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Purified human MDR 1 modulates membrane potential in reconstituted proteoliposomes
1Department of Chemistry, Lombardi Cancer Center Program in Tumor Biology, Georgetown University, 37th and O Streets, Washington, DC 20057, USA.
Biochemistry
|March 26, 2003
Summary
Human multidrug resistance (MDR1) protein, when reconstituted, does not pump protons. Instead, it facilitates ATP-dependent chloride transport, suggesting a passive anion transport mechanism rather than active pumping.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Transport
Background:
- The human multidrug resistance (MDR1) protein is a P-glycoprotein involved in drug efflux.
- Its precise transport mechanism, particularly ion transport, remains debated.
- Understanding MDR1 function is crucial for overcoming multidrug resistance in cancer therapy.
Purpose of the Study:
- To investigate the ion transport capabilities of purified and reconstituted human multidrug resistance (MDR1) protein.
- To elucidate the role of ATP in MDR1-mediated transport.
- To clarify the mechanism of MDR1 in relation to existing transport models.
Main Methods:
- Heterologous overexpression and purification of a human MDR1-transcarboxylase biotin acceptor domain (MDR-TCBD) fusion protein in yeast.
- Reconstitution of the purified protein into proteoliposomes (PLs) using E. coli lipids.
- Measurement of ATPase activity and membrane potential using fluorescent probes (oxonol and carbocyanine dyes) and ion gradients (K+/Na+).
- Assessment of ATP and chloride ion dependency for transport activity.
Main Results:
- The purified and reconstituted MDR-TCBD protein exhibited functional ATPase activity.
- MDR1-TCBD proteoliposomes did not catalyze measurable H+ pumping.
- A membrane potential dissipation was observed in MDR-TCBD PLs, dependent on ATP and chloride ions.
- Replacing chloride with glutamate abolished this dissipation, indicating chloride transport.
Conclusions:
- The human MDR1 protein functions as an electrically passive anion transporter, specifically for chloride ions.
- Transport is dependent on ATP, but not in an ATP-catalyzed pumping manner.
- These findings challenge active pumping models and support a model of ATP-modulated passive transport, potentially resolving conflicting data on MDR1 ion transport.