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The secondary multidrug transporter LmrP contains multiple drug interaction sites
M Putman1, L A Koole, H W van Veen
1Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Abstract:
The secondary multidrug transporter LmrP of Lactococcus lactis mediates the efflux of Hoechst 33342 from the cytoplasmic leaflet of the membrane. Kinetic analysis of Hoechst 33342 transport in inside-out membrane vesicles of L. lactis showed that the LmrP-mediated H(+)/Hoechst 33342 antiport reaction obeyed Michaelis-Menten kinetics, with a low apparent affinity constant of 0.63 microM Hoechst 33342 (= 0.5 mmol Hoechst 33342/mol phospholipid). Several drugs significantly inhibited LmrP-mediated Hoechst 33342 transport through a direct interaction with the protein rather than through dissipation of the proton motive force or reduction of the membrane partitioning of Hoechst 33342. The characterization of the mechanism of inhibition of LmrP-mediated Hoechst 33342 transport indicated competitive inhibition by quinine and verapamil, noncompetitive inhibition by nicardipin and vinblastin, and uncompetitive inhibition by TPP(+). The three types of inhibition of LmrP-mediated Hoechst 33342 transport in inside-out membrane vesicles indicate for the first time the presence of multiple drug interaction sites in a secondary multidrug transporter.
Insights
The secondary multidrug transporter LmrP in Lactococcus lactis facilitates Hoechst 33342 efflux. This study reveals LmrP has multiple drug interaction sites, demonstrated by varied inhibition patterns from different drugs.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- The secondary multidrug transporter LmrP from Lactococcus lactis is known to export molecules like Hoechst 33342.
- Understanding the substrate interaction and inhibition mechanisms of multidrug transporters is crucial for combating antimicrobial resistance and improving drug efficacy.
Purpose of the Study:
- To characterize the kinetic properties of LmrP-mediated Hoechst 33342 transport.
- To investigate the interaction sites and inhibition mechanisms of various drugs on LmrP function.
Main Methods:
- Utilized inside-out membrane vesicles from Lactococcus lactis to study LmrP activity.
- Performed kinetic analysis of Hoechst 33342 transport and inhibition assays with different drugs.
- Determined the type of inhibition (competitive, noncompetitive, uncompetitive) for selected inhibitors.
Main Results:
- LmrP-mediated H+/Hoechst 33342 antiport followed Michaelis-Menten kinetics with a low apparent affinity for Hoechst 33342 (0.63 microM).
- Several drugs, including quinine, verapamil, nicardipine, vinblastine, and TPP+, inhibited Hoechst 33342 transport through direct interaction with LmrP.
- Different drug classes exhibited distinct inhibition patterns: competitive (quinine, verapamil), noncompetitive (nicardipine, vinblastine), and uncompetitive (TPP+).
Conclusions:
- LmrP exhibits multiple drug binding sites, as evidenced by the diverse inhibition mechanisms observed.
- This finding provides novel insights into the allosteric regulation and substrate interaction of secondary multidrug transporters.
- The characterization of LmrP's multiple interaction sites has implications for the design of novel inhibitors and understanding multidrug resistance mechanisms.