Repository logo
 

Complexities of a protonatable substrate in measurements of Hoechst 33342 transport by multidrug transporter LmrP

Published version
Peer-reviewed

Change log

Authors

Swain, Brendan M. 
Guo, Dawei 
Singh, Himansha 
Rawlins, Philip B. 
McAlister, Mark 

Abstract

Abstract: Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior. In transport assays, Hoechst 33342 (referred to as Hoechst) is a commonly used substrate, the fluorescence of which changes in the transport process. With three basic nitrogen atoms that can be protonated, Hoechst can exist as cationic and neutral species that have different fluorescence emissions and different abilities to diffuse across cell envelopes and interact with lipids and intracellular nucleic acids. Due to this complexity, the mechanism of Hoechst transport by multidrug transporters is poorly characterised. We investigated Hoechst transport by the bacterial major facilitator superfamily multidrug-proton antiporter LmrP in Lactococcus lactis and developed a novel assay for the direct quantitation of cell-associated Hoechst. We observe that changes in Hoechst fluorescence in cells do not always correlate with changes in the amount of Hoechst. Our data indicate that chemical proton gradient-dependent efflux by LmrP in cells converts populations of highly fluorescent, membrane-intercalated Hoechst in the alkaline interior into populations of less fluorescent, cell surface-bound Hoechst in the acidic exterior. Our methods and findings are directly relevant for the transport of many amphiphilic antibiotics, antineoplastic agents and cytotoxic compounds that are differentially protonated within the physiological pH range.

Description

Funder: AstraZeneca PhD studentship


Funder: China Scholarship Council; doi: http://dx.doi.org/10.13039/501100004543


Funder: Cambridge Trust; doi: http://dx.doi.org/10.13039/501100003343

Keywords

Article, /631/326/22/1434, /631/326/88, article

Journal Title

Scientific Reports

Conference Name

Journal ISSN

2045-2322

Volume Title

10

Publisher

Nature Publishing Group UK
Sponsorship
Biotechnology and Biological Sciences Research Council (BB/R00224X/1, BB/R00224X/1)
Relationships
Supplements: