Amino acid substitutions in the first transmembrane domain (TM1) of P-glycoprotein that alter substrate specificity

Recently, we showed that the amino acid at position 61 in TM1 of human P-glycoprotein is important in deciding the substrate specificity of this protein. In this work, we investigated whether the amino acids other than His(61) in TM1 of P-glycoprotein are also essential in the function of this protein, Nine amino acids residues, from Ala(57) to Leu(65) in TM1, were independently substituted to Arg, and analyzed the drug resistance of cells stably expressing each of these mutant P-glycoproteins, The mutant P-glycoproteins Ile(60) --> Arg, His(61) --> Arg, Ala(63) --> Arg, Gly(64) --> Arg, and Leu(65) --> Arg were normally processed and expressed in the plasma membrane, Substrate specificities of mutant P-glycoproteins Gly(64) --> Arg and Leu(65) --> Arg were quite different from that of the wild type, and similar to that of the His(61) --> Arg mutant, while the Ile(60) --> Arg and Ala(63) --> Arg mutant P-glycoproteins showed similar substrate specificities to that of the wild-type P-glycoprotein, suggesting that not only the amino acid residue at position 61 but also those at position 64 and 65 are also important in deciding the substrate specificity of P-glycoprotein. These three amino acids His(61), Gly(64), and Leu(65) would form a compact region on an alpha-helix arrangement of TM1, These results suggest that a region consisting of His(61), Gly(64), and Leu(65) in TM1 would participate in the formation of the recognition site for substrates of P-glycoprotein. (C) 1997 Federation of European Biochemical Societies.