Article Figures & Data
Figures
- Figure 1.
Schematic representation of Azo‐NZ1 and cumulative curves of glycine-induced dose-response dependencies for different subtypes of GlyRs. A, Azo‐NZ1 in its trans- and cis‐configurations. B, Cumulative dose/response curves for homomeric α1Z (filled squares, n = 6) and α2Z GlyRs (empty circles, n = 6). C, Cumulative dose/response curves for heteromeric α1H/βM (diamonds, n = 7) and α2M/β M (triangles, n = 7).
- Figure 2.
Interaction of Azo-NZ1 with homomeric α1 and heteromeric α1/β GlyRs. A, Representative trace of the α1Z GlyRs-mediated current induced by application of glycine 20 μm (below EC50) and by mixture of glycine 20 μm with Azo-NZ1 50 μm at visible light and on illumination with UV light (here and in other figures, UV illumination is shown by violet rectangles). B, Representative trace of α1Z current induced by saturating glycine concentration, 300 μm, and by a mixture of glycine 300 μm with Azo-NZ1 50 μm at visible light and on illumination with UV light. Note the absence of Azo-NZ1 inhibitory effect. C, Cumulative dose–response curve for Azo-NZ1 action on α1Z GlyRs at glycine concentration of 20 μm. IC50 = 78 ± 8 μm, nH = 1.4 ± 0.25 (n = 6); Vhold = –30 mV. D, Representative trace of α1/β-mediated current induced by application of glycine 30 μm and by a mixture of glycine with Azo-NZ1 (50 μm). I1 and I2 are the amplitudes of currents used to calculate the Azo-NZ1 inhibitory effect, I1 and I3 were used to calculate the UV effect. E, Relative amplitudes of α1H and α1H/βM currents induced by application of glycine 30 μm or by mixture of glycine with Azo-NZ1 50 μm. Data from three to seven cells.
- Figure 3.
Azo-NZ1 is a potent UV-controllable inhibitor of α2 GlyRs. A, Representative trace of α2Z GlyRs current induced by application of glycine 50 μm or by a mixture of glycine with Azo-NZ1 50 μm at visible light or at UV illumination. B, Representative trace of α2Z current induced by application of glycine 300 μm or by a mixture of glycine with Azo-NZ1 50 μm at visible light or on UV illumination. C, Cumulative data on relative amplitude of Azo-NZ1 inhibition (50 μm) of glycine-induced currents (50 and 300 μm) at visible light and on UV illumination. D, Cumulative dose-response curve for Azo-NZ1; sigmoidal fitting yielded IC50 = 9 ± 2 μm; nH = 1.02 ± 0.3 (n = 4); Vhold = –30 mV. E, Cumulative data on the voltage dependence of Azo-NZ1 interaction with α2Z GlyRs (black column, relative amplitude of the current in control; gray column, on application of Azo-NZ1 20 μm at Vhold = –30 mV; dashed column, on application of Azo-NZ1 20 μm at Vhold = +30 mV); n = 6–8 cells, ***p ≤ 0.001. F, Representative trace of α2M/βM-mediated current induced by glycine 100 μm or by mixture of glycine with Azo-NZ1 (50 μm) at visible light or on UV illumination. G, Relative amplitudes of current mediated by α2M and α2M/βM receptors during the application of Azo-NZ1 50 μm at visible light and on illumination with UV light (n = 8–10 cells, ***p ≤ 0.001).
- Figure 4.
Single-channels recording of α1H and α2M GlyRs interaction with Azo-NZ1 in outside-out configuration of patch-clamp technique. A, Representative recording of single-channel activity of α1Z GlyRs in control, currents were induced by application of 5 μm glycine, Vhold = –30 mV. B, Channel activity in the solution containing a mixture of glycine 5 μm with trans-Azo-NZ1 50 μm at visible light and (C) on UV illumination (cis-Azo-NZ1). D, NPo for α1H GlyRs in control conditions (white column), when glycine 5 μm was co-applied with Azo-NZ1 50 μm under visible light (gray column), or on UV illumination (violet column). E, Channel activation was induced by application of 5 μm glycine, Vhold = –30 mV. F, A mixture of glycine 5 μm with Azo-NZ1 20 μm was applied, Vhold = –30 mV; the part of the trace recorded on illumination with UV light is colored in violet. G, A mixture of glycine 5 μm with Azo-NZ1 50 μm was applied, Vhold = –30 mV; the part of the trace recorded on UV illumination is colored in violet. H, NPo for α2M GlyRs in control conditions (white column), when glycine 5 μm was co-applied with Azo-NZ1 20 μm or 50 μm under visible light (gray columns), or on UV illumination (violet columns).
- Figure 5.
Azo-NZ1 is a photoswitchable modulator of the evoked glycinergic IPSCs (eIPSCs) in the mouse hypoglossal motoneurons. A, Relative eIPSC amplitudes under the action of Azo-NZ1 (15 μm) at visible light and on UV illumination, summary of data from eight neurons (mean percentage ± SEM). *, significant difference with p < 0.05 (paired sample Wilcoxon signed-rank test). B, Superimposed traces of eIPSCs in control condition, after addition to aCSF of 15 μm Azo‐NZ1, during illumination of Azo‐NZ1 containing aCSF by UV (365 nm) and after washing. Each trace represents the average of 18 individual eIPSCs, obtained from a P8 mouse. C, Superimposed traces illustrating an example of eIPSC amplitudes decreasing under 15, 120, and 300 μm Azo‐NZ1 and complete suppression of events by 0.2 μm strychnine. Each trace represents average of 12 individual eIPSCs, obtained from a P6 mouse. D, Decreasing of glycinergic eIPSC amplitude under the action of Azo-NZ1 at the concentrations of 15, 120, and 300 μm, summary of data from six neurons (mean percentage ± SEM). *, significant difference with p < 0.05 (paired sample Wilcoxon signed-rank test).
- Figure 6.
Comparison of the interaction of α1 WT GlyRs and α1 G254A GlyR with Azo-NZ1. A, Amino acid sequences of TM2 domains of α1, α2, and β subunits of GlyR. B, Representative trace of the α1H GlyR current induced by application of glycine 30 μm and by a mixture of glycine 30 μm with Azo-NZ1 50 μm at visible light and on UV illumination. C, Representative trace of the α1H G254A GlyRs current induced by application of glycine 20 μm and by mixture of glycine with Azo-NZ1 50 μm at visible light and on UV illumination. D, Relative amplitudes of α1H and α1H G254A GlyR currents in control (black column), and during application of Azo-NZ1 50 μm at visible light (gray and red columns, respectively) and at UV illumination (violet columns). E, Representative single-channel recording of α1H G254A currents in control; during application of Azo-NZ1 50 μm at visible light (F); on illumination with UV light (G). H, NPo for α1H G254A GlyRs in control conditions (white column), when glycine 5 μm was co-applied with Azo-NZ1 50 μm under visible light (gray column) or on UV illumination (violet column).
- Figure 7.
Results of the docking simulation of Azo-NZ1 in the transmembrane part domain of the α1 G254A GlyR mutant mimicking the α2 GlyR pore. Results for wild-type α1 GlyR and the heteromeric α2/β GlyR are shown in Extended Data Figures 7-1, 7-2, 7-3, 7-4, 7-5, 7-6. A, Tridimensional view of the α1 G254A mutant GlyR structure (open state). The A2’ and T13’ positions are marked with dashed lines. The sulfonate group of Azo-NZ1 can bind with two orientations: up (bound at 13’) and down (bound at 2’); see Extended Data Figures 7-1, 7-3. B, Binding pose of the trans isomer in the transmembrane domain. The sulfonate group is in the down configuration; see Extended Data Figure 7-3 for the corresponding poses in wild-type α1 GlyR and the heteromeric α2/β GlyR. Hydrophobic interactions between Azo-NZ1 and the receptor residues are marked as yellow transparent surfaces and hydrogen bond interactions are represented with red dashed lines. C, Binding pose of the cis isomer in the transmembrane domain. The sulfonate group is in the down configuration; see Extended Data Figure 7-6 for the corresponding poses in wild-type α1 GlyR and the heteromeric α2/β GlyR. The representations used for hydrophobic and hydrogen bond interactions are the same as for the trans isomer. D, Relative number of intermolecular interactions formed by the cis and trans isomers of Azo-NZ1 with the pore-lining residues in α1 G254A mutant GlyR.
- Figure 8.
Proposed scheme of the passage of a hydrated chloride ion through the 2’ pore region in α1 G254A and α1 GlyRs. The hydrated chloride ion is represented in greyscale. Electrostatic repulsion between the negatively charged sulfonate group and the chloride ion is represented with a blue arrow, the steric hindrance between the chloride ion and the methyl side chain of A2’ is shown as curved red dashed lines, and the hydrophilic interactions between the water molecules in the chloride ion solvation shell and the G2’ backbone are represented as black dashed lines. A, In the α1G254A GlyR mutant, the electrostatic and steric barrier created by the combined effect of the sulfonate group of trans-Azo-NZ1 and A2’, respectively, does not let the chloride ion go through this narrow part of the pore. B, In wild-type α1 GlyR, the electrostatic repulsion with the sulfonate group is not enough to prevent chloride conduction, as the residue at the 2’ position is now glycine, so that the pore has more space and the chloride ion can also form hydrophilic interactions with the G2’ backbone.
Extended Data
Extended Data Figure 7-1
Density map of the sulfonate group of trans-Azo-NZ1 bound in the transmembrane part of the pore of α1, α1G254A, and α2/β GlyRs. Regions of continuous density correspond to higher sulfonate occupancy and indicate tighter sulfonate binding. Each contour line corresponds to one particle/nm3, where “particle” refers to the sulfonate group in a given binding pose and is used to follow Azo-NZ1 binding. Longitudinal view of the pore: the front subunit is not displayed to reveal the interior of the pore. The S/T16’ and G/A/P2’ regions are marked with dashed lines. A higher percentage of binding poses is observed in these two regions; the percentage of poses (between parentheses) where the sulfonate is placed in the 16’ region (up configuration) is 40%, 47%, and 34%, whereas 21%, 21%, and 26% of binding poses placed the sulfonate in the 2’ region (down configuration) for α1, α1G254A, and α2/β GlyRs, respectively. Download Figure 7-1, TIF file.
Extended Data Figure 7-2
Interactions of trans-Azo-NZ1 in the “up” binding pose for (A) α1, (B) α1G254A, and (C) α2/β GlyRs. A longitudinal view of the pore is shown, with the pore lining residues represented as ball-and-sticks and their carbon atoms colored in gray. Trans-Azo-NZ1 is represented as sticks with carbon atoms in orange. Nitrogen and oxygen atoms are colored in blue and red, respectively. Hydrogen bonds are marked with red dashed lines and hydrophobic interactions are represented as yellow surfaces. Download Figure 7-2, TIF file.
Extended Data Figure 7-3
Interactions of trans-Azo-NZ1 in the “down” binding pose for (A) α1, (B) α1G254A, and (C) α2/β GlyRs. Representations and color code are the same as in Extended Data Figure 7-2. Download Figure 7-3, TIF file.
Extended Data Figure 7-4
Density map of the sulfonate group of cis-Azo-NZ1 bound in the transmembrane part of the pore for (A) α1, (B) α1G254A, and (C) α2/β GlyR’s. A longitudinal view of the pore is shown; the front subunit is not displayed to reveal the interior of the pore. The T/S13’ and G/A/P2’ regions are marked with dashed lines. A higher percentage of binding poses is observed in the 13’ region for the first two receptors, while for the heteromeric α2/β GlyR binding poses are more dispersed all over the 16’–2’ region. The percentage of binding poses (between parentheses) where the sulfonate is placed in the 13’ region is 60%, 60%, and 65% for α1, α1G254A, and α2/β GlyR’s, respectively. Only the the heteromeric α2/β GlyR shows a significant percentage of poses with the sulfonate bound at 2’ (down configuration), i.e., 34%. Download Figure 7-4, TIF file.
Extended Data Figure 7-5
Interactions of cis-Azo-NZ1 in the “up” binding pose for (A) α1, (B) α1G254A, and (C) α2/β GlyRs. A longitudinal view of the pore is shown, with the pore lining residues represented as ball-and-sticks and their carbon atoms are colored in grey. Cis-Azo-NZ1 is represented as sticks with carbon atoms in purple. Nitrogen and oxygen atoms are colored in blue and red, respectively. Hydrogen bonds are marked with red dashed lines and hydrophobic interactions are represented as yellow surfaces. Download Figure 7-5, TIF file.
Extended Data Figure 7-6
Interactions of cis-Azo-NZ1 in the “down” binding pose for (A) α1, (B) α1G254A, and (C) α2/β GlyRs. Representations and color code are the same as in Extended Data Figure 7-5. Note that there is no significant sulfonate density at position 2’ for wild-type α1 GlyR (Extended Data Figure 7-4A), and thus, we expect that the probability of having cis-Azo-NZ1 bound in the down configuration for this receptor is very low. Download Figure 7-6, TIF file.
Extended Data Figure 7-7
Density map of the sulfonate group of Azo-NZ1 resulting from the blind docking to (A, B) α1 and (C, D) α2 GlyRs. Regions of continuous density correspond to higher sulfonate occupancy and indicate tighter sulfonate binding. Each contour line corresponds to 0.0006 particle/nm3, where “particle” refers to the sulfonate group in a given binding pose and is used to follow the putative interaction regions of Azo-NZ1. Longitudinal view of the receptor: the front subunit is not displayed for the sake of clarity. The pore region (between positions S/T16’ and G/A2’), as well as the interface region between the ECD-TMD, are marked with dashed lines. The residue at position 2’ is displayed as black spheres. Trans-Azo-NZ1 binds preferentially inside the 16’–2’ region for both GlyRs (37% and 46% for α1 and α2 GlyRs, respectively). Instead, cis-Azo-NZ1 shows two possible interaction sites, either inside the pore or at the ECD-TMD interface. The relative percentage of poses in the two regions varies significantly between the two GlyR types. For α1 GlyR, cis-Azo-NZ1 has a higher probability to interact with the ECD-TMD interface (35%) than with the pore (28%). In contrast, for α2 GlyR, the sulfonate density in the interface region is more dispersed, and the associated probability is lower (28%) than the one corresponding to binding in the pore (46%). Download Figure 7-7, TIF file.
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