2.3.1. Q725A Improves the Transport of BODIPY-Verapamil in Any Background

The challenge with BODIPY-verapamil gave a clearer indication that the side chain of the native Gln<sup>725</sup> inhibits transport activity (Figure 4). Comparing the raw transport data of the Q725A single mutant with the wild-type transporter, it is statistically clear that Q725A increased the ability to efflux BODIPY-verapamil. This relationship is maintained for all mutants that include Q725A compared to the backbone into which the mutation was introduced. Thus, Q347/725A is statistically more active for the transport of BODIPYverapamil compared to Q347A. Likewise, Q725/990A is more active than Q990A, and the Qtriple mutant is more active than Q347/Q990A.

− ≥ < < **Figure 4.** Functionality of glutamine to alanine mutants for the transport of BODIPY-verapamil. Live HEK293T cells transiently expressing equivalent amounts of wild-type (wt) and mutant ABCB1 were challenged with BODIPY-verapamil. Functionality was measured as the ratio of BODIPY-verapamil accumulation between the ABCB1-expressing and untransfected cells within the population. This was normalized to 100% for wild-type ABCB1 for the bar graph shown. The mean ± SEM was plotted using GraphPad Prism version 8; sample number was ≥3. Selected statistical analysis (ratio of paired Student's *t*-test, two-tailed) performed on the raw data is shown with *p* values: \* <0.05, \*\* <0.01. The full pairwise comparison of the data is given in Appendix A Table A2.
