Journal: The Journal of Biological Chemistry

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

doi: 10.1074/jbc.RA120.013452

Figure Lengend Snippet: 3α5α-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.

Article Snippet: 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 IC 50 = 85 ± 13 μ m , n H = 0.33 ± 0.03. table ft1 table-wrap mode="anchored" t5 caption a7 Steroid a C-5 R [ 3 H]21-pTFDBzoxy-AP b IC 50 ( n ) GABA enhancement c EC 50 μ m μ m 21- p TFDBzox-AP α -COCH 2 OCOBzTFD 0.21 ± 0.02 2.7 3α5α-A (A2150) α -H 8.7 ± 1.0 (6) 0.3 3α5β-P-20-deoxy (P7800) β 4.2 ± 0.5 (4) ND d (0.3) e 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 ( n H = 0.35) (6) ∼3 3α5α-A-17α-ol (A1150) α >300 (4) ND 17-PA (5αA3α-ol,16-ene, 17-C 6 H 5 ) α -C 6 H 5 85 ± 13 ( n H = 0.3 ± 0.03) (4) 29 ± 5 f Open in a separate window a Catalog numbers are indicated for steroids from Steraloids (A/P-xxxx). b IC 50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of GABA A R photolabeling, were determined as described under “Experimental Procedures” from data of B . n , number of experiments. c EC 50 values for steroid enhancement of GABA responses of α1βγ GABA A Rs expressed in oocytes, from the literature: 21 p -TFD-Bzoxy-AP ( 27 ); 3α5α-A ( 67 ); 5α-pregnan-3α,20α-diol and 5β-pregnan-3α,20β-diol ( 43 ); 3α5αA-17-one ( 52 ). d ND, not determined. e EC 50 for GABA A R enhancement by 3α5α-P-20-deoxy ( 67 ). f EC 50 for enhancement of [ 3 H]muscimol binding .

Techniques: Isolation, SDS Page, Purification, Produced, Sequencing

Journal: The Journal of Biological Chemistry

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

doi: 10.1074/jbc.RA120.013452

Figure Lengend Snippet: 3α5α-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 Fig. 5B) 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.

Article Snippet: 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 IC 50 = 85 ± 13 μ m , n H = 0.33 ± 0.03. table ft1 table-wrap mode="anchored" t5 caption a7 Steroid a C-5 R [ 3 H]21-pTFDBzoxy-AP b IC 50 ( n ) GABA enhancement c EC 50 μ m μ m 21- p TFDBzox-AP α -COCH 2 OCOBzTFD 0.21 ± 0.02 2.7 3α5α-A (A2150) α -H 8.7 ± 1.0 (6) 0.3 3α5β-P-20-deoxy (P7800) β 4.2 ± 0.5 (4) ND d (0.3) e 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 ( n H = 0.35) (6) ∼3 3α5α-A-17α-ol (A1150) α >300 (4) ND 17-PA (5αA3α-ol,16-ene, 17-C 6 H 5 ) α -C 6 H 5 85 ± 13 ( n H = 0.3 ± 0.03) (4) 29 ± 5 f Open in a separate window a Catalog numbers are indicated for steroids from Steraloids (A/P-xxxx). b IC 50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of GABA A R photolabeling, were determined as described under “Experimental Procedures” from data of B . n , number of experiments. c EC 50 values for steroid enhancement of GABA responses of α1βγ GABA A Rs expressed in oocytes, from the literature: 21 p -TFD-Bzoxy-AP ( 27 ); 3α5α-A ( 67 ); 5α-pregnan-3α,20α-diol and 5β-pregnan-3α,20β-diol ( 43 ); 3α5αA-17-one ( 52 ). d ND, not determined. e EC 50 for GABA A R enhancement by 3α5α-P-20-deoxy ( 67 ). f EC 50 for enhancement of [ 3 H]muscimol binding .

Techniques: Sequencing, Labeling

Journal: The Journal of Biological Chemistry

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

doi: 10.1074/jbc.RA120.013452

Figure Lengend Snippet: Location of the [3H]21p-TFDBzox-AP labeled residues in the cytoplasmic domain of the α1β3γ2L GABAAR (PDB 6I53). 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 (18). 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 (19). 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 (20). 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 (32, 69)) and a docked etomidate (in stick figure).

Article Snippet: 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 IC 50 = 85 ± 13 μ m , n H = 0.33 ± 0.03. table ft1 table-wrap mode="anchored" t5 caption a7 Steroid a C-5 R [ 3 H]21-pTFDBzoxy-AP b IC 50 ( n ) GABA enhancement c EC 50 μ m μ m 21- p TFDBzox-AP α -COCH 2 OCOBzTFD 0.21 ± 0.02 2.7 3α5α-A (A2150) α -H 8.7 ± 1.0 (6) 0.3 3α5β-P-20-deoxy (P7800) β 4.2 ± 0.5 (4) ND d (0.3) e 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 ( n H = 0.35) (6) ∼3 3α5α-A-17α-ol (A1150) α >300 (4) ND 17-PA (5αA3α-ol,16-ene, 17-C 6 H 5 ) α -C 6 H 5 85 ± 13 ( n H = 0.3 ± 0.03) (4) 29 ± 5 f Open in a separate window a Catalog numbers are indicated for steroids from Steraloids (A/P-xxxx). b IC 50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of GABA A R photolabeling, were determined as described under “Experimental Procedures” from data of B . n , number of experiments. c EC 50 values for steroid enhancement of GABA responses of α1βγ GABA A Rs expressed in oocytes, from the literature: 21 p -TFD-Bzoxy-AP ( 27 ); 3α5α-A ( 67 ); 5α-pregnan-3α,20α-diol and 5β-pregnan-3α,20β-diol ( 43 ); 3α5αA-17-one ( 52 ). d ND, not determined. e EC 50 for GABA A R enhancement by 3α5α-P-20-deoxy ( 67 ). f EC 50 for enhancement of [ 3 H]muscimol binding .

Techniques: Labeling, Sequencing, Membrane, Binding Assay, Residue

Journal: The Journal of Biological Chemistry

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

doi: 10.1074/jbc.RA120.013452

Figure Lengend Snippet: Structural 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 Fig. 4. 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 Tables 3 and ​and4.4. 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 Equation 2 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).

Article Snippet: 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 IC 50 = 85 ± 13 μ m , n H = 0.33 ± 0.03. table ft1 table-wrap mode="anchored" t5 caption a7 Steroid a C-5 R [ 3 H]21-pTFDBzoxy-AP b IC 50 ( n ) GABA enhancement c EC 50 μ m μ m 21- p TFDBzox-AP α -COCH 2 OCOBzTFD 0.21 ± 0.02 2.7 3α5α-A (A2150) α -H 8.7 ± 1.0 (6) 0.3 3α5β-P-20-deoxy (P7800) β 4.2 ± 0.5 (4) ND d (0.3) e 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 ( n H = 0.35) (6) ∼3 3α5α-A-17α-ol (A1150) α >300 (4) ND 17-PA (5αA3α-ol,16-ene, 17-C 6 H 5 ) α -C 6 H 5 85 ± 13 ( n H = 0.3 ± 0.03) (4) 29 ± 5 f Open in a separate window a Catalog numbers are indicated for steroids from Steraloids (A/P-xxxx). b IC 50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of GABA A R photolabeling, were determined as described under “Experimental Procedures” from data of B . n , number of experiments. c EC 50 values for steroid enhancement of GABA responses of α1βγ GABA A Rs expressed in oocytes, from the literature: 21 p -TFD-Bzoxy-AP ( 27 ); 3α5α-A ( 67 ); 5α-pregnan-3α,20α-diol and 5β-pregnan-3α,20β-diol ( 43 ); 3α5αA-17-one ( 52 ). d ND, not determined. e EC 50 for GABA A R enhancement by 3α5α-P-20-deoxy ( 67 ). f EC 50 for enhancement of [ 3 H]muscimol binding .

Techniques: Binding Assay, Isolation, SDS Page, Concentration Assay, Inhibition

Journal: The Journal of Biological Chemistry

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

doi: 10.1074/jbc.RA120.013452

Figure Lengend Snippet: Inhibition of [ 3 H]21- p TFDBzox-AP photolabeling by C-17 substituted 3α-OH steroid GABA A R PAMs

Article Snippet: 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 IC 50 = 85 ± 13 μ m , n H = 0.33 ± 0.03. table ft1 table-wrap mode="anchored" t5 caption a7 Steroid a C-5 R [ 3 H]21-pTFDBzoxy-AP b IC 50 ( n ) GABA enhancement c EC 50 μ m μ m 21- p TFDBzox-AP α -COCH 2 OCOBzTFD 0.21 ± 0.02 2.7 3α5α-A (A2150) α -H 8.7 ± 1.0 (6) 0.3 3α5β-P-20-deoxy (P7800) β 4.2 ± 0.5 (4) ND d (0.3) e 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 ( n H = 0.35) (6) ∼3 3α5α-A-17α-ol (A1150) α >300 (4) ND 17-PA (5αA3α-ol,16-ene, 17-C 6 H 5 ) α -C 6 H 5 85 ± 13 ( n H = 0.3 ± 0.03) (4) 29 ± 5 f Open in a separate window a Catalog numbers are indicated for steroids from Steraloids (A/P-xxxx). b IC 50 (±S.E.) values, the total drug concentrations resulting in 50% inhibition of GABA A R photolabeling, were determined as described under “Experimental Procedures” from data of B . n , number of experiments. c EC 50 values for steroid enhancement of GABA responses of α1βγ GABA A Rs expressed in oocytes, from the literature: 21 p -TFD-Bzoxy-AP ( 27 ); 3α5α-A ( 67 ); 5α-pregnan-3α,20α-diol and 5β-pregnan-3α,20β-diol ( 43 ); 3α5αA-17-one ( 52 ). d ND, not determined. e EC 50 for GABA A R enhancement by 3α5α-P-20-deoxy ( 67 ). f EC 50 for enhancement of [ 3 H]muscimol binding .

Techniques: Inhibition

Journal: Reproductive biology

Article Title: A change in the steroid metabolic pathway in human testes showing deteriorated spermatogenesis.

doi: 10.1016/j.repbio.2020.02.008

Figure Lengend Snippet: Fig. 2. Autoradiograms of radioactive steroid meta- bolites. (1) Steroid metabolites from 14C-pregnenolone in human testicular samples after TLC development in benzene:acetone (4:1, v/v) solvent systems. A) High-JS group. B) Low-JS group. (2) Steroid metabolites in spot 2 in first TLC in human testicular samples after second TLC de- velopment in cyclohexane:ethyl acetate (1:1, v/v) solvent system. A) High-JS group. B) Low-JS group. (3) Steroid metabolites in spot 3 in first TLC in human testicular samples of Low-JS group after second TLC development in benzene:ethylaceta- te:acetone (8:2:1, v/v) solvent system. Acetylated steroid metabolites in spot 3 in first TLC in human testicular samples of High-JS group after TLC de- velopment in benzene:acetone (4:1, v/v) solvent system. A) Low-JS group. B) Acetylated steroid metabolites of High-JS group (left lane) and acetylated 5β-pregnan- 3β-ol-20-one (right lane). Asterisk represents acety- lated main metabolites. (4) Steroid metabolites in spot 5 in first TLC of human testicular samples of Low-JS group after second and third TLC development in various solvent systems. A) Third TLC development in cyclohexane:ethyl acetate = 3:7. B) Second TLC development in chloroform:MeOH:H2O = 188:12:1. (5) Steroid metabolites with NADPH for in vitro conversion in first TLC of human testicular sam- ples of High- and Low-JS groups after develop- ment in benzene:acetone (4:1, v/v) solvent system. A) High-JS group. B) Low-JS group. Asterisk re- presents Spot 7-1. P4: progesterone; AD: androstenedione; 17α-P4: 17α- hydroxyprogesterone; T: testosterone; 17a, 20b-P: 17α,20β-hydroxy-4-pregnen-3-one.

Article Snippet: For recrystallisation analysis of the radioactive metabolites on spot 7, 5α-pregnan-3β, 20β-diol (Steraloids, Newport, RI, USA) and 5αpregnan-3α-21diol-20-one (Steraloids, Newport, RI, USA) were the standards.

Techniques: Solvent, In Vitro