Mutations in the sixth transmembrane domain of P-glycoprotein that alter the pattern of cross-resistance also alter sensitivity to cyclosporin A reversal.
The expression of a P-glycoprotein (Pgp1) cDNA encoding two amino acid substitutions in the sixth transmembrane domain of the protein (G338A339 to A338P339) confers a unique cross-resistance profile that displays preferential resistance to actinomycin D and diminished resistance to colchicine and daunorubicin. We report here that this multidrug-resistant phenotype is also insensitive to reversal by cyclosporin A (CsA) but not verapamil (VRP). However, the ability of VRP to increase the accumulation of [3H]vincristine is poor in both wild-type and mutant transfectants. In contrast, the accumulation of [3H]vincristine in wild-type versus mutant transfectants in the presence of CsA is dramatically increased. It is the substitution of the alanine residue at position 339 with proline that is primarily responsible for the lowered sensitivity to CsA and for the altered drug accumulation levels. Both substitutions are required to confer the unique cross-resistance profile of the double mutant, although each independently confers a specific profile of its own. These results indicate that alterations in Pgp1 structure can differentially affect the activity of CsA and VRP to mediate drug accumulation in multidrug-resistant cells and support the conclusion that the sixth transmembrane domain of the Pgp1 transporter plays important roles, in both the specificity of drug efflux and the sensitivity of the transporter to reversal agents.
Ma
J F
JF
Department of Biochemistry and Molecular Biology, The Graduate Program in Molecular and Cell Biology, University of Maryland School of Medicine, Baltimore 21201, USA.
Grant
G
G
Melera
P W
PW
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000493:pharmacokinetics; D001665:Binding Sites; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D016572:Cyclosporine / Q000494:pharmacology*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D018432:Drug Resistance, Multiple; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D014162:Transfection; D014316:Tritium; D014750:Vincristine / Q000493:pharmacokinetics
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000972:Antineoplastic Agents, Phytogenic; D018076:DNA, Complementary; D014316:Tritium; D014750:Vincristine; D016572:Cyclosporine