Proton Motive Force-Dependent Hoechst 33342 Transport by the ABC Transporter LmrA of Lactococcus lactis

The fluorescent compound Hoechst 33342 is a substrate for many multidrug resistance (MDR) transporters and is widely used to characterize their transport activity. We have constructed mutants of the adenosine triphosphate (ATP) binding cassette (ABC)-type MDR transporter LmrA of Lactococcus lactis that are defective in ATP hydrolysis. These mutants and wild-type LmrA exhibited an atypical behavior in the Hoechst 33342 transport assay. In membrane vesicles, Hoechst 33342 transport was shown to be independent of the ATPase activity of LmrA, and it was not inhibited by orthovanadate but sensitive to uncouplers that collapse the proton gradient and to N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. In contrast, transport of Hoechst 33342 by the homologous, heterodimeric MDR transporter LmrCD showed a normal ATP dependence and was insensitive to uncouplers of the proton gradient. With intact cells, expression of LmrA resulted in an increased rate of Hoechst 33342 influx while LmrCD caused a decrease in the rate of Hoechst 33342 influx. Cellular toxicity assays using a triple knockout strain, i.e., L. lactis ΔlmrA ΔlmrCD, demonstrate that expression of LmrCD protects cells against the growth inhibitory effects of Hoechst 33342, while in the presence of LmrA, cells are more susceptible to Hoechst 33342. Our data demonstrate that the LmrA-mediated Hoechst 33342 transport in membrane vesicles is influenced by the transmembrane pH gradient due to a pH-dependent partitioning of Hoechst 33342 into the membrane.

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters.

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance