Transport of polypeptide ionophores into proteoliposomes reconstituted with rat liver P-glycoprotein.
The aim of this study was to examine the peptide transport activity of a naturally occurring P-glycoprotein such as that present in rat liver canalicular membrane vesicles. The peptide ionophores valinomycin and gramicidin D, which are known substrates of P-glycoprotein, served to monitor the P-glycoprotein activity indirectly as the ATP-dependent uptake of 86Rb+ mediated by these ionophores. Canalicular membrane vesicles proved inherently permeable to K+ ions, which prevented assay of transport ionophore activity. Therefore, P-glycoprotein was extracted from canalicular membrane vesicles and reconstituted into proteoliposomes that are relatively impermeable to cations. P-glycoprotein activity in the proteoliposomes was dependent on ATP hydrolysis since it was not observed with non-hydrolyzable analogs of ATP. Maximal ATP-dependent 86Rb+ uptake occurred at 50 nM gramicidin D and at 500 nM valinomycin thus possibly reflecting higher affinity of P-glycoprotein for gramicidin D. Nigericin, which does not participate in the multidrug resistance phenomenon, did not support an ATP-dependent uptake of 86Rb+. ATP hydrolysis increased the amount of 86RB+ transported into the proteoliposomes. Furthermore, preincubation of the proteoliposomes in the presence of gramicidin D and 86Rb+, allowing for maximal ATP-independent 86Rb+ uptake to occur, did not interfere with subsequent ATP-dependent uptake, indicating the latter to constitute an active transport mechanism. The ATP-dependent component of 86Rb+ uptake occurred neither with liposomes nor with proteoliposomes reconstituted with proteins extracted from sinusoidal vesicles that lack P-glycoprotein. The ATP-dependent uptake was blocked by the known inhibitors of the ATPase activity associated with P-glycoprotein, oligomycin and vanadate, as well as by its established substrates, daunorubicin, doxorubicin, vinblastine, and the tripeptide N-acetyl-leucyl-leucyl-norleucinal. Thus, the reconstituted P-glycoprotein catalyzes the ATP-dependent 86Rb+ uptake that appears to occur by an energy-dependent translocation of the 86Rb(+)-ionophore complex. In this case, the actual substrate of P-glycoprotein is the ionophore-cation complex, which is both hydrophobic and positively charged as are most of the substrates of P-glycoprotein. This is the first demonstration of transport of a naturally occurring polypeptide by proteoliposomes reconstituted with physiologically expressed P-glycoprotein.
Eytan
G D
GD
Department of Biology, Technion-Israel Institute of Technology, Haifa.
Borgnia
M J
MJ
Regev
R
R
Assaraf
Y G
YG
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D006096:Gramicidin / Q000493:pharmacokinetics*; D008099:Liver / Q000378:metabolism*; D008297:Male; D011510:Proteolipids / Q000378:metabolism*; D051381:Rats; D012414:Rubidium Radioisotopes / Q000378:metabolism; D014634:Valinomycin / Q000493:pharmacokinetics*; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011510:Proteolipids; D012414:Rubidium Radioisotopes; C020485:proteoliposomes; D006096:Gramicidin; D014634:Valinomycin; D000255:Adenosine Triphosphate; D014700:Verapamil