Expression of Mutant Glycine Receptors in Xenopus Oocytes Using Canonical and Non-Canonical Amino Acids Reveals Distinct Roles of Conserved Proline Residues
Abstract
:1. Introduction
2. Materials and Methods
2.1. Molecular Biology
2.2. Oocyte Preparation and RNA Injection
2.3. Electrophysiology
2.4. Structures
3. Results
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gly | Tau | |||||||
---|---|---|---|---|---|---|---|---|
pEC50, M | EC50 μM | nH | pEC50, M | EC50 μM | nH | % ImaxTau /ImaxGly | n | |
WT | 4.282 ± 0.05 | 52 | 2.3 ± 0.5 | 3.235 ± 0.05 | 583 | 1.7 ± 0.3 | 50 ± 4 | 5 |
P96A | 3.518 ± 0.03 * | 304 | 2.2 ± 0.3 | 2.612 ± 0.03 * | 2440 | 1.9 ± 0.2 | 36 ± 2 * | 5 |
P146A | 3.932 ± 0.05 * | 117 | 2.4 ± 0.7 | 2.850 ± 0.06 * | 1410 | 1.5 ± 0.3 | 39 ± 3 * | 5 |
P230A | 3.579 ± 0.03 * | 264 | 2.2 ± 0.4 | 2.925 ± 0.03 * | 1190 | 2.2 ± 0.4 | 20 ± 1 * | 5 |
Site | Substitution | pEC50, M | EC50, μM | nH | n |
---|---|---|---|---|---|
P30(α1β1L) | Pro | 3.948 ± 0.04 | 113 | 2.9 ± 1.2 | 6 |
CFP | 3.825 ± 0.06 | 150 | 2.8 ± 0.7 | 6 | |
TFP | 4.013 ± 0.05 | 97 | 2.7 ± 1.3 | 5 | |
Pip | 4.439 ± 0.03 * | 36 | 2.7 ± 0.7 | 4 | |
2MeP | 3.434 ± 0.06 * | 368 | 1.6 ± 0.3 | 5 | |
3MeP | 5.046 ± 0.05 * | 8.9 | 3.0 ± 2.7 | 4 | |
Vah | 4.684 ± 0.03 * | 21 | 3.8 ± 0.5 | 4 | |
P96 (loopA) | Pro | 3.951 ± 0.04 | 112 | 2.7 ± 1.1 | 5 |
CFP | 3.840 ± 0.05 | 145 | 3.2 ± 0.7 | 4 | |
TFP | 3.898 ± 0.03 | 127 | 2.9 ± 0.7 | 4 | |
Pip | 3.667 ± 0.02 * | 215 | 2.9 ± 0.3 | 4 | |
2MeP | 3.596 ± 0.03 * | 254 | 2.8 ± 0.5 | 4 | |
3MeP | 3.289 ± 0.02 * | 514 | 3.0 ± 0.2 | 4 | |
Vah | 4.029 ± 0.05 | 94 | 1.5 ± 0.3 | 4 | |
P146 (CysL) | Pro | 4.052 ± 0.02 | 89 | 2.5 ± 0.4 | 6 |
CFP | 4.350 ± 0.03 * | 45 | 3.7 ± 0.6 | 6 | |
TFP | 3.806 ± 0.05 * | 156 | 2.3 ± 0.4 | 5 | |
Pip | 4.279 ± 0.11 * | 53 | 1.9 ± 0.7 | 5 | |
2MeP | 3.568 ± 0.04 * | 270 | 2.2 ± 0.5 | 6 | |
3MeP | ND | ||||
Vah | NR | 6 | |||
P230 (M1) | Pro | 3.922 ± 0.06 | 119 | 2.5 ± 0.9 | 4 |
CFP | 4.131 ± 0.04 | 74 | 1.4 ± 0.2 | 4 | |
TFP | 3.904 ± 0.06 | 125 | 3.0 ± 1.4 | 4 | |
Pip | 4.166 ± 0.08 | 68 | 1.1 ± 0.2 | 4 | |
2MeP | 3.751 ± 0.08 | 177 | 1.6 ± 0.5 | 4 | |
3MeP | 4.471 ± 0.08 * | 34 | 1.1 ± 0.4 | 4 | |
Vah | 4.015 ± 0.02 | 97 | 2.6 ± 0.4 | 4 |
Gly | Tau | |||||||
---|---|---|---|---|---|---|---|---|
pEC50, M | EC50 μM | nH | pEC50, M | EC50 μM | nH | % ImaxTau /ImaxGly | n | |
Pro | 3.999 ± 0.03 | 100 | 3.2 ± 1.6 | 2.984 ± 0.07 | 1030 | 1.5 ± 0.3 | 43 ± 5 | 4 |
CFP | 4.213 ± 0.04 * | 61 | 3.0 ± 0.5 | 2.139 ± 0.03 * | 579 | 2.1 ± 0.2 | 71 ± 3 * | 3 |
Pip | 4.103 ± 0.05 * | 79 | 2.7 ± 0.9 | 3.121 ± 0.05 * | 757 | 1.8 ± 0.3 | 64 ± 7 * | 3 |
2MeP | 3.582 ± 0.02 * | 261 | 3.6 ± 0.2 | 2.497 ± 0.04 * | 3184 | 2.4 ± 0.6 | 18 ± 2 * | 3 |
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Lummis, S.C.R.; Dougherty, D.A. Expression of Mutant Glycine Receptors in Xenopus Oocytes Using Canonical and Non-Canonical Amino Acids Reveals Distinct Roles of Conserved Proline Residues. Membranes 2022, 12, 1012. https://doi.org/10.3390/membranes12101012
Lummis SCR, Dougherty DA. Expression of Mutant Glycine Receptors in Xenopus Oocytes Using Canonical and Non-Canonical Amino Acids Reveals Distinct Roles of Conserved Proline Residues. Membranes. 2022; 12(10):1012. https://doi.org/10.3390/membranes12101012
Chicago/Turabian StyleLummis, Sarah C. R., and Dennis A. Dougherty. 2022. "Expression of Mutant Glycine Receptors in Xenopus Oocytes Using Canonical and Non-Canonical Amino Acids Reveals Distinct Roles of Conserved Proline Residues" Membranes 12, no. 10: 1012. https://doi.org/10.3390/membranes12101012
APA StyleLummis, S. C. R., & Dougherty, D. A. (2022). Expression of Mutant Glycine Receptors in Xenopus Oocytes Using Canonical and Non-Canonical Amino Acids Reveals Distinct Roles of Conserved Proline Residues. Membranes, 12(10), 1012. https://doi.org/10.3390/membranes12101012