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Figure 1
Reduction in glycine gated GlyRα1 currents in oocytes co-expressed with GlyTs. GlyRα1 is activated by 10 μM glycine (white bars). When flow of the solution is stopped on cells (black bars) expressing only GlyRα1 there is no change in current, however stopping the flow on oocytes co-expressing GlyRα1 with either GlyT2 or GlyT1 immediately causes a substantial decrease in GlyRα1 current amplitude. Resumption of flow restores the full amplitude of the currents. Peak current values are also decreased in co-expressed oocytes (note scales).
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Figure 2
Reduction of current amplitude by GlyTs in co-expressed cells is glycine concentration dependent. Glycine concentration-dependent modulation of GlyRs by GlyTs. (A,D) Raw Istop current trace examples of cells expressing GlyRα1/GlyT1 or GlyRα1/GlyT2 in the presence of varying concentrations of glycine. (B,E) Istop/Iflow ratios for varying concentrations of glycine. (C,F) Stop-flow and fast-flow (same cell) glycine dose-responses for co-expressed cells. Currents were normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM (n = 5).
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Figure 3
The impact of GlyT expression levels on GlyRα1 currents. Example traces from co-expressed oocytes expressing different (A) GlyT1 and (D) GlyT2 densities showing normalised Istop/Iflow reduction of GlyRα1 currents (B,E) Histograms show increasing the ratio of GlyRα1: GlyT cRNA injected in oocytes increases the normalised Istop/Iflow ratio when co-expressed with GlyT2 but not GlyT1. Significance was tested using a one-way ANOVA and Dunnett’s post-hoc test and **** indicates p < 0.0001. No significant difference was found between values for different GlyT1 ratios. (C,F) Glycine dose responses for GlyRα1 and GlyRα1/GlyT1 or GlyRα1/GlyT2 at 1:3 and 1:10 injected cRNA ratios. Fast flow currents were normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM, n = 5.
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Figure 4
Reduction of stop-flow and fast-flow current amplitudes in co-expressed cells is dependent on GlyT transportable substrate. β-alanine dose-response curves in cells expression GlyRα1, GlyRα1/GlyT1 (A) or GlyRα1/GlyT2 (B) are superimposed, showing no shift in GlyRα1 sensitivity when a non-substrate of GlyTs is used as an agonist for GlyR. Currents were normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM (n ≥ 5)
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Figure 5
Reducing Na+ in the superfusing solution prevents stop-flow and fast-flow (peak) current reduction of by GlyTs. Example traces from cells co-expressing (A) GlyRα1/GlyT1 or (D) GlyRα1/GlyT2 showing stop-flow and fast-flow changes in current when 10 µM glycine (grey bars) is superfused in solutions of different Na+ concentrations. (B,E) Fast-flow currents are larger in low Na+ perfusate for both co-expressed cell types, however the absolute change is larger in GlyRα1/GlyT1. In GlyRα1/GlyT1, GlyT1 can still uptake glycine when flow is stopped on the same oocyte (black bar) in 10 mM Na+ solution and transport is reversed when flow is stopped (grey bar) in 1 mM Na+ solution (A, centre and right, C). In contrast, stop-flow uptake by GlyT2 is prevented when glycine is applied to the same GlyRα1/GlyT2 oocyte in 10 mM and 1 mM Na+ superfusing solutions (D, centre and right, F). Data is mean ± SEM, n ≥ 5. 10 mM and 1 mM Na+ values were compared to 96 mM Na+ values and were significance was tested using a paired t-test. * denotes p ≤ 0.05, ** denotes p ≤ 0.01, and **** denotes p ≤ 0.0001.
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Figure 6
GlyTs also modulate the activity of GlyRα3 and GlyRα3β. Glycine dose responses are shifted to the right and EC50 values for (A) GlyRα1, (B) GlyRα1β, (C) GlyRα3 and (D) GlyRα3β are increased when co-expressed with GlyT1 or GlyT2. Currents were normalised to Imax and fit to the Hill equation. Symbols represent mean ± SEM, n = 5.
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Figure 7
Glycine concentration sensed at the membrane of oocytes expressing GlyRα1/GlyTs. Since currents are reduced under fast-flow conditions, the glycine concentration at the membrane under fast-flow conditions in co-expressed oocytes was estimated by substituting EC50 and Hill coefficient values from cells expressing only GlyRα1, and current values from GlyRα1/GlyT1 or GlyRα1/GlyT2 co-expressed oocytes into the reversed Hill equation. The light blue line represents the glycine concentration sensed at the membrane when no transporters are present. Curves for GlyRα1/GlyT1 and GlyRα1/GlyT2 were fit by linear regression and values represent mean ± SEM, n ≥ 5.
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Figure 8
Pharmacological manipulation of stop-flow and fast-flow currents in GlyRα1/GlyT co-expressed cells. Example traces from cells co-expressing (A) GlyRα1/GlyT1 or (D) GlyRα1/GlyT2 showing co-application of glycine with GlyT1 inhibitor ALX-5407 or GlyT2 inhibitor ORG-25543, respectively (grey bars) prevents stop flow reduction (black bars) of glycine gated currents. Fast-flow currents are also larger when GlyT inhibitors are applied for both co-expressed cell types, suggesting GlyTs also reduce extracellularly available glycine under fast-flow conditions. Istop/Iflow values were compared between application of 10 µM glycine and 10 µM glycine in the presence of a GlyT inhibitor in the same cell expressing (B) GlyRα1/GlyT1 or (E) GlyRα1/GlyT2. ALX5-407 and ORG-25543 reduce the EC50s for glycine for cells co-expressing GlyRα1 and (C) GlyT1 and (F) GlyT2, respectively. Glycine dose-response curves for co-expressed cells in the presence of GlyT inhibitors are superimposed on dose-response curves for GlyRα1 alone, suggesting the effects of GlyTs in this system can be pharmacologically reversed. Currents are normalised to Imax and fit to the Hill equation. Data are presented as mean ± SEM (n ≥ 5). Values in the same cell were compared using a paired t-test. ** denotes p ≤ 0.01 and *** denotes p ≤ 0.001.
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Figure 9
GlyTs create an apparent change in GlyRα1 Hill co-efficient. Glycine dose response curves for GlyRα1, GlyT1 and GlyT2 showing the region in which GlyTs are most effective, i.e., their EC50, is asymmetrical across the GlyRα1 curve. Currents are normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM (n ≥ 5).
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Figure 10
Activity of C18-cis-ω9-L-methionine, C18-cis-ω7-glycine and C18-cis-ω9-glycine on GlyT2 and GlyRα1 separately. (A) 30 µM glycine transport currents mediated by GlyT2 were measured in the presence of lipids in a range of concentrations. Concentration-inhibition curves for C18-cis-ω9-L-methionine, C18-cis-ω7-glycine and C18-cis-ω9-glycine. Normalised response in the presence of 1 µM lipid is marked by dashed blue, green and red lines respectively. Data from [15]. (B) Modulation of GlyRα1 currents at glycine EC5 by 1 µM C18-cis-ω9-L-methionine, C18-cis-ω7-glycine and C18-cis-ω9-glycine were previously measured. (C) Example traces of GlyRα1 modulation by lipids. Currents were normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM (n ≥ 3). Data from [18].
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Figure 11
Pharmacological manipulation of fast-flow currents in GlyRα1 and GlyRα1/GlyT co-expressed cells by bioactive lipids. (A) C18-cis-ω9-L-methionine, (B) C18-cis-ω7-glycine and (C) C18-cis-ω9-glycine modulate the EC50s for glycine in cells expressing GlyRα1 alone and co-expressed with GlyT2. Currents were normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM (n = 5).
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Figure 12
Glycine concentration sensed at the membrane of cells expressing GlyRα1/GlyT2 in the presence of GlyT2 specific inhibitors. Dashed line indicates reference 1:1 relationship of [Gly]bath and [Gly]membrane. Since currents are reduced under fast-flow conditions, the glycine concentration at the membrane under fast-flow conditions in co-expressed cells was estimated by substituting EC50 and Hill co-efficient values from cells expressing only GlyRα1, and current values from GlyRα1/GlyT2 co-expressed cells into the Hill equation (pink lines). Similarly, the glycine concentration at the membrane under fast-flow conditions in co-expressed cells and in the presence of (A) the GlyT2 inhibitor, C18-cis-ω9-L-methionine (orange), (B) the GlyR PAM, C18-cis-ω7-glycine (purple) and (C) the dual action modulator C18-cis-ω9-glycine (magenta) was estimated by substituting EC50 and Hill co-efficient values from cells expressing GlyRα1/GlyT2, and current values from GlyRα1/GlyT2 + lipid into the Hill equation. Curves for GlyRα1/GlyT2 and GlyRα1/GlyT2 + lipid were fit by linear regression and values represent mean ± SEM, n ≥ 5.
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Figure 13
Schematic of inhibitory glycinergic synapse showing effects of modulation at GlyT2 and GlyRs. (A) Prolonged complete inhibition of GlyT2 (yellow) leads to steep reductions in presynaptic glycine concentrations, the lack of presynaptic vesicle filling and termination of subsequent glycine release. (B) Partial, non-competitive GlyT2 inhibition slows the re-uptake of glycine (red spheres), which will improve the activation of glycine gated GlyRs (blue) while still allowing refilling of vesicles to occur and subsequent glycine release into the synapse to be maintained. (C) Partial GlyT2 inhibition and GlyR potentiation by dual-action compounds (black spheres) will increase synaptic glycine concentrations and allow the recycling of glycine through the presynaptic terminal by GlyT2, as in B, while also directly increasing activation of GlyRs.
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Figure 1. Reduction in glycine gated GlyRα1 currents in oocytes co-expressed with GlyTs. GlyRα1 is activated by 10 μM glycine (white bars). When flow of the solution is stopped on cells (black bars) expressing only GlyRα1 there is no change in current, however stopping the flow on oocytes co-expressing GlyRα1 with either GlyT2 or GlyT1 immediately causes a substantial decrease in GlyRα1 current amplitude. Resumption of flow restores the full amplitude of the currents. Peak current values are also decreased in co-expressed oocytes (note scales).
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Figure 2. Reduction of current amplitude by GlyTs in co-expressed cells is glycine concentration dependent. Glycine concentration-dependent modulation of GlyRs by GlyTs. (A,D) Raw Istop current trace examples of cells expressing GlyRα1/GlyT1 or GlyRα1/GlyT2 in the presence of varying concentrations of glycine. (B,E) Istop/Iflow ratios for varying concentrations of glycine. (C,F) Stop-flow and fast-flow (same cell) glycine dose-responses for co-expressed cells. Currents were normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM (n = 5).
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Figure 3. The impact of GlyT expression levels on GlyRα1 currents. Example traces from co-expressed oocytes expressing different (A) GlyT1 and (D) GlyT2 densities showing normalised Istop/Iflow reduction of GlyRα1 currents (B,E) Histograms show increasing the ratio of GlyRα1: GlyT cRNA injected in oocytes increases the normalised Istop/Iflow ratio when co-expressed with GlyT2 but not GlyT1. Significance was tested using a one-way ANOVA and Dunnett’s post-hoc test and **** indicates p < 0.0001. No significant difference was found between values for different GlyT1 ratios. (C,F) Glycine dose responses for GlyRα1 and GlyRα1/GlyT1 or GlyRα1/GlyT2 at 1:3 and 1:10 injected cRNA ratios. Fast flow currents were normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM, n = 5.
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Figure 4. Reduction of stop-flow and fast-flow current amplitudes in co-expressed cells is dependent on GlyT transportable substrate. β-alanine dose-response curves in cells expression GlyRα1, GlyRα1/GlyT1 (A) or GlyRα1/GlyT2 (B) are superimposed, showing no shift in GlyRα1 sensitivity when a non-substrate of GlyTs is used as an agonist for GlyR. Currents were normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM (n ≥ 5).
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Figure 5. Reducing Na+ in the superfusing solution prevents stop-flow and fast-flow (peak) current reduction of by GlyTs. Example traces from cells co-expressing (A) GlyRα1/GlyT1 or (D) GlyRα1/GlyT2 showing stop-flow and fast-flow changes in current when 10 µM glycine (grey bars) is superfused in solutions of different Na+ concentrations. (B,E) Fast-flow currents are larger in low Na+ perfusate for both co-expressed cell types, however the absolute change is larger in GlyRα1/GlyT1. In GlyRα1/GlyT1, GlyT1 can still uptake glycine when flow is stopped on the same oocyte (black bar) in 10 mM Na+ solution and transport is reversed when flow is stopped (grey bar) in 1 mM Na+ solution (A, centre and right, C). In contrast, stop-flow uptake by GlyT2 is prevented when glycine is applied to the same GlyRα1/GlyT2 oocyte in 10 mM and 1 mM Na+ superfusing solutions (D, centre and right, F). Data is mean ± SEM, n ≥ 5. 10 mM and 1 mM Na+ values were compared to 96 mM Na+ values and were significance was tested using a paired t-test. * denotes p ≤ 0.05, ** denotes p ≤ 0.01, and **** denotes p ≤ 0.0001.
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Figure 6. GlyTs also modulate the activity of GlyRα3 and GlyRα3β. Glycine dose responses are shifted to the right and EC50 values for (A) GlyRα1, (B) GlyRα1β, (C) GlyRα3 and (D) GlyRα3β are increased when co-expressed with GlyT1 or GlyT2. Currents were normalised to Imax and fit to the Hill equation. Symbols represent mean ± SEM, n = 5.
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Figure 7. Glycine concentration sensed at the membrane of oocytes expressing GlyRα1/GlyTs. Since currents are reduced under fast-flow conditions, the glycine concentration at the membrane under fast-flow conditions in co-expressed oocytes was estimated by substituting EC50 and Hill coefficient values from cells expressing only GlyRα1, and current values from GlyRα1/GlyT1 or GlyRα1/GlyT2 co-expressed oocytes into the reversed Hill equation. The light blue line represents the glycine concentration sensed at the membrane when no transporters are present. Curves for GlyRα1/GlyT1 and GlyRα1/GlyT2 were fit by linear regression and values represent mean ± SEM, n ≥ 5.
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Figure 8. Pharmacological manipulation of stop-flow and fast-flow currents in GlyRα1/GlyT co-expressed cells. Example traces from cells co-expressing (A) GlyRα1/GlyT1 or (D) GlyRα1/GlyT2 showing co-application of glycine with GlyT1 inhibitor ALX-5407 or GlyT2 inhibitor ORG-25543, respectively (grey bars) prevents stop flow reduction (black bars) of glycine gated currents. Fast-flow currents are also larger when GlyT inhibitors are applied for both co-expressed cell types, suggesting GlyTs also reduce extracellularly available glycine under fast-flow conditions. Istop/Iflow values were compared between application of 10 µM glycine and 10 µM glycine in the presence of a GlyT inhibitor in the same cell expressing (B) GlyRα1/GlyT1 or (E) GlyRα1/GlyT2. ALX5-407 and ORG-25543 reduce the EC50s for glycine for cells co-expressing GlyRα1 and (C) GlyT1 and (F) GlyT2, respectively. Glycine dose-response curves for co-expressed cells in the presence of GlyT inhibitors are superimposed on dose-response curves for GlyRα1 alone, suggesting the effects of GlyTs in this system can be pharmacologically reversed. Currents are normalised to Imax and fit to the Hill equation. Data are presented as mean ± SEM (n ≥ 5). Values in the same cell were compared using a paired t-test. ** denotes p ≤ 0.01 and *** denotes p ≤ 0.001.
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Figure 9. GlyTs create an apparent change in GlyRα1 Hill co-efficient. Glycine dose response curves for GlyRα1, GlyT1 and GlyT2 showing the region in which GlyTs are most effective, i.e., their EC50, is asymmetrical across the GlyRα1 curve. Currents are normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM (n ≥ 5).
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Figure 10. Activity of C18-cis-ω9-L-methionine, C18-cis-ω7-glycine and C18-cis-ω9-glycine on GlyT2 and GlyRα1 separately. (A) 30 µM glycine transport currents mediated by GlyT2 were measured in the presence of lipids in a range of concentrations. Concentration-inhibition curves for C18-cis-ω9-L-methionine, C18-cis-ω7-glycine and C18-cis-ω9-glycine. Normalised response in the presence of 1 µM lipid is marked by dashed blue, green and red lines respectively. Data from [15]. (B) Modulation of GlyRα1 currents at glycine EC5 by 1 µM C18-cis-ω9-L-methionine, C18-cis-ω7-glycine and C18-cis-ω9-glycine were previously measured. (C) Example traces of GlyRα1 modulation by lipids. Currents were normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM (n ≥ 3). Data from [18].
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Figure 11. Pharmacological manipulation of fast-flow currents in GlyRα1 and GlyRα1/GlyT co-expressed cells by bioactive lipids. (A) C18-cis-ω9-L-methionine, (B) C18-cis-ω7-glycine and (C) C18-cis-ω9-glycine modulate the EC50s for glycine in cells expressing GlyRα1 alone and co-expressed with GlyT2. Currents were normalised to Imax and fit to the Hill equation. Symbols are mean ± SEM (n = 5).
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Figure 12. Glycine concentration sensed at the membrane of cells expressing GlyRα1/GlyT2 in the presence of GlyT2 specific inhibitors. Dashed line indicates reference 1:1 relationship of [Gly]bath and [Gly]membrane. Since currents are reduced under fast-flow conditions, the glycine concentration at the membrane under fast-flow conditions in co-expressed cells was estimated by substituting EC50 and Hill co-efficient values from cells expressing only GlyRα1, and current values from GlyRα1/GlyT2 co-expressed cells into the Hill equation (pink lines). Similarly, the glycine concentration at the membrane under fast-flow conditions in co-expressed cells and in the presence of (A) the GlyT2 inhibitor, C18-cis-ω9-L-methionine (orange), (B) the GlyR PAM, C18-cis-ω7-glycine (purple) and (C) the dual action modulator C18-cis-ω9-glycine (magenta) was estimated by substituting EC50 and Hill co-efficient values from cells expressing GlyRα1/GlyT2, and current values from GlyRα1/GlyT2 + lipid into the Hill equation. Curves for GlyRα1/GlyT2 and GlyRα1/GlyT2 + lipid were fit by linear regression and values represent mean ± SEM, n ≥ 5.
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Figure 13. Schematic of inhibitory glycinergic synapse showing effects of modulation at GlyT2 and GlyRs. (A) Prolonged complete inhibition of GlyT2 (yellow) leads to steep reductions in presynaptic glycine concentrations, the lack of presynaptic vesicle filling and termination of subsequent glycine release. (B) Partial, non-competitive GlyT2 inhibition slows the re-uptake of glycine (red spheres), which will improve the activation of glycine gated GlyRs (blue) while still allowing refilling of vesicles to occur and subsequent glycine release into the synapse to be maintained. (C) Partial GlyT2 inhibition and GlyR potentiation by dual-action compounds (black spheres) will increase synaptic glycine concentrations and allow the recycling of glycine through the presynaptic terminal by GlyT2, as in B, while also directly increasing activation of GlyRs.
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