MUTAGENESIS OF TRANSMEMBRANE DOMAIN-11 OF P-GLYCOPROTEIN BY ALANINE SCANNING

The biochemical and genetic analyses of P-glycoprotein (P-gp) have ind icated that the membrane-associated regions of P-gp play an important role in drug recognition and drug transport. Predicted transmembrane d omain 11 (TM11) maps near a major drug binding site revealed by photoa ffinity labeling, and mutations in this domain alter the substrate spe cificity of P-gp. To investigate further the role of TM11 in P-gp func tion in general, and substrate specificity in particular, each of the 21 residues of TM11 of the P-gp isoform encoded by the mouse mdr3 gene was independently mutated to alanine, or to glycine in the case of en dogenous alanines. After transfection and overexpression in Chinese ha mster ovary cells, pools of stable transfectants were analyzed for qua litative or quantitative deviations from the profile of resistance to vinblastine, adriamycin, colchicine, and actinomycin D displayed by th e wild-type protein. While mutations at eight of the positions had no effect on P-gp function, 13 mutants showed a 2-10-fold reduction of ac tivity against one of the four drugs tested, Although the phenotype of individual mutants was varied, replacements at most mutation-sensitiv e positions seemed to affect the drug resistance profiles rather than the overall activity of the mutant P-gp. When TM11 was projected in a alpha-helical configuration, the distribution of deleterious and neutr al mutations was not random but segregated with a more hydrophobic (mu tation-insensitive) face and a more hydrophilic (mutation-sensitive) f ace of a putative amphipathic helix. The alternate clustering pattern of deleterious vs neutral mutations in TM11 together with the altered drug resistance profile of deleterious mutants suggest that the more h ydrophilic face of the TM11 helix may play an important structural or functional role in drug recognition and transport by P-gp. Finally, th e conservation of the two residues most sensitive to mutations (Y949 a nd F953) in TM11, and in the homologous TM5, of all mammalian P-gps an d also in other ABC transporters, suggests that these residues and dom ains may play an important role in structural as well as mechanistic a spects common to this family of proteins.