A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Wild Type (WT) and Mutant RNA Transcription
2.3. Oocyte Preparation and Injection
2.4. Two-Electrode Voltage Clamp Electrophysiology
2.5. Substrate and Agonist Concentration Responses
2.6. Stop-Flow Recording
2.7. Data Analysis
3. Results
3.1. Glycine-Gated GlyR Peak Currents and Stopped-Flow Currents Are Reduced in Co-expressed Oocytes
3.2. Stop-Flow and Fast-Flow Reduction of Glycine Gated Currents Are Concentration Dependent
3.3. Glycine Transporter Density Affects Degree of GlyR Modulation and Is Dependent on the Presence of a GlyT Transportable Substrate
3.4. Stop-Flow and Fast-Flow Reduction of Current in GlyRα1 Is Reliant on Na+ Dependent GlyT Driving Force
3.5. Estimation of Glycine Sensed at the Membrane by GlyRs in Cells Co-Expressing GlyTs
3.6. Pharmacological Inhibition of GlyTs Reverses Stop-Flow and Fast-Flow Changes to GlyR Activation Profiles
3.7. Co-Expression of GlyTs with GlyRs Changes Hill Co-Efficient Values
3.8. Novel, Bioactive Lipid Modulators of GlyRα1 and GlyT2
3.9. Estimation of the Apparent Glycine Concentration Sensed at the Membrane by GlyRα1 in GlyRα1/GlyT2 Co-Expressed Cells, in the Presence of Lipid Modulators
4. Discussion
4.1. The Impact of GlyTs on GlyR Function
4.2. Pharmacological Modulation of the GlyR/GlyT Co-Expression System
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Istop/Iflow | Peak Current (nA) | |
---|---|---|
GlyRα1 | 0.98 ± 0.01 | 740.6 ± 105.9 |
GlyRα1/GlyT1 | 0.29 ± 0.04 **** | 62.3 ± 7.9 **** |
GlyRα1/GlyT2 | 0.23 ± 0.02 **** | 150.7 ± 14.2 **** |
Fast-Flow Glycine EC50 (µM) | 95% CI | nH | Stop-Flow Glycine EC50 (µM) | 95% CI | nH | |
---|---|---|---|---|---|---|
GlyRα1 | 13.1 | 12.1–14.4 | 3.2 ± 0.4 | – | – | – |
GlyRα1/ GlyT1 | 47.3 | 45.0–49.7 | 3.7 ± 0.2 | 88.3 **** | 85.2–91.4 | 4.9 ± 0.4 |
GlyRα1/ GlyT2 | 30.9 | 27.8–34.6 | 3.0 ± 0.47 | 83.1 **** | 80.0–86.2 | 4.0 ± 0.3 |
Peak Current (nA) | 96 mM Na+ | 10 mM Na+ | 1 mM Na+ |
---|---|---|---|
GlyRα1/GlyT1 | 395.2 ± 27.0 | 664.8 ± 52.6 * | 723.2 ± 46.6 ** |
GlyRα1/GlyT2 | 204.1 ± 88.6 | 596.4 ± 120.8 ** | 1084 ± 182.6 ** |
Istop/Iflow | 96 mM Na+ | 10 mM Na+ | 1 mM Na+ |
GlyRα1/GlyT1 | 0.31 ± 0.06 | 0.58 ± 0.05 * | 1.32 ± 0.06 **** |
GlyRα1/GlyT2 | 0.23 ± 0.03 | 0.95 ± 0.01 **** | 0.97 ± 0.01 **** |
Fast-Flow Glycine EC50 (µM) | 95% CI | |
---|---|---|
GlyRα1 | 13.2 | 12.9–14.3 |
GlyRα1/GlyT1 | 48.8 **** | 46.6–51.0 |
GlyRα1/GlyT2 | 41.6 **** | 38.8–44.7 |
GlyRα1β | 14.7 | 12.6–16.9 |
GlyRα1β/GlyT1 | 39.1 **** | 37.0–41.4 |
GlyRα1β/GlyT2 | 36.8 **** | 34.7–39.0 |
GlyRα3 | 64.8 | 59.0–70.9 |
GlyRα3/GlyT1 | 153.9 **** | 143.7–164.6 |
GlyRα3/GlyT2 | 160.2 **** | 145.7–175.8 |
GlyRα3β | 109.4 | 102.4–117.1 |
GlyRα3β/GlyT1 | 219.7 **** | 209.2–230.8 |
GlyRα3β/GlyT2 | 224.5 **** | 208.6–241.5 |
Istop/Iflow | Glycine EC50 (μM) | 95% CI | nH | |
---|---|---|---|---|
GlyRα1 | 11.2 | 10.0–12.4 | 2.2 ± 0.2 | |
GlyRα1/GlyT1 | 0.39 ± 0.05 | 48.8 **** | 46.6–51.0 | 3.8 ± 0.2 *** |
+ ALX-5407 | 0.97 ± 0.01 ** | 11.3 ns | 10.0–12.3 | 2.0 ± 0.2 ns |
GlyRα1/GlyT2 | 0.33 ± 0.04 | 41.6 **** | 38.8–44.7 | 3.2 ± 0.3 ns |
+ ORG-25543 | 0.90 ± 0.04 *** | 13.2 ns | 11.7–14.6 | 2.6 ± 0.4 ns |
Lipid | Structure | GlyRα1 Potentiation at EC5 (%) | GlyT2 Activity |
---|---|---|---|
C18-cis-ω9-L-methionine | 2.2 ± 4.2 (6) | % Inhibition (1 µM) 84.9 ± 0.07 (4) IC50 29.2 nM Max inhibition (%) 91.2 | |
C18-cis-ω7-glycine | 184.7 ± 41.3 (5) | % Inhibition (1 µM) 10.5 ± 0.02 (4) IC50 >10 µM Max inhibition (%) – | |
18-cis-ω9-glycine | 84.7 ± 14.0 (8) | % Inhibition (1 µM) 89.6 ± 0.01 (10) IC50 31 nM Max inhibition (%) 95.0 |
Glycine EC50 (µM) | 95% CI | nH | |
---|---|---|---|
GlyRα1 | 14.8 | 14.1–15.6 | 3.4 ± 0.2 |
+ C18-cis-ω9-L-methionine | 12.8 ns | 11.2–14.7 | 2.2 ± 0.3 ns |
GlyRα1/GlyT2 | 27.9 **** | 26.5–29.3 | 3.3 ± 0.2 |
+ C18-cis-ω9-L-methionine | 17.4 * | 15.7–19.5 | 2.6 ± 0.4 ns |
GlyRα1 | 13.2 | 12.2–14.3 | 3.2 ± 0.3 |
+ C18-cis-ω7-glycine | 9.3 **** | 8.6–10.0 | 2.8 ± 0.3 ns |
GlyRα1/GlyT2 | 27.3 **** | 23.6–31.4 | 3.1 ± 0.5 |
+ C18-cis-ω7-glycine | 21.7 **** | 19.7–24.4 | 3.0 ± 0.5 ns |
GlyRα1 | 15.7 | 15.1–16.4 | 3.5 ± 0.2 |
+ C18-cis-ω9-glycine | 13.4 ns | 12.4–14.5 | 2.4 ± 0.2 ns |
GlyRα1/GlyT2 | 34.1 **** | 29.6–39.4 | 2.6 ± 0.3 |
+ C18-cis-ω9-glycine | 18.0 * | 16.1–20.2 | 2.7 ± 0.4 ns |
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Sheipouri, D.; Gallagher, C.I.; Shimmon, S.; Rawling, T.; Vandenberg, R.J. A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors. Biomolecules 2020, 10, 1618. https://doi.org/10.3390/biom10121618
Sheipouri D, Gallagher CI, Shimmon S, Rawling T, Vandenberg RJ. A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors. Biomolecules. 2020; 10(12):1618. https://doi.org/10.3390/biom10121618
Chicago/Turabian StyleSheipouri, Diba, Casey I. Gallagher, Susan Shimmon, Tristan Rawling, and Robert J. Vandenberg. 2020. "A System for Assessing Dual Action Modulators of Glycine Transporters and Glycine Receptors" Biomolecules 10, no. 12: 1618. https://doi.org/10.3390/biom10121618