2.2.3. The Q725A Mutation Improves the Efficiency of Transport of Calcein-AM

The most surprising result is that the mutation of glutamine 725 to alanine improves the functionality of ABCB1 for the transport of Calcein-AM. This is most clearly evident when the Q725A mutation is introduced into the Q347/990A background to generate the Qtriple mutant. The Qtriple mutant has a transport activity of 50.7 ± 4.0% while the Q347/990A double mutant has a transport activity of 8.8 ±1.9%. The *p* value for this comparison by Student's *t*-test is 0.0055, strongly suggesting that the inclusion of the Q725A mutation has made the Qtriple mutant more active than the Q347/990A double mutant and at the same time emphasizing that neither Gln<sup>347</sup> nor Gln<sup>990</sup> are absolutely necessary for ABCB1 to transport Calcein-AM. The ostensible increase in the mean activities of the other three constructs that include the Q725A mutation when compared to their respective backbones (wild-type versus Q725A, Q347A versus Q347/725A and Q990A versus Q725/990A) fail to reach statistical significance (Table A1).
