P-glycoprotein is a dimer in the kidney and brain capillary membranes: effect of cyclosporin A and SDZ-PSC 833.
Radiation-inactivation studies were performed in order to elucidate the oligomeric nature of P-glycoprotein (P-gp) expressed in brain capillaries and renal brush border membranes (BBMs). Irradiation of renal BBMs resulted in a dose-dependent loss of P-gp, which corresponded to a target size (TS) of 255 and 211 kDa, as detected by Western blot and [125I]arylazidoprazosin labeling, respectively. Similar TSs were determined for P-gp expressed in brain capillaries. These TSs correspond to approximately twice the size (120 kDa) of deglycosylated P-gp. Furthermore, the estimated TS for P-gp was not significantly different when renal BBMs were incubated with SDZ-PSC 833 (PSC) prior and during exposure to ionizing radiation. To confirm these results, the size of P-gp was evaluated from its mobility on blue-native polyacrylamide gels followed by Western blot analysis. Using this method, an apparent molecular size of 334 and 264 kDa was determined for P-gp in brain capillaries and renal BBMs, respectively. This corresponds to approximately twice the size of the glycosylated monomeric subunit of P-gp in brain capillaries (162 kDa) or renal BBMs (140 kDa). P-gp expressed in renal BBMs isolated from rats which had been treated daily with cyclosporin A (CsA) or PSC also migrated as a 264 kDa protein. These results suggest that P-gp exists mainly as a dimer in normal tissues and that resistance modulators such as CsA and PSC do not alter its oligomeric state.
Jetté
L
L
Département de chimie-biochimie, Université du Québec à Montréal, Canada.
Potier
M
M
Béliveau
R
R
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000187:drug effects* / Q000528:radiation effects; D000818:Animals; D001921:Brain / Q000098:blood supply* / Q000187:drug effects / Q000201:enzymology; D002196:Capillaries / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology; D016572:Cyclosporine / Q000494:pharmacology*; D003524:Cyclosporins / Q000494:pharmacology*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005720:Gamma Rays; D007668:Kidney / Q000737:chemistry* / Q000187:drug effects; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D011487:Protein Conformation / Q000187:drug effects / Q000528:radiation effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D005723:gamma-Glutamyltransferase / Q000737:chemistry
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D003524:Cyclosporins; D046911:Macromolecular Substances; D016572:Cyclosporine; D005723:gamma-Glutamyltransferase; C070272:valspodar