fully occupies those sites without inhibiting [3H]21-pTFDBzox-AP photolabeling, and consistent with the functional studies, ivermectin binds with higher affinity to the α++–β sites than to the β+–α sites. Furthermore, the concentration dependence of inhibition of [3H]R-mTFD-MPAB photolabeling (nH = 0.6) established that ivermectin binds nonequivalently to the α+–β and γ+–β sites in the presence of GABA. This is not the case for R-mTFD-MPAB, but it is for other GABAAR PAMs including the anesthetic p-benzoyl-propofol and the sedative/anticonvulsant loreclezole (
      • Jayakar S.S.
      • Zhou X.J.
      • Chiara D.C.
      • Jarava-Barrera C.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Tortosa M.
      • Miller K.W.
      • Cohen J.B.
      Identifying drugs that bind selectively to intersubunit general anesthetic sites in the α1β3γ2 GABAAR transmembrane domain.
      ). Based upon a fit of the inhibition data to a two-site model, ivermectin binds to the β sites with IC50 values of 3.1 ± 1.1 and 150 ± 65 nm. Further studies would be required to determine whether it is the α+–β or γ+–β site that binds with highest affinity.
      Figure thumbnail gr10
      Figure 10Ivermectin binds to the α++–β ([3H]R-mTFD-MPAB) and β+–α ([3H]azietomidate) intersubunit anesthetic sites without altering binding of [3H]21p-TFDBzox-AP to its β+–α intersubunit site. α1β3γ2 GABAARs were photolabeled in the presence of GABA with varying concentrations of ivermectin. After SDS-PAGE, 3H incorporation into GABAAR subunits was determined by liquid scintillation counting. For each independent experiment, nonspecific photolabeling was determined in the presence of 30 μm 3α5β-P ([3H]21p-TFDBzox-AP (n = 4)), 60 μm nonradioactive R-mTFD-MPAB ([3H]R-mTFD-MPAB (n = 3)), or 300 μm etomidate ([3H]azietomidate (n = 3)). For each photoprobe, specific binding in 3H cpm was determined for each independent experiment and normalized to the specific binding in the control condition, and data from the independent experiments were pooled. The plotted data are the mean ± S.D. from the independent experiments. When fit to , for [3H]azietomidate, IC50 = 6.4 ± 1.0 μm, nH =1 (R2 = 0.93). For [3H]R-mTFD-MPAB, IC50 = 21 ± 5 nm and nH = 0.55 ± 0.08 (dots, R2 = 0.92) or when fit to a two site model ( Bx=0.5(Bo-Bns)1+xIC50H+0.5(Bo-Bns)1+xIC50L+Bns; dashed line), IC50H = 3 ± 1 nm, IC50L = 140 ± 64 nm (R2 = 0.91).

      Discussion

      In this report we show that a novel photoreactive steroid, [3H]21-pTFDBzoxy-AP, binds with high affinity to a site in the TMD of heteromeric GABAARs at the cytoplasmic end of the β+–α subunit interface, and we use a photolabeling inhibition assay to provide a first definition of the structure-affinity relationships for a GABAAR steroid-binding site. In α1β3 and α1β3γ2 GABAARs, pharmacologically specific [3H]21-pTFD-Bzoxy-AP photolabeling was primarily within the β subunit, with the photolabeled amino acids located in the GABAAR structure near the cytoplasmic ends of the βM4 (β3Pro-415, β3Leu-417, and β3Thr-418) and βM3 (β3Arg-309) helices that contribute to the base of a pocket at the β+–α subunit interface. This binding site extends upward to the level of α1Gln-242, a position identified by mutational analysis as a major determinant for steroid enhancement of GABA responses. Many 3α-OH pregnane and androstane GABAAR PAMs bind to this site at concentrations similar to those necessary for GABAAR enhancement, but we also identified potent steroid GABAAR PAMs that do not bind to this site. High affinity binding depends on the presence of a free 3α-OH and is highly sensitive to the nature of the substitution at the C-17 position. 3-Deoxy-5α-P and steroids with an –OH in place of the carbonyl at C-20 of 3α5α-P enhance GABA responses with potencies similar to 3α5α-P (
      • Akk G.
      • Li P.
      • Bracamontes J.
      • Reichert D.E.
      • Covey D.F.
      • Steinbach J.H.
      Mutations of the GABA-A receptor α1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids.
      ,
      • Park-Chung M.
      • Malayev A.
      • Purdy R.H.
      • Gibbs T.T.
      • Farb D.H.
      Sulfated and unsulfated steroids modulate γ-aminobutyric acidA receptor function through distinct sites.
      ,
      • Belelli D.
      • Lambert J.J.
      • Peters J.A.
      • Gee K.W.
      • Lan N.C.
      Modulation of human recombinant GABAA receptors by pregnanediols.
      ), but their binding affinities for the β+–α steroid site are reduced by more than 1000-fold. These potent steroid PAMs that do not bind to the β+–α steroid site should serve as useful lead compounds for the development of novel reagents to identify additional GABAAR steroid-binding sites.

      Structural determinants for binding to the β+–α subunit interface steroid site

      Because the β subunit amino acids photolabeled by [3H]21-pTFDBzoxy-AP were located within a common binding pocket at the β+–α subunit interface, characterization of the effects of nonradioactive steroids on GABAAR photolabeling at the level of the β subunit could be used to determine the affinities (IC50 values) of nonradioactive drugs for that site. We did not identify any nonsteroidal GABAAR PAMs that inhibited photolabeling, including drugs varying in size from propofol to ivermectin that bind to the adjacent etomidate site at the β+–α subunit interface. Many steroid 3α-OH GABAAR PAMs were potent inhibitors of [3H]21-pTFDBzoxy-AP photolabeling, reducing β subunit photolabeling maximally to the same extent as 30 μm 3α5α-P with a concentration dependence characterized by a Hill coefficient of 1. The simplest interpretation of this pattern of inhibition is that it results from competitive interactions at a common binding site. For α1β3 and α1β3γ2 GABAARs, the IC50 values for 5β-isomers differed by less than a factor of 2 from those for 3α5α-P, 3α5α-THDOC, and alphaxalone (Table 1, Table 2, Table 3, Table 4), and the IC50 values for inhibition of photolabeling were within a factor of 2 of EC50 values reported for enhancement of GABA responses (
      • Wang M.D.
      • He Y.J.
      • Eisenman L.N.
      • Fields C.
      • Zeng C.M.
      • Mathews J.
      • Benz A.
      • Fu T.
      • Zorumski E.
      • Steinbach J.H.
      • Covey D.F.
      • Zorumski C.F.
      • Mennerick S.
      3β-Hydroxypregnane steroids are pregnenolone sulfate-like GABA(A) receptor antagonists.
      ,
      • Miller P.S.
      • Scott S.
      • Masiulis S.
      • De Colibus L.
      • Pardon E.
      • Steyaert J.
      • Aricescu A.R.
      Structural basis for GABAA receptor potentiation by neurosteroids.
      ,
      • Savechenkov P.Y.
      • Chiara D.C.
      • Desai R.
      • Stern A.T.
      • Zhou X.N.
      • Ziemba A.M.
      • Szabo A.L.
      • Zhang Y.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.
      ). A similar good correlation between photolabeling inhibition IC50 and GABAAR enhancement EC50 was seen for many substituted pregnanolones, including those acting in vivo as an anesthetic (Org20599), anti-convulsant (ganaxolone), or antidepressant (SAGE-217) (Table 3, Table 4, Table 5).
      Our results establish that high affinity binding to the GABAAR β+–α-binding site depends critically on the presence of a free 3α-OH, consistent with the inactivity of 3β-OH steroids, 3-oxo-5α-P, and 3α-acetyl-5α-P as GABAAR PAMs (
      • Harrison N.L.
      • Majewska M.D.
      • Harrington J.W.
      • Barker J.L.
      Structure-activity relationships for steroid interaction with the γ-aminobutyric acidA receptor complex.
      ,
      • Cottrell G.A.
      • Lambert J.J.
      • Peters J.A.
      Modulation of GABAA receptor activity by alphaxalone.
      ,
      • Purdy R.H.
      • Morrow A.L.
      • Blinn J.R.
      • Paul S.M.
      Synthesis, metabolism, and pharmacological activity of 3α-hydroxy steroids which potentiate GABA-receptor-mediated chloride ion uptake in rat cerebral cortical synaptoneurosomes.
      ,
      • Peters J.A.
      • Kirkness E.F.
      • Callachan H.
      • Lambert J.J.
      • Turner A.J.
      Modulation of the GABAA receptor by depressant barbiturates and pregnane steroids.
      ). We found that they bound at least 1000-fold more weakly than 3α5α-P. 3-Deoxy-5α-P also did not bind to this site, despite the fact that it acts as a GABAAR PAM with a potency similar to 3α5α-P (
      • Akk G.
      • Li P.
      • Bracamontes J.
      • Reichert D.E.
      • Covey D.F.
      • Steinbach J.H.
      Mutations of the GABA-A receptor α1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids.
      ). Thus, 3-deoxy-5α-P enhances GABA responses without binding to this site.
      We found substitutions at the steroid C-17 position that were unexpectedly important determinants of binding to the β+–α site. Early studies of 3α-OH steroids as GABAAR PAMs established that the C-20 carbonyl of 3α5α-P was not essential, because androsterone (3α5αA-17-one) and pregnan-3α,20-diols were potent PAMs (
      • Purdy R.H.
      • Morrow A.L.
      • Blinn J.R.
      • Paul S.M.
      Synthesis, metabolism, and pharmacological activity of 3α-hydroxy steroids which potentiate GABA-receptor-mediated chloride ion uptake in rat cerebral cortical synaptoneurosomes.
      ,
      • Park-Chung M.
      • Malayev A.
      • Purdy R.H.
      • Gibbs T.T.
      • Farb D.H.
      Sulfated and unsulfated steroids modulate γ-aminobutyric acidA receptor function through distinct sites.
      ,
      • Belelli D.
      • Lambert J.J.
      • Peters J.A.
      • Gee K.W.
      • Lan N.C.
      Modulation of human recombinant GABAA receptors by pregnanediols.
      ). The C-20 carbonyl is also not essential for binding to the β+–α site, because 3α5α-A and 3α5β-P-20-deoxy, PAMs with an –H or β-ethyl at C-17, bound with high affinity (Table 4). However, two potent PAMs, 5α-pregnan-3α,20α-diol and 5β-pregnan-3α,20β-diol, did not bind to the β+–α site at concentrations 100-fold higher than necessary for GABAAR enhancement. 3α5α-A-17α-ol, with an α–OH at C-17, also did not bind to the β+–α site. However, this may not be simply a consequence of the -OH, because 3α5α-A-17β-ol is a PAM (
      • Reddy D.S.
      • Jian K.H.
      The testosterone-derived neurosteroid androstanediol is a positive allosteric modulator of GABAA receptors.
      ), as are other steroids with a C-17 side chain in a β-configuration (
      • Purdy R.H.
      • Morrow A.L.
      • Blinn J.R.
      • Paul S.M.
      Synthesis, metabolism, and pharmacological activity of 3α-hydroxy steroids which potentiate GABA-receptor-mediated chloride ion uptake in rat cerebral cortical synaptoneurosomes.
      ,
      • Hawkinson J.E.
      • Kimbrough C.L.
      • Belelli D.
      • Lambert J.J.
      • Purdy R.H.
      • Lan N.C.
      Correlation of neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding and γ-aminobutyric acidA receptor function.
      ).
      Although many 3α-OH steroid PAMs potently inhibited GABAAR photolabeling to the same extent as 3α5α-P and with a concentration dependence characterized by a Hill coefficient of 1, ganaxalone and 3α5αA-17-one were exceptions. Ganaxolone was a potent inhibitor (IC50 = 0.3 μm), but at high concentrations, maximal inhibition (Bns = 29 ± 2%) was less than that seen in the presence of 3α5α-P (Bns = 0%), whereas other PAMs with 3β-substituents inhibited fully. For 3α5αA-17-one, which enhances α1β2γ GABAAR responses with an EC50 of ∼3 μm (
      • Katona B.W.
      • Krishnan K.
      • Cai Z.Y.
      • Manion B.D.
      • Benz A.
      • Taylor A.
      • Evers A.S.
      • Zorumski C.F.
      • Mennerick S.
      • Covey D.F.
      Neurosteroid analogues: 12. potent enhancement of GABA-mediated chloride currents at GABAA receptors by ent-androgens.
      ), inhibition was fit equally well either to nH = 1 and variable Bns (IC50 = 5 ± 2 μm, Bns = 63 ± 3%) or with Bns equal to 0 and a variable Hill coefficient (IC50 = 700 ± 390 μm, nH = 0.32 ± 0.05). There was no evidence that the partial inhibition resulted from limited solubility of these two steroids in the detergent/lipid environment used for GABAAR purification, and further studies are required to clarify the mechanism of inhibition.

      Mode of steroid binding at the β+–α steroid site

      Our photolabeling results establish that 21-pTFDBzoxy-AP binds in heteromeric GABAARs at the β+–α subunit interface. Based upon computational docking, in its most energetically favorable binding mode, 21-pTFDBzoxy-AP's photoreactive diazirine is in proximity to the photolabeled amino acids at the cytoplasmic surface of the TMD and the 3α-OH is in proximity to α1Gln-242 and α1Trp-246. Thus, 21-pTFDBzoxy-AP binds at the β+–α subunit interface site in an orientation similar to that of 3α5α-THDOC, 3α5β-P, or alphaxalone at α+–α subunit interfaces in the crystal structures of homopentameric, chimeric receptors with GABAAR α subunit TMDs (
      • Laverty D.
      • Thomas P.
      • Field M.
      • Andersen O.J.
      • Gold M.G.
      • Biggin P.C.
      • Gielen M.
      • Smart T.G.
      Crystal structures of a GABAA-receptor chimera reveal new endogenous neurosteroid-binding sites.
      ,
      • Miller P.S.
      • Scott S.
      • Masiulis S.
      • De Colibus L.
      • Pardon E.
      • Steyaert J.
      • Aricescu A.R.
      Structural basis for GABAA receptor potentiation by neurosteroids.
      ,
      • Chen Q.
      • Wells M.M.
      • Arjunan P.
      • Tillman T.S.
      • Cohen A.E.
      • Xu Y.
      • Tang P.
      Structural basis of neurosteroid anesthetic action on GABA(A) receptors.
      ). Consistent with this mode of binding, positive modulation of GABA responses by 21-pTFDBzoxy-AP is lost in the α1Q242W mutant receptor (
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      ). Although direct interactions between the free 3α-OH and αGln-242 were predicted based upon the loss of 3α5α-P PAM activity seen for substitutions at αGln-242 (19), substitutions at αGln-242 also caused loss of PAM activity for 3-deoxy-5α-P (
      • Akk G.
      • Li P.
      • Bracamontes J.
      • Reichert D.E.
      • Covey D.F.
      • Steinbach J.H.
      Mutations of the GABA-A receptor α1 subunit M1 domain reveal unexpected complexity for modulation by neuroactive steroids.
      ), a PAM that did not inhibit [3H]21-pTFD-Bzoxy-AP photolabeling. This discrepancy indicates that substitutions at αGln-242 can interfere with PAM activity even for steroids that do not bind to the β+–α site, and the GABAAR amino acids interacting directly with the 3α-OH remain to be determined.
      Based upon competition photolabeling, the presence of a free –OH at C-20 is as deleterious for binding to the β+–α site as is its absence at the C-3 position, even though a free hydroxyl group at C-21 (3α5α-THDOC) or certain bulky substitutions (21-pTFDBzoxy-AP, SAGE-217) are well-tolerated. Just as the high affinity binding associated with the 3α-OH must result from specific interactions with GABAAR amino acids, the 1000-fold reduction of binding affinity (IC50) for the pregnane diols compared with 3α5α/β-P is likely to result from energetically unfavorable interactions between a C-20 hydroxyl and GABAAR amino acids in the β+–α interface steroid-binding site. If a pregnan-3α,20α/β-diol binds to the β+–α steroid site in the same orientation as alphaxalone or pregananolone in the crystal structures of chimeric receptors with α subunit TMDs (
      • Miller P.S.
      • Scott S.
      • Masiulis S.
      • De Colibus L.
      • Pardon E.
      • Steyaert J.
      • Aricescu A.R.
      Structural basis for GABAA receptor potentiation by neurosteroids.
      ,
      • Chen Q.
      • Wells M.M.
      • Arjunan P.
      • Tillman T.S.
      • Cohen A.E.
      • Xu Y.
      • Tang P.
      Structural basis of neurosteroid anesthetic action on GABA(A) receptors.
      ), the C-20 hydroxyl would be in proximity to β3Phe-301 in βM3, the position equivalent to α1Thr-206/α5Thr-309 that is in proximity to the C-20 carbonyl of alphaxalone and pregnanolone. Simple solubility considerations cannot account for the loss of binding, because incorporation of a hydroxyl residue instead of a carbonyl group increases the octanol-water partition coefficient calculated by the ALOGPS 2.1 program (RRID:SCR_018786) for any of the steroids tested by less than a factor of two, whereas 3α5β-P binds 7-fold more tightly and 5β-pregnan-3α,20β-diol binds >100-fold more weakly than 3α5β-P-20-deoxy (Table 4). Thus, the carbonyl at C-20 allows for favorable energetic interactions with GABAAR amino acids not possible for a hydroxyl function.
      In contrast to the differential effect on binding affinity seen for incorporation of a carbonyl or hydroxyl at C-20, direct incorporation of a carbonyl (androsterone) or a hydroxyl (3α5αA-17α-ol) on the steroid ring system at C-17 weakens binding by more than 50-fold compared with 3α5α-A. Likewise, the presence of a carbonyl (renanolone) or a hydroxyl (3α5β-P-11β-OH) at C-11 weakens binding by 20- and 300-fold compared with 3α5β-P. Because incorporation of a carbonyl or a hydroxyl will decrease the steroid partition coefficient by 10-30–fold, the decreased affinities (increased IC50 values, measured as the total steroid concentration) for these steroids may result in large measure from their decreased hydrophobicity and lipid partitioning rather than from energetically unfavorable specific interactions of the carbonyl or hydroxyl with the GABAAR.

      Additional binding sites for steroid PAMs

      Our sequencing results established that the β subunit amino acids photolabeled most efficiently by [3H]21-pTFDBzoxy-AP all contribute to the steroid-binding site at the β+–α subunit interface. Although we did not identify photolabeled amino acids that would contribute to other steroid-binding sites, our competition photolabeling results identified three potent PAMs (EC50 <10 μm, 3-deoxy-5α-P, 5α-pregnan-3α,20α-diol, and 5β-pregnan-3α,20β-diol) that did not bind to this site. The absence of an α hydroxyl at C-3 at one end of the steroid ring system or the presence of a hydroxyl at C-20, 10 Å away at the other end of the ring system, prevents binding to the β+–α steroid site. The presence of the 3α-OH is insufficient to overcome unfavorable interactions at the other end of the molecule. It remains to be determined whether one or more of these “orphan” PAMs bind to the intrasubunit sites near the extracellular end of the TMD recently identified by photolabeling in αM4 by steroids containing photoreactive groups at C-21 or C-6 and in βM3 by a steroid containing a C-3α photoreactive group (
      • Chen Z.W.
      • Bracamontes J.R.
      • Budelier M.M.
      • Germann A.L.
      • Shin D.J.
      • Kathiresan K.
      • Qian M.X.
      • Manion B.
      • Cheng W.W.L.
      • Reichert D.E.
      • Akk G.
      • Covey D.F.
      • Evers A.S.
      Multiple functional neurosteroid binding sites on GABAA receptors.
      ).

      Antagonist steroids

      Although early studies suggested that 3β-OH steroids competitively antagonize steroid enhancement of GABAAR function (
      • Prince R.J.
      • Simmonds M.A.
      5β-Pregnan-3β-ol-20-one, a specific antagonist at the neurosteroid site of the GABAA receptor-complex.
      ,
      • Maitra R.
      • Reynolds J.N.
      Modulation of GABAA receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABAA receptor isoforms.
      ,
      • Garrett K.M.
      • Gan J.
      Enhancement of γ-aminobutyric acidA receptor activity by α-chloralose.
      ), subsequent studies indicate that they noncompetitively inhibit GABA responses in the absence of enhancing steroids, acting in a manner more similar to the sulfated 3β-OH neurosteroids PS and DHEAS (
      • Wang M.D.
      • He Y.J.
      • Eisenman L.N.
      • Fields C.
      • Zeng C.M.
      • Mathews J.
      • Benz A.
      • Fu T.
      • Zorumski E.
      • Steinbach J.H.
      • Covey D.F.
      • Zorumski C.F.
      • Mennerick S.
      3β-Hydroxypregnane steroids are pregnenolone sulfate-like GABA(A) receptor antagonists.
      ,
      • Wang M.-D.
      • Rahman M.
      • Zhu D.
      • Johansson I.-M.
      • Bäckström T.
      3β-Hydroxysteroids and pregnenolone sulfate inhibit recombinant rat GABAA receptor through different channel property.
      ). Although steroid PAMs generally enhance [3H]muscimol or [3H]flunitrazepam equilibrium binding (
      • Hawkinson J.E.
      • Kimbrough C.L.
      • Belelli D.
      • Lambert J.J.
      • Purdy R.H.
      • Lan N.C.
      Correlation of neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding and γ-aminobutyric acidA receptor function.
      ,
      • Turner D.M.
      • Ransom R.W.
      • Yang J.S.J.
      • Olsen R.W.
      Steroid anesthetics and naturally occurring analogs modulate the γ-aminobutyric acid receptor complex at a site distinct from barbiturates.
      ,
      • Prince R.J.
      • Simmonds M.A.
      Differential antagonism by epipregnanolone of alphaxalone and pregnanolone potentiation of [3H]flunitrazepam binding suggests more than one class of binding site for steroids at GABAA receptors.
      ), our results indicate that inhibitory 3β-OH steroids can modulate [3H]muscimol binding in 3 different ways. (i) The enhancement of binding seen for betaxalone and 3β5β-P indicates that those steroids stabilize the GABAAR in a desensitized state with high affinity for [3H]muscimol. 17-PA, a 3α-OH androstene that does not inhibit GABA responses in the absence of a steroid PAM (
      • Mennerick S.
      • He Y.J.
      • Jiang X.
      • Manion B.D.
      • Wang M.D.
      • Shute A.
      • Benz A.
      • Evers A.S.
      • Covey D.F.
      • Zorumski C.F.
      Selective antagonism of 5α-reduced neurosteroid effects at GABAA receptors.
      ,
      • Kelley S.P.
      • Alan J.K.
      • O'Buckley T.K.
      • Mennerick S.
      • Krishnan K.
      • Covey D.F.
      • Leslie Morrow A.
      Antagonism of neurosteroid modulation of native γ-aminobutyric acid receptors by (3α,5α)-17-phenylandrost-16-en-3-ol.
      ), also enhanced [3H]muscimol binding. (ii) The lack of modulation seen for 3β5α-P and PS at concentrations as high as 100 μm suggests that they do not perturb the receptor conformational state. (iii) That DHEAS partially inhibits binding indicates that it stabilizes the receptor in a state with low affinity for [3H]muscimol, potentially a resting, closed channel state. The effects we observed for 3β5α-P, PS, and DHEAS are consistent with previous studies of [3H]muscimol binding to rat brain membranes (
      • Demirgören S.
      • Majewska M.D.
      • Spivak C.E.
      • London E.D.
      Receptor binding and electrophysiological effects of dehydroepiandrosterone sulfate, an antagonist of the GABAA receptor.
      ,
      • Goodnough D.B.
      • Hawkinson J.E.
      Neuroactive steroid modulation of [3H]muscimol binding to the GABAA receptor complex in rat cortex.
      ).
      Our competition photolabeling results are consistent with functional studies indicating that free and sulfated 3β-OH steroids inhibit GABA responses without binding to the same site as steroid PAMs. Thus, PS, 3β5α-P, and 3β5β-P inhibit GABA responses with IC50 values of less than 5 μm (
      • Wang M.D.
      • He Y.J.
      • Eisenman L.N.
      • Fields C.
      • Zeng C.M.
      • Mathews J.
      • Benz A.
      • Fu T.
      • Zorumski E.
      • Steinbach J.H.
      • Covey D.F.
      • Zorumski C.F.
      • Mennerick S.
      3β-Hydroxypregnane steroids are pregnenolone sulfate-like GABA(A) receptor antagonists.
      ,
      • Rahman M.
      • Lindblad C.
      • Johansson I.M.
      • Backstrom T.
      • Wang M.D.
      Neurosteroid modulation of recombinant rat α5β2γ2L and α1β2γ2L GABAA receptors in Xenopus oocyte.
      ), but any inhibition of [3H]21-pTFDBzoxy-AP, photolabeling, if it occurred, would be characterized by IC50 values greater than 300 μm. Betaxalone was a possible exception, because inhibition (IC50 = 175 μm) occurred at similar concentrations as enhancement of [3H]muscimol binding (EC50 = 50 μm). However, the concentration dependence of inhibition of photolabeling (nH = 0.5) was inconsistent with a simple model of direct completion for the [3H]21-pTFDBzoxy-AP–binding site.

      Conclusions

      We have shown that [3H]21-pTFDBzoxy-AP, a photoreactive steroid and GABAAR PAM, binds with high affinity in the β+–α subunit interface of heteromeric, human, full-length α1β3 and α1β3γ2L GABAARs in a site homologous to that revealed in crystal structures of chimeric homomeric pentameric ligand-gated ion channels of the same superfamily. We used competition photolabeling to establish that the steroid structure–activity relationships of this site parallel that observed in many functional pharmacological studies. These studies also reveal that some potent PAMs, such as 3α-deoxy-5α-P and pregnan-3α,20-diols, bind to a different site or sites, Thus, [3H]21-pTFD-Bzoxy-AP is a useful tool for the development of steroids that selectively target specific sites on GABAARs including those with other subunit compositions.

      Experimental procedures

      Materials

      Nonradioactive 21-pTFDBzox-AP and [3H]21-pTFDBzox-AP (21.8 Ci/mmol, stored at −20 °C in ethanol at 1 mCi/ml) were prepared as described previously (
      • Wu B.
      • Jayakar S.S.
      • Zhou X.
      • Titterton K.
      • Chiara D.C.
      • Szabo A.L.
      • Savechenkov P.Y.
      • Kent D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Inhibitable photolabeling by neurosteroid diazirine analog in the β3-subunit of human hetereopentameric type A GABA receptors.
      ). The 21-pTFDBzox-AP UV spectrum was characterized by a major absorption peak at 241 nm (ε = 16,160 m−1 cm−1) with a secondary, diazirine band with absorption maximum at 347 nm (ε = 360 m−1 cm−1). [3H]Azietomidate (19 Ci/mmol, 53 μm in ethanol) and nonradioactive and [3H]R-mTFD-MPAB (38 Ci/mmol, 26 μm) were also synthesized and tritiated previously (
      • Savechenkov P.Y.
      • Zhang X.
      • Chiara D.C.
      • Stewart D.S.
      • Ge R.
      • Zhou X.
      • Raines D.E.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Allyl m-trifluoromethyldiazirine mephobarbital: an unusually potent enantioselective and photoreactive barbiturate general anesthetic.
      ,
      • Jayakar S.S.
      • Zhou X.
      • Chiara D.C.
      • Dostalova Z.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Dailey W.
      • Miller K.W.
      • Eckenhoff R.G.
      • Cohen J.B.
      Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.
      ). 11-Azi-AP and 11-F4N3Bzox-AP were prepared as described previously (
      • Savechenkov P.Y.
      • Chiara D.C.
      • Desai R.
      • Stern A.T.
      • Zhou X.N.
      • Ziemba A.M.
      • Szabo A.L.
      • Zhang Y.
      • Cohen J.B.
      • Forman S.A.
      • Miller K.W.
      • Bruzik K.S.
      Synthesis and pharmacological evaluation of neurosteroid photoaffinity ligands.
      ). UCI-50027 (
      • Hogenkamp D.J.
      • Tran M.B.
      • Yoshimura R.F.
      • Johnstone T.B.
      • Kanner R.
      • Gee K.W.
      Pharmacological profile of a 17β-heteroaryl-substituted neuroactive steroid.
      ) and CCD-3693 (
      • Edgar D.M.
      • Seidel W.F.
      • Gee K.W.
      • Lan N.C.
      • Field G.
      • Xia H.
      • Hawkinson J.E.
      • Wieland S.
      • Carter R.B.
      • Wood P.L.
      CCD-3693: an orally bioavailable analog of the endogenous neuroactive steroid, pregnanolone, demonstrates potent sedative hypnotic actions in the rat.
      ) were gifts from Drs. Derk Hogenkamp and Kelvin Gee (Department of Pharmacology, College of Medicine, University of California, Irvine). 3β-CH3OCH2-THDOC (
      • Tsai S.E.
      • Lee J.C.
      • Uramaru N.
      • Takayama H.
      • Huang G.J.
      • Wong F.F.
      Synthesis and antiproliferative activity of 3α-hydroxyl-3β-methoxymethyl-5α-pregnan-20-one with a C-21 hydrophilic substituent.
      ) was a gift from Drs. Shuo enTsai and Fung Fuh Wong (School of Pharmacy, China Medical University, Taichung, Taiwan). Other nonradioactive steroids were from commercial sources, most from Research Plus or Steraloids, but also from Tocris (3α5α-P, Org20599, and 17-PA), Santa Cruz Biotechnology (PS), Sigma-Aldrich (ganaxolone, DHEAS, 3α5β-P, and alphaxalone), and MedChemExpress (Sage-217). Chemical structures of steroids tested are presented in Fig. S1. Ivermectin was from Tocris. o-Phthalaldehyde and cyanogen bromide were from TCI Chemicals and Alfa Aesar, respectively. Endoproteinase Lys-C was from New England Biolabs.
      Human α1β3 and α1β3γ2 GABAARs with the α1 subunits containing a FLAG epitope at the N terminus of the mature subunit were expressed in tetracycline-inducible, stably transfected HEK293-TetR cell lines, and purified from detergent extracts as described previously (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ,
      • Jayakar S.S.
      • Zhou X.
      • Chiara D.C.
      • Dostalova Z.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Dailey W.
      • Miller K.W.
      • Eckenhoff R.G.
      • Cohen J.B.
      Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.
      ,
      • Dostalova Z.
      • Liu A.P.
      • Zhou X.J.
      • Farmer S.L.
      • Krenzel E.S.
      • Arevalo E.
      • Desai R.
      • Feinberg-Zadek P.L.
      • Davies P.A.
      • Yamodo I.H.
      • Forman S.A.
      • Miller K.W.
      High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors.
      ,
      • Dostalova Z.
      • Zhou X.
      • Liu A.
      • Zhang X.
      • Zhang Y.
      • Desai R.
      • Forman S.A.
      • Miller K.W.
      Human α1β3γ2L γ-aminobutyric acid type A receptors: high-level production and purification in a functional state.
      ) by use of an anti-FLAG antibody column. In brief, cells were grown for 72 h on 15-cm plates at 37 °C, induced with tetracycline and 5 mm sodium butyrate for 24 h, then harvested and lysed as described (
      • Dostalova Z.
      • Liu A.P.
      • Zhou X.J.
      • Farmer S.L.
      • Krenzel E.S.
      • Arevalo E.
      • Desai R.
      • Feinberg-Zadek P.L.
      • Davies P.A.
      • Yamodo I.H.
      • Forman S.A.
      • Miller K.W.
      High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors.
      ), with membrane pellet suspensions flash frozen in liquid nitrogen and stored at –80 °C. α1β3 and α1β3γ2 GABAARs were expressed at ∼30 and 5-10 pmol of [3H]muscimol-binding sites per mg of membrane protein, respectively. For GABAAR purifications, membranes (1 mg of protein/ml) were solubilized for 2.5 h in purification buffer (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ) supplemented with 30 mm n-dodecyl β-d-maltopyranoside. Column wash and elution buffers contained 0.2 mm asolectin and 5 mm CHAPS. After elution from columns in the presence of 5 mm FLAG peptide (elutions 1 and 2, 13 ml each), aliquots were characterized for [3H]muscimol binding. Membranes from 30 plates of α1β3 GABAARs (10-15 nmol of binding sites) yielded 2-3 nmol of purified receptor (60-110 and 30-40 nm binding sites in elutions 1 and 2). Membranes from 60 to 70 plates of α1β3γ2 GABAARs contained 4-5 nmol of binding sites and yielded ∼1 nmol of purified receptor (50-60 nm and 20-30 nm binding sites in elutions 1 and 2). Aliquots of purified GABAARs were stored at –80 °C until use.

      [3H]Muscimol binding

      Membrane homogenates were prepared as described (
      • Dostalova Z.
      • Liu A.P.
      • Zhou X.J.
      • Farmer S.L.
      • Krenzel E.S.
      • Arevalo E.
      • Desai R.
      • Feinberg-Zadek P.L.
      • Davies P.A.
      • Yamodo I.H.
      • Forman S.A.
      • Miller K.W.
      High-level expression and purification of Cys-loop ligand-gated ion channels in a tetracycline-inducible stable mammalian cell line: GABAA and serotonin receptors.
      ) from HEK 293 TetR cells expressing α1β3 GABAARs. Membrane suspensions (50 µg of protein/ml in 2 ml of assay buffer (200 mm KCl, 1 mm EDTA, 10 mm phosphate buffer, pH 7.4)) were equilibrated with 2 nm [3H]muscimol (PerkinElmer Life Sciences) and various concentrations of steroid for 1 h at 4 °C and then filtered in quadruplicate on Whatman GF/B glass fiber filters that had been pretreated with 0.5% polyethyleneimine for 1 h. After being washed twice with 5 ml of cold assay buffer, filters were dried and 3H retention was determined by liquid scintillation counting. Nonspecific binding was determined in the presence of 1 mm GABA. The modulation results are presented as the percentage of the specifically bound [3H]muscimol over that without steroid. The plotted data are the mean ± S.D. of pooled data from 2 to 4 independent experiments, and the full data sets were fit to Equation 1 to determine values of EC50, nH, and maximal enhancement.
      B%x=Bmax-1001+EC50xnH+100
      (Eq. 1)


      GABAAR photolabeling

      Purified α1β3 or α1β3γ2 GABAARs were photolabeled with [3H]21-pTFDBzox-AP in the presence of 300 μm GABA on an analytical scale (50 µl/gel lane, ∼2 pmol of [3H]muscimol-binding sites) or for α1β3 GABAARs on a preparative scale (1.5-2 ml, 60-170 pmol of [3H]muscimol sites per condition). Appropriate volumes of [3H]21-pTFDBzox-AP were dried down under an argon stream and resuspended with gentle vortexing for 30 min at 4 °C in freshly thawed GABAAR aliquots. Final concentrations of [3H]21-pTFDBzox-AP varied between 0.5 and 1 μm for α1β3 GABAAR photolabelings and 0.5 and 1.5 μm for α1β3γ2 GABAARs. For preparative scale labeling, the resuspended sample was divided into two equal aliquots for determination of photolabeled amino acids in the absence or presence of 30 μm 3α5α-P. Both samples contained 0.5% (v/v) methanol. Samples were incubated at 4 °C for 30 min, placed into 3.5 cm diameter plastic Petri dishes, and irradiated using a Spectroline model EN-280L 365-nm lamp for 30 min on ice from a distance of 1 cm. For analytical photolabeling assays, a 1-µl glass syringe (Hamilton 86200) was used to add 0.25 µl (0.5%) of the steroid/drug to be tested to a 10-µl aliquot of GABAAR in glass vials (CERT5000-69LV, ThermoFisher Scientific) followed by addition of 40-µl aliquots of GABAAR equilibrated with [3H]21-pTFDBzox-AP. Samples were vortexed, incubated on ice for 45 min, transferred to 96-well plates, and then irradiated for 30 min at 365 nm. Most stock solutions of nonradioactive steroids were prepared in ethanol at 60 mm, or at 150 (DHEAS and PS), 20 (3α5β-THDOC), or 6 mm (Org20599). Stock solutions of 11-aziAP (11 mm) and 11-F4N3Bzox-AP (7.6 mm) were prepared in methanol. Stock solutions in DMSO were prepared at 150 (3β5β-P, 5β-pregnan-3α,20β-diol, 3α5α-A, 3α5αA-17α-ol, and 3α5α-A-17-one), 60 (3-acetyl-5α-P, 3-deoxy-5α-P, 5α-pregnan-3α,20α-diol, 5β-pregnan-3α,11β-diol-20-one, and SAGE-217), or 22 mm (21-pTFDBzox-AP). The final concentrations of ethanol, methanol, or DMSO during labeling were 0.5, 0.5, or 0.2% (v/v), respectively.
      After photolabeling, GABAAR subunits were separated by SDS-PAGE as described (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ), and gel bands containing α/γ (56 kDa) and β (59/61 kDa) subunits were identified by Gel Code Blue Safe Protein Stain (ThermoFisher Scientific) for analytical labelings (
      • Jayakar S.S.
      • Zhou X.J.
      • Chiara D.C.
      • Jarava-Barrera C.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Tortosa M.
      • Miller K.W.
      • Cohen J.B.
      Identifying drugs that bind selectively to intersubunit general anesthetic sites in the α1β3γ2 GABAAR transmembrane domain.
      ). For analytical scale experiments, [3H]21-pTFDBzox-AP incorporation was measured by scintillation counting of excised gel bands (in 3H cpm) or by fluorography (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ). For preparative scale experiments, gel bands of interest were excised and eluted passively in elution buffer (100 mm NH4HCO3, 2.5 mm DTT, 0.1% SDS, pH 8.4) for 3 days at room temperature. The eluates were filtered and concentrated, and the proteins in the eluate were precipitated (75% acetone, overnight at −20 °C) and then resuspended in digestion buffer (15 mm Tris, 0.5 mm EDTA, 0.1% SDS, pH 8.4).

      Quantitation of concentration dependence of inhibition of photolabeling

      The concentration dependence of inhibition of 3H incorporation into GABAAR subunits was fit by nonlinear least squares to Equation 2,
      Bx=B0-Bns1+xIC50nH+Bns
      (Eq. 2)


      where, B(x) is the 3H cpm incorporated into a subunit gel band at a total inhibitor concentration of x. B0 is incorporation in the absence of inhibitor, Bns is the nonspecific incorporation, IC50 is total inhibitor concentration that reduces the specific incorporation by 50%, and nH is the Hill coefficient. For [3H]21-pTFDBzox-AP, nonspecific photolabeling was determined in the presence of 30 μm 3α5α-P. IC50 values were determined for inhibition of [3H]21-pTFDBzox-AP incorporation in the β subunit gel bands (59/61 kDa) that reflects photolabeling of residues at the β+–α subunit interface at the cytoplasmic end of the TMD (see “Results”). For [3H]azietomidate and [3H]R-mTFD-MPAB, IC50 values were determined for inhibition of photolabeling in the α (56 kDa) and β (59/61 kDa) subunit gel bands, respectively, which reflect labeling of α1Met-236 and β3Met-227 (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ,
      • Jayakar S.S.
      • Zhou X.
      • Chiara D.C.
      • Dostalova Z.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Dailey W.
      • Miller K.W.
      • Eckenhoff R.G.
      • Cohen J.B.
      Multiple propofol-binding sites in a γ-aminobutyric acid type A receptor (GABAAR) identified using a photoreactive propofol analog.
      ). For each drug tested, data from 4 to 6 experiments, using at least 2 different GABAAR purifications, were combined by normalizing (as %) the specific incorporation (BxBns) at each concentration to the specific incorporation in the absence of drug (B0Bns) for each experiment individually. The data plotted in the figures are the mean ± S.D. values of the normalized specific data from n experiments. The full normalized data sets were fit (GraphPad Prism 7.0 or SigmaPlot 11.0) using Equation 2. For all fits, the best fit values (± S.E.) of the variable parameters and the number of experiments are reported in the tables, with the plotted curves calculated from those parameters. Unless noted otherwise, the reported IC50 values and calculated curves are for fits with Bns = 0 and nH = 1. Parameters from fits with variable nH or variable Bns are not reported unless nH was less than 0.8 or Bns was greater than 15%. The extra sum of the squares principle (F test, α = 0.05) was used to determine whether a variable nH provided a statistically favored fit compared with nH = 1 (null hypothesis), or whether IC50 values for a drug were the same (null hypothesis) or different for α1β3 and α1β3γ2 GABAARs.

      Enzymatic and chemical fragmentation

      α1 and β3 subunits isolated by SDS-PAGE from α1β3 GABAARs photolabeled on a preparative scale were digested with Endo Lys-C (3-5 µg, 3 days, 20 °C), which produces fragments beginning at the N termini of each subunit's M1, M3 and M4 helices that can be separated and purified by rpHPLC (
      • Chiara D.C.
      • Jayakar S.S.
      • Zhou X.
      • Zhang X.
      • Savechenkov P.Y.
      • Bruzik K.S.
      • Miller K.W.
      • Cohen J.B.
      Specificity of intersubunit general anesthetic-binding sites in the transmembrane domain of the human α1β3γ2 γ-aminobutyric acid Type A (GABAA) receptor.
      ). To cleave at the C-terminal side of methionines, samples already loaded onto sequencing supports were treated with cyanogen bromide as described (
      • Scott M.G.
      • Crimmins D.L.
      • McCourt D.W.
      • Tarrand J.J.
      • Eyerman M.C.
      • Nahm M.H.
      A simple in situ cyanogen bromide cleavage method to obtain internal amino acid sequence of proteins electroblotted to polyvinyldifluoride membranes.
      ,
      • Hamouda A.K.
      • Kimm T.
      • Cohen J.B.
      Physostigmine and galanthamine bind in the presence of agonist at the canonical and noncanonical subunit interfaces of a nicotinic acetylcholine receptor.
      ).

      HPLC purification and protein microsequencing

      Subunit digests were fractionated by rpHPLC on an Agilent 1100 binary pump system using a Brownlee C4 Aquapore column (100 × 2.1 mm, 7-µm particle size) at 40 °C with an upstream guard column (Newguard RP-2). The aqueous solvent contained 0.08% TFA and the organic solvent contained 60% acetonitrile, 40% isopropyl alcohol, 0.05% TFA. Elution was achieved using a nonlinear gradient increasing from 5 to 100% organic solvent over 80 min at a flow rate of 0.2 ml/min. Fractions of 0.5 ml were collected, and 10% aliquots were assayed for determination of 3H. Fractions of interest were pooled for sequencing and drop-loaded at 45 °C onto Micro TFA glass fiber sequencing filters (Applied Biosytems) that were treated after loading with Biobrene Plus (Applied Biosystems).
      Samples were sequenced on an Applied Biosystems Procise 492 Protein sequencer programed to use 2/3 (∼80 of 120 µl) of the material from each cycle of Edman degradation for PTH-derivative identification and quantitation and to collect 1/3 for 3H determination by liquid scintillation counting. For some samples, sequencing was interrupted at a designated cycle for treatment of the sequencing filter with o-phthalaldehyde (
      • Brauer A.W.
      • Oman C.L.
      • Margolies M.N.
      Use of o-phthalaldehyde to reduce background during automated Edman degradation.
      ,
      • Middleton R.E.
      • Cohen J.B.
      Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label.
      ) to prevent further sequencing of any peptide not containing a proline at that cycle. The amount of PTH-derivative released (in picomoles) for a given residue was quantified using their peak height in the chromatogram, background-subtracted, compared with a standard peak, and the PTH-derivatives released for the detected peptide were fit to the equation,
      F(x) = I0×Rx
      (Eq. 3)


      where F(x) is the pmol of the amino acid in cycle x, I0 is the calculated initial amount of the peptide, and R is the repetitive yield. The 1st residue in the peptide as well as Cys, Trp, His, and Ser were not used in the calculation due to known problems with their quantitation. The efficiency of photolabeling (E in cpm/pmol) at a labeled amino acid in cycle x was calculated by the equation,
      Ex=2×cpmx-cpmx-1I0×Rx
      (Eq. 4)


      where cpmx is the 3H released in cycle x.

      Molecular modeling and computational docking

      For computational docking studies, we used the recently solved cryo-EM structure of a desensitized state of α1β3γ2L GABAAR (PDB 6I53) (
      • Laverty D.
      • Desai R.
      • Uchański T.
      • Masiulis S.
      • Stec W.J.
      • Malinauskas T.
      • Zivanov J.
      • Pardon E.
      • Steyaert J.
      • Miller K.W.
      • Aricescu A.R.
      Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer.
      ) in a lipid-nanodisc with a bound positive allosteric modulator megabody in the extracellular domain. This structure was determined from GABAARs purified from the same cell line as that used in our photolabeling studies, a cell line expressing full-length receptor subunits with intact cytoplasmic domains. Although most of the ∼120 amino acids comprising each subunit cytoplasmic domain were not resolved in this structure, the locations of 4 of the 5 amino acids specifically photolabeled by [3H]21-pTFDBzox-AP were resolved. In contrast, only the photolabeled β3Arg-309 was resolved in 5 other structures using the same source of GABAARs that were determined in the presence of GABA, picrotoxinin, or bicuculline (
      • Masiulis S.
      • Desai R.
      • Uchański T.
      • Serna Martin I.
      • Laverty D.
      • Karia D.
      • Malinauskas T.
      • Zivanov J.
      • Pardon E.
      • Kotecha A.
      • Steyaert J.
      • Miller K.W.
      • Aricescu A.R.
      GABAA receptor signalling mechanisms revealed by structural pharmacology.
      ).
      Docking of 21-pTFDBzox-AP and other steroids to the PDB 6I53 model was performed using the Discovery Studio CDOCKER module. Potential binding sites at each subunit interface of the PDB 6I53 structure were defined by 14-Å radius binding site spheres centered by the position of 3α5α-THDOC molecules overlaid (Discovery Studio: Tools: Superimpose Proteins: Sequence Alignment) from the PDB 5OSB structure (after removal of the extracellular domain and cytoplasmic linker). For docking, a structure of 21-pTFDBzox-AP was created by appropriate additions at the 21 position of 3α5α-THDOC (PubChem structure CID No. 101,771). Four copies of 21-pTFDBzox-AP, differing by rotations of ∼180°, were seeded into the binding site spheres. The 50 lowest energy solutions (simulated annealing with full potential minimization) were collected for each molecule from 50 random conformations (high temperature molecular dynamics) and 50 randomized orientations within the sphere (i.e. 2,500 attempted dockings per molecule). In two independent docking runs, we found that 21-pTFDBzox-AP was predicted to bind most favorably at the γβ+–α subunit interface with the lowest CDocker interaction energy (−49.17 kcal/mol) at that site 4.5 kcal/mol more favorable than at the β+–αγ–binding site and more than 10 kcal/mol more favorable than at the homologous α+–γ, α+–β, or γ+–β intersubunit sites. At the γβ+–α site, for the energetically most favored solution and 56% of all collected solutions, 21-pTFDBzox-AP adopted a common orientation with the 3α-OH directed toward α1Gln-242 and the aromatic diazirine extending linearly from the steroid backbone into a groove between β3Arg-309 and β3Leu-417, residues photolabeled by [3H]21-pTFDBzox-AP (see “Results”). 3α5α-THDOC and 3α5α-P were also predicted to bind in a similar orientation at the γβ+–α site, with most favorable CDOCKER interaction energies of −40.6 and −35.0 kcal/mol. Although both molecules were predicted to bind in an orientation with the 3α-OH in proximity to α1Gln-242, no consistent prediction was made concerning the energetic importance of a 3α-OH. For THDOC, the CDOCKER interaction energy for the 3α-OH isomer was 1.8 kcal/mol more favorable than for the 3β-epimer, whereas the interaction energy was 1.4 kcal/mol more favorable for 3β5α-P than for 3α5α-P.

      Data availability

      All data are contained within the article.

      Acknowledgments

      We dedicate this article to the memory of David C. Chiara, our wonderful colleague who passed away recently. We thank Drs. Derk Hogenkamp and Kelvin Gee for the gifts of UCI-50027 (
      • Hogenkamp D.J.
      • Tran M.B.
      • Yoshimura R.F.
      • Johnstone T.B.
      • Kanner R.
      • Gee K.W.
      Pharmacological profile of a 17β-heteroaryl-substituted neuroactive steroid.
      ) and CCD-3693 (
      • Edgar D.M.
      • Seidel W.F.
      • Gee K.W.
      • Lan N.C.
      • Field G.
      • Xia H.
      • Hawkinson J.E.
      • Wieland S.
      • Carter R.B.
      • Wood P.L.
      CCD-3693: an orally bioavailable analog of the endogenous neuroactive steroid, pregnanolone, demonstrates potent sedative hypnotic actions in the rat.
      ) and Drs. Shuo en Tsai and Fung Fuh Wong for the resynthesis and gift of 3β-CH3OCH2-THDOC (
      • Tsai S.E.
      • Lee J.C.
      • Uramaru N.
      • Takayama H.
      • Huang G.J.
      • Wong F.F.
      Synthesis and antiproliferative activity of 3α-hydroxyl-3β-methoxymethyl-5α-pregnan-20-one with a C-21 hydrophilic substituent.
      ).

      Supplementary Material

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