Functional modulation of multidrug resistance-related P-glycoprotein by Ca(2+)-calmodulin.
Studies with inside-out plasma membrane vesicles from multidrug-resistant (MDR 3) murine erythroleukemia (MEL/VCR-6) cells have provided evidence for down-modulation of P-glycoprotein (P-gp) function by Ca(2+)-calmodulin (CLM). These studies showed that CLM in the presence or absence of Ca2+ had no effect on binding of [3H]vinblastine (VBL) by P-gp in inside-out plasma membrane vesicles. However, profound inhibition of ATP-dependent [3H]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 microM Ca2+ reduced the inhibition of [3H]VBL efflux by CLM, shifting the concentration required for inhibition to the nM range (IC50 = 2.55 +/- 0.35 nM). The inhibition of as 0.01 mM Ca2+, and no inhibition occurred with concentrations greater than 0.2 mM Ca2+. Binding of CLM, itself, to P-gp was demonstrated in two ways. The P-gp content 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 Western blotting with anti-P-gp antibody (C219) before and after CLM-Sepharose treatment. Also, depletion of P-gp from solution by CLM was less in the presence of 1 mM Ca2+. Blotting of P-gp after SDS-PAGE of plasma membrane from MEL/VCR-6 cells was also obtained using 125I-CLM as a probe. These results strongly suggest that the MDR 3 homolog of P-gp is a CLM-binding protein and that direct interaction of Ca(2+)-CLM with P-gp, while not affecting its binding of [3H]VBL, down-modulates the translocation of this agent in the presence of ATP.
Schlemmer
S R
SR
Program of Molecular Pharmacology and Experimental Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Yang
C H
CH
Sirotnak
F M
FM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D018432:Drug Resistance, Multiple; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014747:Vinblastine / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002147:Calmodulin; D014747:Vinblastine; D002118:Calcium