RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities
Abstract
:1. Introduction
2. Result
2.1. Identification of New Mutations That Enhance Aequorin Ca2+ Sensitivity
2.2. Generating Redquorin Mutants with Ca2+ Sensitivity and Light Emission Rate That Match Those of GA
2.3. Effects of Key Amino Acid Mutations on Redquorin Thermostability
2.4. RedquorinXS Mutants Efficiently Report the Activation of P2Y2 Receptor in CHO Cells
2.5. Imaging Neuronal Network Activities in Brain Slice with a RedquorinXS Mutant
3. Discussion
3.1. Apoaequorin Mutations That Enhance Aequorin Ca2+ Sensitivity
3.2. AequorinXS Mutations Primarily Enhance Redquorin Sensitivity to Low Ca2+
3.3. RedquorinXS Sensors Report Intracellular Ca2+ Signals with High Sensitivity in Cell Lines and in Brain Slice
4. Materials and Methods
4.1. Site-Directed Mutagenesis of Apo-Aequorin and Redquorin
4.2. Production and Purification of Apophotoproteins from Mammalian HEK Cells
4.3. Protein Expression and Purification from E. coli
4.4. Aequorin Reconstitution for In Vitro Assays
4.5. Functional Analysis of Aequorin Mutants and Fusions In Vitro
4.6. Functional Analysis of Photoproteins in CHO Cell Lines
4.7. Bioluminescence Imaging of Photoproteins in Mouse Neocortical Slices
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Mutation | Ca2+ Sensitivity, EC50 (nM) a | Relative Intensity a | Decay Kinetics, t1/2 (ms) b | Emission Peak Wavelength (nm) | ||
---|---|---|---|---|---|---|
at pCa 6.5 | at pCa 7.2 | |||||
Aeq-wt | 659 ± 23 | 1 | 1 | 906 ± 53 | 476 | |
Single mutants with medium-high to high Ca2+ sensitivity | Q159D | 330 ± 8 | 25.1 | 23.4 | 794 ± 42 | 478 |
Q159T | 310 ± 13 | 16.6 | 4.8 | 866 ± 23 | 477 | |
S157D | 322 ± 20 | 6.0 | 2.2 | 835 ± 47 | 475 | |
N121D | 400 ± 14 | 4.9 | 2.1 | 957 ± 30 | 477 | |
A123D | 374 ± 9 | 3.6 | 1.6 | 862 ± 48 | 476 | |
Q159G | 492 ± 10 | 3.3 | 3.2 | 830 ± 37 | 478 | |
A179T | 330 ± 19 | 3.0 | 1.3 | 779 ± 33 | 477 | |
Double mutants with high Ca2+ sensitivity | QD+AT | 216 ± 26 | 58.5 | 60.8 | 750 ± 50 | 478 |
QD+ND | 295 ± 17 | 29.0 | 41.7 | 738 ± 28 | 477 | |
QD+AD | 271 ± 15 | 26.2 | 15.0 | 612 ± 26 | 475 | |
QT+AT | 281 ± 25 | 29.0 | 14.0 | 650 ± 68 | 478 | |
QT+ND | 337 ± 10 | 14.4 | 12.2 | 680 ± 55 | 478 | |
QT+AD | 279 ± 16 | 22.8 | 10.0 | 620 ± 33 | 476 |
Mutation | Ca2+ Sensitivity, EC50 (nM) a | Relative Intensity a | Decay Kinetics, t1/2 (ms) b | Emission Peak Wavelength (nm) | ||
---|---|---|---|---|---|---|
at pCa 6,5 | at pCa 7,2 | |||||
CLZ-native | Redq | 859 ± 45 | 1.0 | 1.0 | 1 203 ± 70 | 582 |
Redq/Q159D | 680 ± 33 | 9.1 | 13.2 | 980 ± 66 | 582 | |
Redq/QD+AT | 515 ± 20 | 32.1 | 24.5 | 913 ± 39 | 582 | |
Redq/QD+AD | 621 ± 44 | 10.8 | 12.8 | 1 120 ± 46 | 582 | |
Redq/Q159T | 478 ± 39 | 13.0 | 7.1 | 1 103 ± 50 | 582 | |
Redq/QT+AT | 605 ± 16 | 18.3 | 14.1 | 990 ± 87 | 582 | |
Redq/QT+AD | 577 ± 33 | 14.4 | 10.0 | 950 ± 56 | 582 | |
CitA | 573 ± 45 | 24 | 9.5 | 794 ± 46 | 529 | |
GA | 630 ± 29 | 6.8 | 3.2 | 852 ± 64 | 509 | |
CLZ-f | Redq | 600 ± 35 | 1.0 | 1.0 | 880 ± 68 | 582 |
Redq/Q159D | 290 ± 18 | 23.9 | 14.9 | 770 ± 88 | 582 | |
Redq/QD+AT | 252 ± 40 | 58.5 | 48.3 | 740 ± 55 | 582 | |
Redq/QD+AD | 300 ± 26 | 22.8 | 15.0 | 750 ± 66 | 582 | |
Redq/Q159T | 296 ± 13 | 11.0 | 2.8 | 705 ± 36 | 582 | |
Redq/QT+AT | 284 ± 30 | 25.6 | 9.0 | 810 ± 61 | 582 | |
Redq/QT+AD | 336 ± 11 | 14.4 | 7.4 | 670 ± 50 | 582 | |
CitA | 266 ± 18 | 41.1 | 18.7 | 680 ± 72 | 529 | |
GA | 463 ± 46 | 8.6 | 2.9 | 700 ± 48 | 509 |
Ca2+ Sensor Variant | [ATP] CL (nM) | EC50 (µM) | Z-Factor |
---|---|---|---|
Redq/Q159T | 310 | 1.7 ± 0.2 | 0.76 |
Redq/Q159D | 320 | 2.2 ± 0.1 | 0.68 |
Redq/QD+AT | 548 | 2.0 ± 0.2 | 0.67 |
Redq/QT+AD | 1025 | 3.0 ± 0.4 | 0.82 |
Redq | 2033 | 4.0 ± 0.3 | 0.56 |
GA | 640 | 1.5 ± 0.2 | 0.69 |
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Bakayan, A.; Picaud, S.; Malikova, N.P.; Tricoire, L.; Lambolez, B.; Vysotski, E.S.; Peyriéras, N. RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities. Int. J. Mol. Sci. 2020, 21, 7846. https://doi.org/10.3390/ijms21217846
Bakayan A, Picaud S, Malikova NP, Tricoire L, Lambolez B, Vysotski ES, Peyriéras N. RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities. International Journal of Molecular Sciences. 2020; 21(21):7846. https://doi.org/10.3390/ijms21217846
Chicago/Turabian StyleBakayan, Adil, Sandrine Picaud, Natalia P. Malikova, Ludovic Tricoire, Bertrand Lambolez, Eugene S. Vysotski, and Nadine Peyriéras. 2020. "RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities" International Journal of Molecular Sciences 21, no. 21: 7846. https://doi.org/10.3390/ijms21217846