P-GLYCOPROTEIN IS A DIMER IN THE KIDNEY AND BRAIN CAPILLARY MEMBRANES- EFFECT OF CYCLOSPORINE-A AND SDZ-PSC-833

Radiation-inactivation studies were performed in order to elucidate th e oligomeric nature of P-glycoprotein (P-gp) expressed in brain capill aries and renal brush border membranes (BBMs), Irradiation of renal BB Ms 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 [ I-125]arylazidoprazosin labeling, respectively. Similar TSs were deter mined for P-gp expressed in brain capillaries. These TSs correspond to approximately twice the size (120 kDa) of deglycosylated P-gp. Furthe rmore, the estimated TS for P-gp was not significantly different when renal BBMs were incubated with SDZ-PSC 833 (PC) prior and during expos ure to ionizing radiation. To confirm these results, the size of P-gp was evaluated from its mobility on blue-native polyacrylamide gels fol lowed by Western blot analysis. Using this method, an apparent molecul ar size of 334 and 264 kDa was determined for P-gp in brain capillarie s and renal BBMs, respectively. This corresponds to approximately twic e the size of the glycosylated monomeric subunit of P-gp in brain capi llaries (162 kDa) or renal BBMs (140 kDa). P-gp expressed in renal BBM s 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 resistan ce modulators such as CsA and PSC do not alter its oligomeric state.