Single Amino Acid Substitution in Loop1 Switches the Selectivity of α-Conotoxin RegIIA towards the α7 Nicotinic Acetylcholine Receptor
Abstract
:1. Introduction
2. Results
2.1. Synthesis and Identification of the Native RegIIA and Its Analogues
2.2. Alanine Scanning of Loop1 Revealed His5 and Pro6 as a Critical Point for RegIIA’s Selectivity
2.3. H5S and H5V Mutation Led to Significant Selectivity Increase of RegIIA towards rα7 nAChR
2.4. Molecular Modeling of RegIIA Binding to Rat α3β2, α3β4, α7 nAChRs
2.5. Circular Dichroism Analysis of the Native RegIIA and Its Analogues
2.6. Serum Stability of RegIIA and Its Analogues
3. Discussion
4. Materials and Methods
4.1. Materials and Animals
4.2. Synthesis of α-Conotoxin RegIIA and Its Analogues
4.3. cRNA Preparation and Injection
4.4. Electrophysiology
4.5. Date Analysis
4.6. Circular Dichroism Spectroscopy
4.7. Serum Stability Assay
4.8. AlphaFold 3-Based Molecular Modeling
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
References
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Peptides | rα3β2 | rα3β4 | rα7 | |||
---|---|---|---|---|---|---|
IC50 (nM) a | Hillslope a | IC50 (nM) a | Hillslope a | IC50 (nM) a | Hillslope a | |
RegIIA | 36 (28–44) | 1.0 (0.8–1.2) | 102 (78–129) | 0.9 (0.8–1.2) | 43 (26–62) | 0.8 (0.6–1.0) |
[G1A]RegIIA | 90 (51–138) | 0.7 (0.5–0.9) | 132 (102–165) | 1.0 (0.8–1.3) | 286 (226–364) | 1.1 (0.9–1.4) |
[S4A]RegIIA | 34 (26–42) | 0.9 (0.7–1.0) | 95 (73–121) | 0.9 (0.7–1.1) | 39 (25–56) | 0.8 (0.6–1.0) |
[H5A]RegIIA | >10,000 b | ND c | >10,000 b | ND c | 446 (327–574) | 1.0 (0.8–1.4) |
[P6A]RegIIA | >10,000 b | ND c | >10,000 b | ND c | 459 (317–617) | 1.2 (0.8–2.2) |
[A7G]RegIIA | >10,000 b | ND c | 964 (814–1152) | 1.1 (0.9–1.5) | 600 (488–731) | 0.9 (0.8–1.1) |
Peptides | rα3β2 | rα3β4 | rα7 | |||
---|---|---|---|---|---|---|
IC50 (nM) a | Hillslope a | IC50 (nM) a | Hillslope a | IC50 (nM) a | Hillslope a | |
[H5S]RegIIA | >10,000 b | ND c | 5732 (3975–8006) | 0.8 (0.6–1.0) | 100 (76–128) | 0.9 (0.7–1.1) |
[H5V]RegIIA | >10,000 b | ND c | 8800 (6094–12,240) | 0.9 (0.7–1.4) | 97 (77–119) | 1.0 (0.8–1.3) |
[H5L]RegIIA | >10,000 b | ND c | >10,000 b | ND c | >10,000 b | ND c |
[H5G]RegIIA | >10,000 b | ND c | >10,000 b | ND c | 3539 (2846–4416) | 1.2 (1.0–1.4) |
[H5E]RegIIA | >10,000 b | ND c | >10,000 b | ND c | 441 (335–552) | 1.1 (0.9–1.5) |
[P6O]RegIIA | >10,000 b | ND c | >10,000 b | ND c | >10,000 b | ND c |
Peptides | α-Helix (%) | β-Sheet (%) | β-Turn (%) | Random Coil (%) |
---|---|---|---|---|
RegIIA | 10% | 31% | 25% | 33% |
[H5A]RegIIA | 19% | 21% | 26% | 35% |
[H5L]RegIIA | 9% | 31% | 25% | 34% |
[H5S]RegIIA | 18% | 22% | 26% | 34% |
[H5V]RegIIA | 20% | 20% | 28% | 32% |
[P6A]RegIIA | 12% | 30% | 24% | 33% |
[P6O]RegIIA | 13% | 29% | 25% | 34% |
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Yu, J.; Xie, J.; Ma, Y.; Wei, P.; Zhang, P.; Tang, Z.; Zhu, X.; Zhangsun, D.; Luo, S. Single Amino Acid Substitution in Loop1 Switches the Selectivity of α-Conotoxin RegIIA towards the α7 Nicotinic Acetylcholine Receptor. Mar. Drugs 2024, 22, 390. https://doi.org/10.3390/md22090390
Yu J, Xie J, Ma Y, Wei P, Zhang P, Tang Z, Zhu X, Zhangsun D, Luo S. Single Amino Acid Substitution in Loop1 Switches the Selectivity of α-Conotoxin RegIIA towards the α7 Nicotinic Acetylcholine Receptor. Marine Drugs. 2024; 22(9):390. https://doi.org/10.3390/md22090390
Chicago/Turabian StyleYu, Jinpeng, Junjie Xie, Yuting Ma, Pengcheng Wei, Panpan Zhang, Zepei Tang, Xiaopeng Zhu, Dongting Zhangsun, and Sulan Luo. 2024. "Single Amino Acid Substitution in Loop1 Switches the Selectivity of α-Conotoxin RegIIA towards the α7 Nicotinic Acetylcholine Receptor" Marine Drugs 22, no. 9: 390. https://doi.org/10.3390/md22090390