ABCB1 Does Not Require the Side-Chain Hydrogen-Bond Donors Gln347, Gln725, Gln990 to Confer Cellular Resistance to the Anticancer Drug Taxol
Abstract
:1. Introduction
2. Results
2.1. Endpoint Two-Colour Flow Cytometry Assay Measures the Function of ABCB1 in Live Cells
2.2. Gln347, Gln725 and Gln990 Modulate the Transport of the Xanthene Dye Calcein-AM
2.2.1. Of the Single Mutants, Only Q347A Shows a Statistically Significant Reduction in Calcein-AM Transport
2.2.2. Q347A and Q990A Act Synergistically to Reduce the Transport of Calcein-AM
2.2.3. The Q725A Mutation Improves the Efficiency of Transport of Calcein-AM
2.3. Gln347, Gln725 and Gln990 Also Modulate the Transport of the Phenylalkylamine BODIPY-Verapamil
2.3.1. Q725A Improves the Transport of BODIPY-Verapamil in Any Background
2.3.2. Q347A and Q990A Also Act Synergistically to Reduce the Transport of BODIPY-Verapamil
2.4. Gln347, Gln725 and Gln990 Have a More Limited Effect on the Transport of the Taxane Diterpenoid Derivative OG-Taxol
2.4.1. Of the Single Mutants Only Q990A Appears to Reduce the Transport of OG-Taxol
2.4.2. The Double Mutants Q347/990A and Q725/990A Reduce the Transport Activity Further but the Triple Mutant Restores Wild-Type Levels of OG-Taxol Transport
2.4.3. There Is No Indication That Gln725 Is Inhibitory for the Transport of OG-Taxol
2.5. The Q347/990A and the Qtriple Mutant Are Indistinguishable from the Wild-Type Transporter in Conferring Taxol Resistance to Cells in Culture
3. Discussion
3.1. A Possible Allosteric Explanation for the Increased Transport Activity of Q725A
3.2. Induced Fit of the TMDs around the Transport Substrate Likely Explains the Lack of Importance of Gln347, Gln725 and Gln990 in Conferring Resistance to Taxol
4. Materials and Methods
4.1. Site-Directed Mutagenesis
4.2. Transient Expression of ABCB1
4.3. Drug Transport Assay
4.4. Stable Expression of ABCB1 in HEK293 Flp-in Cells
4.5. Taxol Survival Curve
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Wild Type 100% | E556/1201Q 1.3 ± 0.6 | Q347A 63.1 ± 8.0 | Q725A 122.3 ± 36.4 | Q990A 90.7 ± 12.8 | Q347/725A 92.1 ± 14.2 | Q347/990A 8.8 ± 1.8 | Q725/990A 75.1 ± 4.0 | |
---|---|---|---|---|---|---|---|---|
E556/1201Q 1.3 ± 0.6 | <0.0001 **** | - | ||||||
Q347A 63.1 ± 8.0 | 0.0363 * | 0.0061 ** | - | |||||
Q725A 122.3 ± 36.4 | ns | 0.0379 * | ns | - | ||||
Q990A 90.7 ± 12.8 | ns | 0.0025 ** | 0.0485 * | ns | - | |||
Q347/725A 92.1 ± 14.2 | ns | 0.0002 *** | ns | ns | ns | - | ||
Q347/990A 8.8 ± 1.8 | 0.0097 ** | 0.0024 ** | 0.0025 ** | 0.0358 * | 0.0031 ** | 0.0010 ** | - | |
Q725/990A 75.1 ± 4.0 | 0.0301 * | 0.0005 *** | ns | ns | ns | ns | 0.0106 * | - |
Qtriple 50.7 ± 4.0 | 0.0131 * | 0.0031 ** | ns | ns | ns | 0.0296 * | 0.0055 ** | ns |
Wild Type 100% | E556/1201Q 10.5 ± 1.6 | Q347A 81.4 ± 7.7 | Q725A 140.5 ± 12.3 | Q990A 105.6 ± 16.3 | Q347/725A 136.3 ± 10.0 | Q347/990A 37.9 ± 10.1 | Q725/990A 151.3 ± 44.3 | |
---|---|---|---|---|---|---|---|---|
E556/1201Q 10.5 ± 1.6 | <0.0001 **** | - | ||||||
Q347A 81.4 ± 7.7 | ns | 0.0007 *** | - | |||||
Q725A 140.5 ± 12.3 | 0.0410 * | <0.0001 **** | 0.0061 ** | - | ||||
Q990A 105.6 ± 16.3 | ns | 0.0026 ** | ns | ns | - | |||
Q347/725A 136.3 ± 10.0 | ns | 0.0040 ** | 0.0089 ** | ns | 0.0422 * | - | ||
Q347/990A 37.9 ± 10.1 | 0.0258 * | 0.0042 ** | ns | 0.0029 ** | ns | ns | - | |
Q725/990A 151.3 ± 44.3 | ns | 0.0032 ** | 0.0018 ** | 0.0286 * | 0.0182 * | ns | 0.0225 * | - |
Qtriple 115.7 ± 15.3 | ns | 0.0008 *** | 0.0022 ** | ns | ns | ns | 0.0300 * | ns |
Wild Type 100 % | E556/1201Q 2.0 ± 0.2 | Q347A 104 ± 25 | Q725A 84 ± 10 | Q990A 66 ± 8 | Q347/725A 69 ± 11 | Q347/990A 48 ± 9 | Q725/990A 50 ± 2 | |
---|---|---|---|---|---|---|---|---|
E556/1201Q 2.0 ± 0.2 | 0.0075 ** | - | ||||||
Q347A 104 ± 25 | ns | 0.0148 * | - | |||||
Q725A 84 ± 10 | ns | 0.0032 ** | ns | - | ||||
Q990A 66 ± 8 | 0.0467 * | 0.0034 ** | ns | ns | - | |||
Q347/725A 69 ± 11 | ns | 0.0003 *** | ns | ns | ns | - | ||
Q347/990A 48 ± 9 | 0.0184 * | 0.0001 *** | ns | ns | ns | 0.0292 * | - | |
Q725/990A 50 ± 2 | 0.0290 * | 0.028 * | 0.0203 * | 0.0305 * | ns | 0.0234 * | ns | - |
Qtriple 82 ± 13 | ns | 0.0231 * | ns | ns | ns | ns | ns | 0.027 * |
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Q347A | |
---|---|
Forward | 5′-ttaattggggcttttagtgttggagcggcatctccaagcat-3′ |
Reverse | 5′-atgcttggagatgccgctccaacactaaaagccccaattaa-3′ |
Q725A | |
Forward | 5′-gtgccattataaatggaggcctggcaccagcatttgcaataatatttt-3′ |
Reverse | 5′-aaaatattattgcaaatgctggtgccaggcctccatttataatggcac-3′ |
Q990A | |
Forward | 5′-gccatggccgtgggggcagtcagttcatttgc-3′ |
Reverse | 5′-gcaaatgaactgactgcccccacggccatggc-3′ |
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Sasitharan, K.; Iqbal, H.A.; Bifsa, F.; Olszewska, A.; Linton, K.J. ABCB1 Does Not Require the Side-Chain Hydrogen-Bond Donors Gln347, Gln725, Gln990 to Confer Cellular Resistance to the Anticancer Drug Taxol. Int. J. Mol. Sci. 2021, 22, 8561. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168561
Sasitharan K, Iqbal HA, Bifsa F, Olszewska A, Linton KJ. ABCB1 Does Not Require the Side-Chain Hydrogen-Bond Donors Gln347, Gln725, Gln990 to Confer Cellular Resistance to the Anticancer Drug Taxol. International Journal of Molecular Sciences. 2021; 22(16):8561. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168561
Chicago/Turabian StyleSasitharan, Keerthana, Hamzah Asad Iqbal, Foteini Bifsa, Aleksandra Olszewska, and Kenneth J. Linton. 2021. "ABCB1 Does Not Require the Side-Chain Hydrogen-Bond Donors Gln347, Gln725, Gln990 to Confer Cellular Resistance to the Anticancer Drug Taxol" International Journal of Molecular Sciences 22, no. 16: 8561. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22168561