Coordinate changes in drug resistance and drug-induced conformational transitions in altered-function mutants of the multidrug transporter P-glycoprotein

The MDRI P-glycoprotein (Pgp), responsible for a clinically important form of multidrug resistance in cancer, is an ATPase efflux pump for multiple li pophilic drugs. The G185V mutation near transmembrane domain 3 of human Pgp increases its relative ability to transport several drugs, including etopo side, but decreases the transport of other substrates. MDRI cDNA with the C 185V substitution was used in a function-based selection to identify mutati ons that would further increase Pgp-mediated resistance to etoposide. This selection yielded the I186N substitution, adjacent to G185V. Pgps with G185 V, I186N, or both mutations were compared to the wild-type Pgp for their ab ility to confer resistance to different drugs in NIH 3T3 cells. In contrast to the differential effects of G185V, I186N mutation increased resistance to all the tested drugs and augmented the effect of G185V on etoposide resi stance. The effects of the mutations on conformational transitions of Pgp i nduced by different drugs were investigated using a conformation-sensitive antibody UIC2. Ligand-binding analysis of the drug-induced increase in UIC2 reactivity was used to determine the K-m value that reflects the apparent affinity of drugs for Pgp, and the Hill number reflecting the apparent numb er of drug-binding sites. Both mutations altered the magnitude of drug-indu ced increases in UIC2 immunoreactivity, the K-m values, and the Hill number s for individual drugs. Mutation-induced changes in the magnitude of UIC2 r eactivity shift did not correlate with the effects of the mutations on resi stance to the corresponding drugs. In contrast, an increase or a decrease i n drug resistance relative to that of the wild type was accompanied by a co rresponding increase or decrease in the K-m or in both the K-m and the Hill number. These results suggest that mutations that alter the ability of Pgp to transport individual drugs change the apparent affinity and the apparen t number of drug-binding sites in Pgp.