FUNCTIONAL MODULATION OF MULTIDRUG RESISTANCE-RELATED P-GLYCOPROTEIN BY CA2-CALMODULIN()

Studies with inside-out plasma membrane vesicles from multidrug-resist ant (MDR 3) murine erythroleukemia (MEL/VCR-6) cells have provided evi dence for down-modulation of P-glycoprotein (P-gp) function by Ca-2+-c almodulin (CLM). These studies showed that CLM in the presence or abse nce of Ca2+ had no effect on binding of [H-3]vinblastine (VBL) by P-gp in inside out plasma membrane vesicles. However, profound inhibition of ATP-dependent [H-3]VBL efflux by these vesicles was demonstrated by the addition of subnanomolar concentrations of CLM (IC50 = 0.15 +/- 0 .02 nM). The addition of 1 mM Ca2+ reduced the inhibition of [H-3]VBL efflux by CLM, shifting the concentration required for inhibition to t he nM range (IC50 = 2.55 +/- 0.35 nM). The inhibition of [H-3]VBL effl ux by 0.6 nM CLM was reduced with as little as 0.01 mM Ca2+, and no in hibition occurred with concentrations greater than 0.2 mM Ca2+. Bindin g of CLM, itself, to P-gp was demonstrated in two ways, The P-gp conte nt of detergent-solubilized plasma membrane from MEL/VCR-6 cells could be appreciably depleted by treating this material with CLM-Sepharose beads as shown by SDS-polyacrylamide gel electrophoresis (PAGE) and We stern blotting with anti-P-gp antibody (C219) before and after CLM-Sep harose treatment. Also, depletion of P-gp from solution by CLM was les s in the presence of 1 mM Ca2+. Blotting of P-gp after SDS-PAGE of pla sma membrane from MEL/VCR-6 cells was also obtained using I-125-CLM as a probe. These results strongly suggest that the MDR 3 homolog of P-g p is a CLM-binding protein and that direct interaction of Ca2+-CLM wit h P-gp, while not affecting its binding of [H-3]VBL, down-modulates th e translocation of this agent in the presence of ATP.