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Hoechst 33342 Is a Hidden "Janus" amongst Substrates for the Multidrug Efflux Pump LmrP.

Neuberger A, van Veen HW - PLoS ONE (2015)

Bottom Line: Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells.Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342.These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden "Janus" amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of "Hoechst 33342-like" drugs to cells and tissues in which these transporters are expressed.

No MeSH data available.


Related in: MedlinePlus

LmrP mediates active efflux of ethidium.The efflux experiments described in Fig 3 were performed with ethidium at a final concentration of 2 μM. Traces represent data obtained in three independent experiments using different batches of cells.
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pone.0141991.g004: LmrP mediates active efflux of ethidium.The efflux experiments described in Fig 3 were performed with ethidium at a final concentration of 2 μM. Traces represent data obtained in three independent experiments using different batches of cells.

Mentions: To measure dye efflux from preloaded cells (Figs 3A–3C and 4), cells were first ATP-depleted by washing once with 50 mM KPi (pH 7.0) supplemented with 5 mM MgSO4, and resuspending in the same KPi buffer containing 0.5 mM 2,4-dinitrophenol (DNP) [19]. Following incubation for 30 min at 30°C, cells were subsequently washed 3 times with ice-cold 50 mM KPi buffer (pH 7.0) containing 5 mM MgSO4, and re-suspended in this buffer to an OD660 of 5.0 and kept on ice. Cells were pre-loaded at 30°C with 0.1 μM Hoechst 33342 or 2 μM ethidium until the fluorescence emission reached a constant level. Glucose (25 mM) was then added as a source of metabolic energy to assess active substrate efflux. After 15 min of incubation in the presence of glucose, the cells were spun down in an Eppendorf 5415 D centrifuge at 16,168g for 5 min to measure the free Hoechst 33342 concentration in the culture supernatant (Fig 3D).


Hoechst 33342 Is a Hidden "Janus" amongst Substrates for the Multidrug Efflux Pump LmrP.

Neuberger A, van Veen HW - PLoS ONE (2015)

LmrP mediates active efflux of ethidium.The efflux experiments described in Fig 3 were performed with ethidium at a final concentration of 2 μM. Traces represent data obtained in three independent experiments using different batches of cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4634932&req=5

pone.0141991.g004: LmrP mediates active efflux of ethidium.The efflux experiments described in Fig 3 were performed with ethidium at a final concentration of 2 μM. Traces represent data obtained in three independent experiments using different batches of cells.
Mentions: To measure dye efflux from preloaded cells (Figs 3A–3C and 4), cells were first ATP-depleted by washing once with 50 mM KPi (pH 7.0) supplemented with 5 mM MgSO4, and resuspending in the same KPi buffer containing 0.5 mM 2,4-dinitrophenol (DNP) [19]. Following incubation for 30 min at 30°C, cells were subsequently washed 3 times with ice-cold 50 mM KPi buffer (pH 7.0) containing 5 mM MgSO4, and re-suspended in this buffer to an OD660 of 5.0 and kept on ice. Cells were pre-loaded at 30°C with 0.1 μM Hoechst 33342 or 2 μM ethidium until the fluorescence emission reached a constant level. Glucose (25 mM) was then added as a source of metabolic energy to assess active substrate efflux. After 15 min of incubation in the presence of glucose, the cells were spun down in an Eppendorf 5415 D centrifuge at 16,168g for 5 min to measure the free Hoechst 33342 concentration in the culture supernatant (Fig 3D).

Bottom Line: Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells.Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342.These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden "Janus" amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of "Hoechst 33342-like" drugs to cells and tissues in which these transporters are expressed.

No MeSH data available.


Related in: MedlinePlus