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

Abstract

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 α-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.