The transmembrane domain 10 of the yeast Pdr5p ABC antifungal efflux pump determines both substrate specificity and inhibitor susceptibility

We have previously shown that a S1360F mutation in transmembrane domain 10 (TMD10) of the Pdr5p ABC transporter modulates substrate specificity and si multaneously leads to a loss of FK506 inhibition. In this study, we have co nstructed and characterized the S1360F/A/T and T1364F/A/S mutations located in the hydrophilic face of the amphipatic Pdr5p TMD10. A T1364F mutation l eads to a reduction in Pdr5p-mediated azole and rhodamine 6G resistance. Li ke S1360F, the T1364F and T1364A mutants were nearly non-responsive to FK50 6 inhibition. Most remarkably, however, the S1360A mutation increases FK506 inhibitor susceptibility, because Pdr5p-S1360A is hypersensitive to FK506 inhibition when compared with either wild-type Pdr5p or the non-responsive S1360F variant. Hence, the Pdr5p TMD10 determines both azole substrate spec ificity and susceptibility to reversal agents. This is the first demonstrat ion of a eukaryotic ABC transporter where a single residue change causes ei ther a loss or a gain in inhibitor susceptibility, depending on the nature of the mutational change. These results have important implications for the design of efficient reversal agents that could be used to overcome multidr ug resistance mediated by ABC transporter overexpression.