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Functional modulation of multidrug resistance-related P-glycoprotein by Ca(2+)-calmodulin
S R Schlemmer1, C H Yang, F M Sirotnak
1Program of Molecular Pharmacology and Experimental Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Abstract:
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.
Insights
Calcium-calmodulin (CLM) down-regulates P-glycoprotein (P-gp) function in multidrug-resistant cells. CLM inhibits vinblastine efflux but not binding, with calcium ions modulating this effect, suggesting direct P-gp interaction.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Multidrug resistance (MDR) in cancer is often mediated by P-glycoprotein (P-gp) efflux pumps.
- Understanding P-gp regulation is crucial for developing effective cancer therapies.
Purpose of the Study:
- To investigate the role of calcium-calmodulin (CLM) in modulating P-gp function.
- To elucidate the mechanism by which CLM affects P-gp-mediated drug transport.
Main Methods:
- Utilized inside-out plasma membrane vesicles from multidrug-resistant murine erythroleukemia (MEL/VCR-6) cells.
- Assessed [3H]vinblastine (VBL) binding and ATP-dependent VBL efflux in the presence of varying CLM and Ca2+ concentrations.
- Employed CLM-Sepharose beads and 125I-CLM for demonstrating direct CLM-P-gp binding via SDS-PAGE and Western blotting.
Main Results:
- CLM significantly inhibited ATP-dependent [3H]VBL efflux in a dose-dependent manner (IC50 = 0.15 nM).
- Calcium ions modulated CLM's inhibitory effect, reducing potency and shifting the IC50 to the nM range (2.55 nM) at 1 µM Ca2+.
- CLM binding to P-gp was confirmed, with reduced binding observed in the presence of 1 mM Ca2+.
- CLM did not affect [3H]VBL binding to P-gp.
Conclusions:
- P-glycoprotein is a direct calmodulin-binding protein.
- Ca2+-calmodulin directly interacts with P-gp to down-modulate vinblastine translocation, independent of drug binding.
- This interaction offers a potential target for overcoming P-gp-mediated multidrug resistance.