Article Figures & Data
Figures
- Fig. 1.
TETS can block closed and open α2β3γ2L receptors. (A) Chloride currents were activated by a 5-second application of 100 μM GABA directly to the patch-clamped cell. GABA was then washed out by a 50-second wash of the chamber with Ringer’s solution. One minute later, 50 μM of TETS was perfused into the chamber and allowed to equilibrate for 3 minutes. GABA (100 μM) was then reapplied directly to the cell with TETS in the bath. A subsequent 50-second wash of the chamber with Ringer’s solution completely reversed the TETS effect. (B) After a control current was elicited by direct application of 100 μM GABA, TETS and GABA were perfused together directly onto the cell with no preincubation.
- Fig. 2.
Generation of a homology model of the α2β3γ2 GABAA receptor. (A) The X-ray structure of the β3ECD-α5TMD served as a template. (B) The Rosetta-generated homology model of the α2β3γ2 in the open state. The receptor is color-coded as follows: α2 (blue), β3 (red), γ2 (yellow). (C) Sequence alignment of α2, β3, and γ2 in the M2 segment. (D) Common numbering of pore-lining residues in GABAA receptors.
- Fig. 3.
Searching for the TETS binding site in the open-state model. The receptor is color-coded as follows: α2 (blue), β3 (red), and γ2 (yellow). (A) “Walk” through the pore of the α2β3γ2 homology model in six boxes of 7-Å diameter in RosettaLigand, which identified site-A and site-C as possible binding sites. (B) TETS binding sites suggested by Glide XP. (C) TETS binding sites suggested by Swissdock.
- Fig. 4.
Site-directed mutagenesis of the β3 (A), γ2 (B), and α2 (C) subunits of the α2β3γ2L receptor. Wild-type and mutant receptor combinations were recombinantly expressed in L929 cells. Scatter plots show the percentage of current inhibition obtained with 50 µM of TETS when chloride currents were elicited by EC90 GABA (100 μM). Cutaways of the homology model are shown next to the graphs to visualize the position of the mutated residues. A representative current trace from a T6′ mutation is included for each subunit. Percentage of current blocked (mean ± S.D. from n = 5 to 8 cells per mutant) was analyzed with one-way ANOVA followed by Dunnett’s test to compare the means with the WT control and to correct for multiple comparisons. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. WT, wild type.
- Fig. 5.
TETS docked in the closed/resting-state homology model of the α2β3γ2 GABAA receptor. (A) Transmembrane view of the dominant low-energy binding pose of TETS identified by RosettaLigand. (B) Alternative low-energy TETS binding pose. In both panels, one β3 subunit has been removed for clarity. Hydrogen bonds are shown in green. The receptor is color-coded as follows: α2 (blue), β3 (red), and γ2 (yellow). (C) The TETS binding site viewed from above the T6′ ring. TETS is shown in stick representation with a transparent molecular surface. The five threonine residues are rendered as spheres. (D) Van der Waals interactions of TETS shown in the same transmembrane view as in (A). (E) Van der Waals interactions of TETS viewed from above the T6′ ring.
- Fig. 6.
Concentration-response curves for TETS inhibition of currents evoked by EC90 GABA (100 μM) comparing wild-type α2β3γ2L receptors with mutant receptors. (A) T6′ mutations: α2β3γ2L (IC50 0.48 µM, 95% CI 0.32–0.64 µM), α2T6′Mβ3γ2L (IC50 438.6 µM, 95% CI 346.2–503.9 µM, P < 0.0001), and α2β3T6′Cγ2L (IC50 326.7 µM, 95% CI 263.3–355.3 µM, P = < 0.0001). (B) T2′ mutations: α2β3γ2L (IC50 0.48 µM, 95% CI 0.32–0.64 µM), α2V2′Wβ3γ2L (IC50 5.70 µM, 95% CI 5.10–6.28 µM, P = 0.03), and α2β3A2′Sγ2L (IC50 299.5 µM, 95% CI 263.3–335.6 µM, P < 0.0001). Individual data points are presented as mean ± S.D. from five to eight independent recordings. Concentration-response curves were compared using an extra sum-of-squares F test (GraphPad Prism8; GraphPad Software). The reported P-values test the null hypothesis that the concentration-response curves for wild-type and mutant channels are identical. CI, confidence interval.
- Fig. 7.
(A) Overlay of the lowest-energy binding poses of TETS and picrotoxinin in stick representation in the closed/resting-state homology model of the α2β3γ2 GABAA receptor. The receptor is color-coded as follows: α2 (blue), β3 (red), and γ2 (yellow). One α2 subunit is removed for clarity. Picrotoxinin is shown in black. Hydrogen bonds are shown in green. The panels on the side show transparent molecular surfaces of picrotoxinin (black) and TETS (green) with pore-lining resides in the L9′, T6′, and 2′ ring rendered as spheres. (B) Concentration-response curves for picrotoxinin inhibition of currents evoked by EC90 GABA (100 μM) comparing wild-type α2β3γ2L receptors with T6′ mutations: α2β3γ2L (IC50 6.8 µM, 95% CI 4.5–8.4 µM), α2T6′Mβ3γ2L (no meaningful IC50 can be determined since the maximal effect is drastically reduced, and we therefore consider this channel insensitive to picrotoxinin), and α2β3T6′Cγ2L (IC50 4.2 µM, 95% CI 1.2–6.9 µM, P = 0.2). Individual data points are presented as mean ± S.D. from five to eight independent recordings. Concentration-response curves were compared using an extra sum-of-squares F test (GraphPad Prism8; GraphPad Software). The reported P-values test the null hypothesis that the concentration-response curves for wild-type and mutant channels are identical. CI, confidence interval.
- Fig. 8.
Concentration-response curves comparing the potency of TETS (black symbols) and three TETS haptens in blocking of currents evoked by EC90 GABA (100 μM) on wild-type α2β3γ2L receptors. TETS (IC50 0.48 µM, 95% CI 0.32–0.64 µM), Hapten 4a (IC50 6.5 µM, 95% CI 5.0–8.0 µM), Hapten 2a (IC50 7.9 µM, 95% CI 7.1–8.7 µM), Hapten 6b (IC50 86.6 µM, 95% CI 83.2–90.1 µM). Individual data points are presented as mean ± S.D. from five to eight independent recordings. The gray inset shows the % of current blocked on the α2β3T6′Cγ2L mutant by 1 mM of Hapten 2a (orange), Hapten 4a (green), and Hapten 6b (purple). CI, confidence interval.
Additional Files
Data Supplement
- Supplemental Data -
Supplementary Figure 1 - A, Dominant binding pose of EBOB identified by RosettaLigand in the pore of ȕ3ECD-Į5TMD
chimera-based homology model of the Į2ȕ3Ȗ2 receptor. B, The two most frequently identified binding poses of picrotoxinin in the same model.Supplementary Figure 2 - Dominant binding pose of TETS identified by RosettaLigand in the pore of the 6HUO-based
homology model of the Į2ȕ3Ȗ2 receptor in the desensitized state.Supplementary Figure 3 - A, TETS at the T6' ring of the open state model of the Į2ȕ3Ȗ2 GABAA receptor based on the
ȕ3ECD-Į5TMD chimera (5O8F). B, TETS at the T6' ring of the desensitized state model of the Į2ȕ3Ȗ2 GABAA receptor based on the Į1ȕ3Ȗ2L receptor structure (6HUO). C, TETS at the T6' ring of the closed/resting state model of the Į2ȕ3Ȗ2 GABAA receptor based on the Į1ȕ3Ȗ2L receptor structure (6HUG).Supplementary Figure 4 - 2D ligand-protein diagram of TETS in the closed/resting state model of the Į2ȕ3Ȗ2 GABAA
receptor.Supplementary Figure 5 - Comparison of the picrotoxinin pose observed in the cryo-EM structure of the picrotoxininĮ1ȕ3Ȗ2L complex (6HUG) with the dominant binding pose of picrotoxinin identified by RosettaLigand in the closed/resting state model of the Į2ȕ3Ȗ2 GABAA receptor. Hydrogen bonds are shown in green.
- Supplemental PDB Files -
Supplementary PDB File 1 - Homology model of the closed/resting stated of the Į2ȕ3Ȗ2 receptor based on the Į1ȕ3Ȗ2 cryo-EM structure with picrotoxinin bound (6HUG).
- Supplemental PDB Files -
Supplementary PDB File 2 - Dominant binding pose of TETS identified by RosettaLigand in the homology model of the
closed/resting stated of the Į2ȕ3Ȗ2 receptor. The extracellular domains are truncated.
- Supplemental Data -
In this issue
The Binding Site of TETS
