P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• P-glycoprotein (P-gp) is a key efflux transporter involved in multidrug resistance.
• Understanding P-gp substrate transport kinetics is crucial for drug development and overcoming resistance.

Purpose of the Study:

• To compare P-glycoprotein ATPase activity in intact cells versus membrane vesicles to elucidate substrate transport mechanisms.
• To investigate how passive membrane influx influences P-gp mediated active efflux.

Main Methods:

• Utilized inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells for comparative analysis.
• Assessed P-glycoprotein ATPase activity using six substrates with varying passive influx rates.

Main Results:

• Compounds with high passive influx showed similar ATPase activity in cells and vesicles, indicating efficient transport.
• Compounds with influx rates similar to efflux exhibited different ATPase profiles, with lower intracellular concentrations in cells.
• P-glycoprotein demonstrated the ability to manage passive influx, preventing substrate accumulation in the cytosol.

Conclusions:

• P-glycoprotein functions as a transporter for all tested compounds, with activity proportional to ATP hydrolysis.
• The transporter effectively limits substrate entry into the cytosol when passive influx is comparable to active efflux.
• This study provides insights into the dynamic interplay between passive diffusion and active transport by P-glycoprotein.