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ATP-binding cassette (ABC) multidrug transporters are large, polytopic membrane proteins that exhibit astonishing promiscuity for their transport substrates. These transporters unidirectionally efflux thousands of structurally and functionally distinct compounds. To preclude the reentry of xenobiotic molecules via the drug-binding pocket, these proteins contain a highly conserved molecular gate, essentially allowing the transporters to function as molecular diodes. However, the structure-function relationship of these conserved gates and gating regions are not well characterized. In this study, we combine recent single-molecule, cryo-EM data with genetic and biochemical analyses of residues in the gating region of the yeast multidrug transporter Pdr5, the founding member of a large group of clinically relevant asymmetric ABC efflux pumps. Unlike the symmetric ABCG2 efflux gate, the Pdr5 counterpart is highly asymmetric, with only four (instead of six) residues comprising the gate proper. However, other residues in the near vicinity are essential for the gating activity. Furthermore, we demonstrate that residues in the gate and in the gating regions have multiple functions. For example, we show that Ile-685 and Val-1372 are required not only for successful efflux but also for allosteric inhibition of Pdr5 ATPase activity. Our investigations reveal that the gating region residues of Pdr5, and possibly other ABCG transporters, play a role not only in molecular gating but also in allosteric regulation, conformational switching, and protein folding.

Photodynamic therapy (PDT) involves light activation of the photosensitizer to generate reactive molecular species that induce cell modulation or death. Based on earlier findings showing that the photosensitizer benzoporphyrin derivative (BPD) is a breast cancer resistance protein (ABCG2) substrate, we investigated the ability of the P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) to transport BPD. In a panel of breast cancer cell lines overexpressing P-gp, MRP1, or ABCG2, BPD transport occurs only in cells overexpressing P-gp and ABCG2. Intracellular BPD fluorescence is not affected by MRP1, as determined by flow cytometry. To bypass P-gp- and ABCG2-mediated efflux of BPD, we introduce a lipidation strategy to create BPD derivatives that are no longer P-gp and ABCG2 substrates. The phospholipid-conjugated BPD and its nanoliposomal formulation evade both P-gp- and ABCG2-mediated transport. In cytotoxicity assays, lipidated BPD and its nanoliposomal formulation abrogate P-gp- and ABCG2-mediated PDT resistance. We verify that P-gp, like ABCG2, plays a role in BPD transport and BPD-PDT resistance. Furthermore, we introduce porphyrin-lipid nanovesicles as a new strategy to escape P-gp and ABCG2-mediated efflux of BPD for improved PDT outcomes in two breast cancer cell lines.

The overexpression of ATP-binding cassette (ABC) transporter ABCB1 (P-glycoprotein) or ABCG2 (BCRP/MXR/ABCP) in cancer cells is frequently associated with the development of multidrug resistance (MDR) in cancer patients, which remains a major obstacle to effective cancer treatment. By utilizing energy derived from ATP hydrolysis, both transporters have been shown to reduce the chemosensitivity of cancer cells by actively effluxing cytotoxic anticancer drugs out of cancer cells. Knowing that there are presently no approved drugs or other therapeutics for the treatment of multidrug-resistant cancers, in recent years, studies have investigated the repurposing of tyrosine kinase inhibitors (TKIs) to act as agents against MDR mediated by ABCB1 and/or ABCG2. SKLB610 is a multi-targeted TKI with potent activity against vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor 2 (FGFR2). In this study, we investigate the interaction of SKLB610 with ABCB1 and ABCG2. We discovered that neither ABCB1 nor ABCG2 confers resistance to SKLB610, but SKLB610 selectively sensitizes ABCG2-overexpressing multidrug-resistant cancer cells to cytotoxic anticancer agents in a concentration-dependent manner. Our data indicate that SKLB610 reverses ABCG2-mediated MDR by attenuating the drug-efflux function of ABCG2 without affecting its total cell expression. These findings are further supported by results of SKLB610-stimulated ABCG2 ATPase activity and in silico docking of SKLB610 in the drug-binding pocket of ABCG2. In summary, we reveal the potential of SKLB610 to overcome resistance to cytotoxic anticancer drugs, which offers an additional treatment option for patients with multidrug-resistant cancers and warrants further investigation.

The ABCG2 transporter plays a pivotal role in multidrug resistance, however, no clinical trial using specific ABCG2 inhibitors have been successful. Although ABC transporters actively extrude a wide variety of substrates, photodynamic therapeutic agents with porphyrinic scaffolds are exclusively transported by ABCG2. In this work, we describe for the first time a porphyrin derivative (4B) inhibitor of ABCG2 and capable to overcome multidrug resistance in vitro. The inhibition was time-dependent and 4B was not itself transported by ABCG2. Independently of the substrate, the porphyrin 4B showed an IC(50) value of 1.6 μM and a mixed type of inhibition. This compound inhibited the ATPase activity and increased the binding of the conformational-sensitive antibody 5D3. A thermostability assay confirmed allosteric protein changes triggered by the porphyrin. Long-timescale molecular dynamics simulations revealed a different behavior between the ABCG2 porphyrinic substrate pheophorbide a and the porphyrin 4B. Pheophorbide a was able to bind in three different protein sites but 4B showed one binding conformation with a strong ionic interaction with GLU446. The inhibition was selective toward ABCG2, since no inhibition was observed for P-glycoprotein and MRP1. Finally, this compound successfully chemosensitized cells that overexpress ABCG2. These findings reinforce that substrates may be a privileged source of chemical scaffolds for identification of new inhibitors of multidrug resistance-linked ABC transporters.

The overexpression of ABCB1 in cancer cells is a major factor contributing to the development of multidrug resistance (MDR) and treatment failure in cancer patients. Therefore, re-sensitization of MDR cancer cells to anticancer drugs remains an important aspect in chemotherapy. The progress in developing clinically applicable synthetic inhibitors of ABCB1 has been slow, mostly due to complications associated with intrinsic toxicities and unforeseen drug-drug interactions. Here, we explored the drug-repositioning approach for cancer therapy by targeting ABCB1-mediated MDR in human cancer cells. We found that DPI-201106, a positive inotropic agent, selectively inhibits the drug efflux function of ABCB1, and in doing so, re-sensitizes ABCB1-overexpressing MDR cancer cells to conventional anticancer drugs. Furthermore, the ATPase activity of ABCB1 and docking analysis of DPI-201106 in the drug-binding pocket of ABCB1 were determined to confirm the interaction between DPI-201106 and ABCB1 protein. In summary, we revealed an additional action and a potential clinical application of DPI-201106 to reverse ABCB1-mediated MDR in human cancer cells, which may be beneficial for cancer patients who have developed multidrug resistance and no longer respond to conventional chemotherapy, and should be further investigated.

One of the major challenges in cancer chemotherapy is the development of multidrug resistance phenomenon attributed to the overexpression of ATP-binding cassette (ABC) transporter ABCB1 or ABCG2 in cancer cells. Therefore, re-sensitizing MDR cancer cells to chemotherapy by directly inhibiting the activity of ABC transporters has clinical relevance. Unfortunately, previous attempts of developing clinically applicable synthetic inhibitors have failed, mostly due to problems associated with toxicity and unforeseen drug-drug interactions. An alternative approach is by repositioning drugs with known pharmacological properties as modulators of ABCB1 and ABCG2. In this study, we discovered that the transport function of ABCB1 and ABCG2 is strongly inhibited by SIS3, a specific inhibitor of Smad3. More importantly, SIS3 enhances drug-induced apoptosis and resensitizes ABCB1- and ABCG2-overexpressing cancer cells to chemotherapeutic drugs at non-toxic concentrations. These findings are further supported by ATPase assays and by a docking analysis of SIS3 in the drug-binding pockets of ABCB1 and ABCG2. In summary, we revealed an additional action of SIS3 that re-sensitizes MDR cancer cells and a combination therapy with this drug and other chemotherapeutic agents may be beneficial for patients with MDR tumors.

Ricolinostat is the first orally available, selective inhibitor of histone deacetylase 6 (HDAC6), currently under evaluation in clinical trials in patients with various malignancies. It is likely that the inevitable emergence of resistance to ricolinostat is likely to reduce its clinical effectiveness in cancer patients. In this study, we investigated the potential impact of multidrug resistance-linked ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 on the efficacy of ricolinostat, which may present a major hurdle to its development as an anticancer drug in the future. We demonstrated that the overexpression of ABCB1 or ABCG2 reduces the intracellular accumulation of ricolinostat, resulting in reduced efficacy of ricolinostat to inhibit the activity of HDAC6 in cancer cells. Moreover, the efficacy of ricolinostat can be fully restored by inhibiting the drug efflux function of ABCB1 and ABCG2 in drug-resistant cancer cells. In conclusion, our results provide some insights into the basis for the development of resistance to ricolinostat and suggest that co-administration of ricolinostat with a modulator of ABCB1 or ABCG2 could overcome ricolinostat resistance in human cancer cells, which may be relevant to its use in the clinic.

The expression of breast cancer resistant protein (BCRP) in lung cancer is correlated with development of multidrug resistance (MDR) and therefore leads to lower response to chemotherapy. ZM323881, a previously developed selective VEGFR-2 inhibitor, was found to have inhibitory effects on BCRP-mediated MDR in this investigation. ZM323881 significantly decreased the cytotoxic doses of mitoxantrone and SN-38 in BCRP-overexpressing NCI-H460/MX20 cells. Mechanistic studies revealed that ZM323881 effected by inhibiting BCRP-mediated drug efflux, leading to intracellular accumulation of BCRP substrates. No significant alteration in the expression levels and localization pattern of BCRP was observed when BCRP-overexpressing cells were exposed to ZM323881. Stimulated bell-shaped ATPase activities were observed. Molecular docking suggested that ZM323881 binds to the modulator site of BCRP and the binding pose is stable validated by 100 ns molecular dynamic simulation. Overall, our results indicated that ZM323881 reversed BCRP-related MDR by inhibiting its efflux function. These findings might be useful in developing combination chemotherapy for MDR cancer treatment.

ATP-binding cassette multidrug efflux pumps transport a wide range of substrates. Current models suggest that a drug binds relatively tightly to a transport site in the transmembrane domains when the protein is in the closed inward facing conformation. Upon binding of ATP, the transporter can switch to an outward facing (drug off or drug releasing) structure of lower affinity. ATP hydrolysis is critically important for remodeling the drug-binding site to facilitate drug release and to reset the transporter for a new transport cycle. We characterized the novel phenotype of an S1368A mutant that lies in the putative drug-binding pocket of the yeast multidrug transporter Pdr5. This substitution created broad, severe drug hypersensitivity, although drug binding, ATP hydrolysis, and intradomain signaling were indistinguishable from the wild-type control. Several different rhodamine 6G efflux and accumulation assays yielded evidence consistent with the possibility that Ser-1368 prevents reentry of the excluded drug.

Cancer cells often become resistant to chemotherapy through a phenomenon known as multidrug resistance (MDR). Several factors are responsible for the development of MDR, preeminent among them being the accelerated drug efflux mediated by overexpression of ATP binding cassette (ABC) transporters. Some small molecule tyrosine kinase inhibitors (TKIs) were recently reported to modulate the activity of ABC transporters. Therefore, the purpose of this study was to determine if motesanib, a multikinase inhibitor, could reverse ABCB1-mediated MDR. The results showed that motesanib significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to its substrate anticancer drugs. Motesanib significantly increased the accumulation of [ 3 H]-paclitaxel in ABCB1 overexpressing cells by blocking the efflux function of ABCB1 transporter. In contrast, no significant change in the expression levels and localization pattern of ABCB1 was observed when ABCB1 overexpressing cells were exposed to 3 μM motesanib for 72 h. Moreover, motesanib stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, the docking studies indicated favorable binding of motesanib within the transmembrane region of homology modeled human ABCB1. Here, we report for the first time, motesanib, at clinically achievable plasma concentrations, antagonizes MDR by inhibiting the efflux activity of the ABCB1 transporter. These findings may be useful for cancer combination therapy with TKIs in the clinic.

Melanoma is the most serious type of skin cancer and one of the most common cancers in the world. Advanced melanoma is often resistant to conventional therapies and has high potential for metastasis and low survival rates. Vemurafenib is a small molecule inhibitor of the BRAF serine-threonine kinase recently approved by the United States Food and Drug Administration to treat patients with metastatic and unresectable melanomas that carry an activating BRAF (V600E) mutation. Many clinical trials evaluating other therapeutic uses of vemurafenib are still ongoing. The ATP-binding cassette (ABC) transporters are membrane proteins with important physiological and pharmacological roles. Collectively, they transport and regulate levels of physiological substrates such as lipids, porphyrins and sterols. Some of them also remove xenobiotics and limit the oral bioavailability and distribution of many chemotherapeutics. The overexpression of three major ABC drug transporters is the most common mechanism for acquired resistance to anticancer drugs. In this review, we highlight some of the recent findings related to the effect of ABC drug transporters such as ABCB1 and ABCG2 on the oral bioavailability of vemurafenib, problems associated with treating melanoma brain metastases and the development of acquired resistance to vemurafenib in cancers harboring the BRAF (V600E) mutation., Graphical abstract This review highlights some of the recent findings related to the effect of ABC drug transporters such as ABCB1 and ABCG2 on the oral bioavailability of vemurafenib, problems associated with treating melanoma brain metastases and the development of acquired resistance to vemurafenib in cancers harboring the BRAF (V600E) mutation.

Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites.

The importance of the trifluoromethyl group in the polypharmacological profile of nilotinib was investigated. Molecular editing of nilotinib led to the design, synthesis and biological evaluation of analogues where the trifluoromethyl group was replaced by a proton, fluorine and a methyl group. While these analogues were less active than nilotinib toward Abl, their activity toward Kit was comparable, with the monofluorinated analogue being the most active. Docking of nilotinib and of analogues 2a-c to the binding pocket of Abl and of Kit showed that the lack of shape complementarity in Kit is compensated by the stabilizing effect from its juxtamembrane region.

Overexpression of the ATP-binding cassette (ABC) drug efflux proteins P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) on malignant cells is associated with inferior chemotherapy outcomes. Both, ABCB1 and ABCG2, are substrates of the serine/threonine kinase Pim-1; Pim-1 knockdown decreases their cell surface expression, but SGI-1776, the first clinically tested Pim inhibitor, was shown to reverse drug resistance by directly inhibiting ABCB1-mediated transport. We sought to characterize Pim-1-dependent and -independent effects of SGI-1776 on drug resistance. SGI-1776 at the Pim-1-inhibitory and non-cytotoxic concentration of 1 μM decreased the IC(50)s of the ABCG2 and ABCB1 substrate drugs in cytotoxicity assays in resistant cells, with no effect on the IC(50) of non-substrate drug, nor in parental cells. SGI-1776 also increased apoptosis of cells overexpressing ABCG2 or ABCB1 exposed to substrate chemotherapy drugs and decreased their colony formation in the presence of substrate, but not non-substrate, drugs, with no effect on parental cells. SGI-1776 decreased ABCB1 and ABCG2 surface expression on K562/ABCB1 and K562/ABCG2 cells, respectively, with Pim-1 overexpression, but not HL60/VCR and 8226/MR20 cells, with lower-level Pim-1 expression. Finally, SGI-1776 inhibited uptake of ABCG2 and ABCB1 substrates in a concentration-dependent manner irrespective of Pim-1 expression, inhibited ABCB1 and ABCG2 photoaffinity labeling with the transport substrate [(125)I]iodoarylazidoprazosin ([(125)I]IAAP) and stimulated ABCB1 and ABCG2 ATPase activity. Thus SGI-1776 decreases cell surface expression of ABCB1 and ABCG2 and inhibits drug transport by Pim-1-dependent and -independent mechanisms, respectively. Decrease in ABCB1 and ABCG2 cell surface expression mediated by Pim-1 inhibition represents a novel mechanism of chemosensitization.

Cancer stem cells are distinguished from normal adult stem cells by their stemness without tissue homeostasis control. Glycosphingolipids (GSLs), particularly globo-series GSLs, are important markers of undifferentiated embryonic stem cells, but little is known about whether or not ceramide glycosylation, which controls glycosphingolipid synthesis, plays a role in modulating stem cells. Here, we report that ceramide glycosylation catalyzed by glucosylceramide synthase, which is enhanced in breast cancer stem cells (BCSCs) but not in normal mammary epithelial stem cells, maintains tumorous pluripotency of BCSCs. Enhanced ceramide glycosylation and globotriosylceramide (Gb3) correlate well with the numbers of BCSCs in breast cancer cell lines. In BCSCs sorted with CD44(+)/ESA(+)/CD24(-) markers, Gb3 activates c-Src/β-catenin signaling and up-regulates the expression of FGF-2, CD44, and Oct-4 enriching tumorigenesis. Conversely, silencing glucosylceramide synthase expression disrupts Gb3 synthesis and selectively kills BCSCs through deactivation of c-Src/β-catenin signaling. These findings highlight the unexploited role of ceramide glycosylation in selectively maintaining the tumorous pluripotency of cancer stem cells. It speculates that disruption of ceramide glycosylation or globo-series GSL is a useful approach to specifically target BCSCs specifically.

OSI-930, a dual c-Kit and KDR tyrosine kinase inhibitor, is reported to have undergone a Phase I dose escalation study in patients with advanced solid tumors. A series of fifteen pyridyl and phenyl analogues of OSI-930 were designed and synthesized. Extensive screening of these compounds led to the discovery that nitropyridyl and ortho-nitrophenyl analogues, VKJP1 and VKJP3, were effective in reversing ABC subfamily G member 2 (ABCG2) transporter-mediated multidrug resistance (MDR). VKJP1 and VKJP3 significantly sensitized ABCG2-expressing cells to established substrates of ABCG2 including mitoxantrone, SN-38, and doxorubicin in a concentration-dependent manner, but not to the non-ABCG2 substrate cisplatin. However, they were unable to reverse ABCB1- or ABCC1-mediated MDR indicating their selectivity for ABCG2. Western blotting analysis was performed to evaluate ABCG2 expression and it was found that neither VKJP1 nor VKJP3 significantly altered ABCG2 protein expression for up to 72 h. [(3)H]-mitoxantrone accumulation study demonstrated that VKJP1 and VKJP3 increased the intracellular accumulation of [(3)H]-mitoxantrone, a substrate of ABCG2. VKJP1 and VKJP3 also remarkably inhibited the transport of [(3)H]-methotrexate by ABCG2 membrane vesicles. Importantly, both VKJP1 and VKJP3 were efficacious in stimulating the activity of ATPase of ABCG2 and inhibited the photoaffinity labeling of this transporter by its substrate [(125)I]-iodoarylazidoprazosin. The results suggested that VKJP1 and VKJP3, specifically inhibit the function of ABCG2 through direct interaction with its substrate binding site(s). Thus VKJP1 and VKJP3 represent a new class of drugs for reducing MDR in ABCG2 over-expressing tumors.

Over-expression of ABCG2 is linked to multidrug resistance in cancer chemotherapy. We have previously shown that functionalized aurones effectively reduced the efflux of pheophorbide A (an ABCG2 substrate) from ABCG2 over-expressing MDA-MB-231/R ("R") cells. In the present report, we investigated the functional relevance of this observation and the mechanisms by which it occurs. Aurones and related analogs were investigated for re-sensitization of R cells to mitoxantrone (MX, a chemotherapeutic substrate of ABCG2) in cell-based assays, accumulation of intracellular MX by cell cytometry, interaction with ABCG2 by biochemical assays and in vivo efficacy in MX resistant nude mice xenografts. We found that methoxylated aurones interacted directly with ABCG2 to inhibit efflux activity, possibly by competing for occupancy of one of the substrate binding sites on ABCG2. The present evidence suggests that they are not transported by ABCG2 although they stimulate ABCG2-ATPase activity. Alteration of ABCG2 protein expression was also discounted. One member was found to re-sensitize R cells to MX in both in vitro and in vivo settings. Our study identified methoxylated aurones as promising compounds associated with low toxicities and potent modulatory effects on the ABCG2 efflux protein. Thus, they warrant further scrutiny as lead templates for development as reversal agents of multidrug resistance.

Previously, we reported sipholenol A, a sipholane triterpenoid from the Red Sea sponge Callyspongia siphonella, as a potent reversal of multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp). Through extensive screening of several related sipholane triterpenoids that have been isolated from the same sponge, we identified sipholenone E, sipholenol L and siphonellinol D as potent reversals of MDR in cancer cells. These compounds enhanced the cytotoxicity of several P-gp substrate anticancer drugs, including colchicine, vinblastine and paclitaxel, and significantly reversed the MDR-phenotype in P-gp-overexpressing MDR cancer cells KB-C2 in a dose-dependent manner. Moreover, these three sipholanes had no effect on the response to cytotoxic agents in cells lacking P-gp expression or expressing MRP1 (ABCC1) or MRP7 (ABCC10) or breast cancer resistance protein (BCRP/ABCG2). All three sipholanes (IC(50) >50 μM) were not toxic to all the cell lines that were used. [(3)H]-Paclitaxel accumulation and efflux studies demonstrated that all three triterpenoids time-dependently increased the intracellular accumulation of [(3)H]-paclitaxel by directly inhibiting P-gp-mediated drug efflux. Sipholanes also inhibited calcein-AM transport from P-gp-overexpressing cells. The Western blot analysis revealed that these three triterpenoids did not alter the expression of P-gp. However, they stimulated P-gp ATPase activity in a concentration-dependent manner and inhibited the photolabeling of this transporter with its transport substrate [(125)I]-iodoarylazidoprazosin. In silico molecular docking aided the virtual identification of ligand binding sites of these compounds. In conclusion, sipholane triterpenoids efficiently inhibit the function of P-gp through direct interactions and may represent potential reversal agents for the treatment of MDR.

Nucleotide binding domains (NBDs) of the multidrug transporter of Candida albicans, CaCdr1p, possess unique divergent amino acids in their conserved motifs. For example, NBD1 (N-terminal-NBD) possesses conserved signature motifs, while the same motif is divergent in NBD2 (C-terminal-NBD). In this study, we have evaluated the contribution of these conserved and divergent signature motifs of CaCdr1p in ATP catalysis and drug transport. By employing site-directed mutagenesis, we made three categories of mutant variants. These included mutants where all the signature motif residues were replaced with either alanines or mutants with exchanged equipositional residues to mimic the conservancy and degeneracy in opposite domain. In addition, a set of mutants where signature motifs were swapped to have variants with either both the conserved or degenerated entire signature motif. We observed that conserved and equipositional residues of NBD1 and NBD2 and swapped signature motif mutants showed high susceptibility to all the tested drugs with simultaneous abrogation in ATPase and R6G efflux activities. However, some of the mutants displayed a selective increase in susceptibility to the drugs. Notably, none of the mutant variants and WT-CaCdr1p showed any difference in drug and nucleotide binding. Our mutational analyses show not only that certain conserved residues of NBD1 signature sequence (S304, G306, and E307) are important in ATP hydrolysis and R6G efflux but also that a few divergent residues (N1002 and E1004) of NBD2 signature motif have evolved to be functionally relevant and are not interchangeable. Taken together, our data suggest that the signature motifs of CaCdr1p, whether it is divergent or conserved, are nonexchangeable and are functionally critical for ATP hydrolysis.

In view of the importance of Candida Drug Resistance Protein (Cdr1p) of pathogenic Candida albicans in azole resistance, we have characterized its ability to efflux variety of substrates by subjecting its entire transmembrane segment (TMS) 5 to site directed mutagenesis. All the mutant variants of putative 21 amino acids of TMS 5 and native CaCdr1p were over expressed as a GFP-tagged protein in a heterologous host Saccharomyces cerevisiae. Based on the drug susceptibility pattern, the mutant variants could be grouped into two categories. The variants belonging to first category were susceptible to all the tested drugs, as compared to those belonging to second category which exhibited resistance to selective drugs. The mutant variants of both the categories were analyzed for their ATP catalysis and drug efflux properties. Irrespective of the categories, most of the mutant variants of TMS 5 showed an uncoupling between ATP hydrolysis and drug efflux. The mutant variants such as M667A, F673A, I675A and P678A were an exception since they reflected a sharp reduction in both Km and Vmax values of ATPase activity when compared with WT CaCdr1p-GFP. Based on the competition experiments, we could identify TMS 5 residues which are specific to interact with select drugs. TMS 5 residues of CaCdr1p thus not only impart substrate specificity but also selectively act as a communication link between ATP hydrolysis and drug transport.

The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is a potent and specific EGFR tyrosine kinase inhibitor (TKI); its promising pre-clinical results have led to clinical trials. Overexpression of ATP-binding cassette (ABC) transporters such as ABCB1, ABCC1 and ABCG2 is one of the main causes of multidrug resistance (MDR) and usually results in the failure of cancer chemotherapy. However, the interaction of AG1478 with these ABC transporters is still unclear. In the present study, we have investigated this interaction and found that AG1478 has differential effects on these transporters. In ABCB1-overexpressing cells, non-toxic doses of AG1478 were found to partially inhibit resistance to ABCB1 substrate anticancer drugs as well as increase intracellular accumulation of [3H]-paclitaxel. Similarly, in ABCG2-overexpressing cells, AG1478 significantly reversed resistance to ABCG2 substrate anticancer drugs and increased intracellular accumulation of [3H]-mitoxantrone as well as fluorescent compound BODIPY-prazosin. AG1478 also profoundly inhibited the transport of [3H]-E(2)17betaG and [3H]-methotrexate by ABCG2. We also found that AG1478 slightly stimulated ABCB1 ATPase activity and significantly stimulated ABCG2 ATPase activity. Interestingly, AG1478 did not inhibit the photolabeling of ABCB1 or ABCG2 with [125I]-iodoarylazidoprazosin. Additionally, AG1478 did not alter the sensitivity of parental, ABCB1- or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drug and had no effect on the function of ABCC1. Overall, we conclude that AG1478 is able to inhibit the function of ABCB1 and ABCG2, with a more pronounced effect on ABCG2. Our findings provide valuable contributions to the development of safer and more effective EGFR TKIs for use as anticancer agents in the clinic.

How can we understand a case in which a given amino acid sequence folds into structurally and functionally distinct molecules? Synonymous single-nucleotide polymorphisms in the MDR1 (multidrug resistance 1 or ABCB1) gene involving frequent-to-rare codon substitutions lead to identical protein sequences. Remarkably, these alternative sequences give a protein product with similar but different structures and functions. Here, we propose that long-enough ribosomal pause time scales may lead to alternate folding pathways and distinct minima on the folding free energy surface. While the conformational and functional differences between the native and alternate states may be minor, the MDR1 case illustrates that the barriers may nevertheless constitute sufficiently high hurdles in physiological time scales, leading to kinetically trapped states with altered structures and functions. Different folding pathways leading to conformationally similar trapped states may be due to swapping of (fairly symmetric) segments. Domain swapping is more likely in the no-pause case in which the chain elongates and folds simultaneously; on the other hand, sufficiently long pause times between such segments may be expected to lessen the chances of swapping events. Here, we review the literature in this light.

Cellular expression of ATP-binding cassette (ABC) transport proteins, such as P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP1), or ABCG2, is known to confer a drug-resistant phenotype. Thus, the development of effective transporter inhibitors could be of value to cancer treatment. CBT-1 is a bisbenzylisoquinoline plant alkyloid currently in development as a Pgp inhibitor. We characterized its interactions with the three major ABC transporters associated with drug resistance - Pgp, MRP1 and ABCG2 - and compared it to other known inhibitors. CBT-1 completely inhibited rhodamine 123 transport from Pgp-overexpressing cells at a concentration of 1muM. Additionally, 1 microM completely reversed Pgp-mediated resistance to vinblastine, paclitaxel and depsipeptide in SW620 Ad20 cells. CBT-1 was found to compete [(125)I]-IAAP labeling of Pgp with an IC(50) of 0.14 microM, and low concentrations of CBT-1 (1 microM) stimulated Pgp-mediated ATP hydrolysis. In MRP1-overexpressing cells, 10 microM CBT-1 was found to completely inhibit MRP1-mediated calcein transport. CBT-1 at 25 microM did not have a significant effect on ABCG2-mediated pheophorbide a transport. Serum levels of CBT-1 in samples obtained from eight patients receiving CBT-1 increased intracellular rhodamine 123 levels in CD56+ cells 2.1- to 5.7-fold in an ex vivo assay. CBT-1 is able to inhibit the ABC transporters Pgp and MRP1, making it an attractive candidate for clinical trials in cancers where Pgp and/or MRP1 might be overexpressed. Further clinical studies with CBT-1 are warranted.

P-glycoprotein (P-gp, ABCB1) is the most studied member of the ATP-Binding Cassette (ABC) transporter superfamily. This transporter utilizes energy from ATP hydrolysis for the efflux of a great variety of compounds, including anticancer drugs. P-gp is expressed at the apical surface of epithelial cells in the intestines, kidney, liver, adrenal gland, blood-brain barrier, and placenta, and therefore affects the pharmacokinetics of many drugs. The expression of P-gp at the surface of tumor cells is also one of the mechanisms of multidrug resistance, which occurs in many cancers. The development of an effective inhibitor requires first the understanding of the mechanism of action of P-gp. In our recent work, mutagenesis and molecular modeling studies led to the identification of a pair of phenylalanine-tyrosine structural motifs that mediate the inhibition of ATP hydrolysis by drugs such as zosuquidar, tariquidar and elacridar. Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues interact with the tyrosines in an edge-to-face conformation, helping the tyrosines to adopt the proper orientation to effectively establish hydrogen-bond contact with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation of tyrosine or phenylalanine residues that disrupts the structural motifs, they bind at lower affinity sites, leading to stimulation of ATP hydrolysis and poor inhibition of transport.

The transport cycle of ABC transporters in general and P-glycoprotein in particular has been extensively studied, but the molecular mechanism remains controversial. We identify stable reaction intermediates in the progression of the P-glycoprotein-mediated ATPase reaction equivalent to the enzyme-substrate (E.S, P-glycoprotein.ATP) and enzyme-product (E.P, P-glycoprotein.ADP.P(i)) reaction intermediates. These have been characterized using the photoaffinity analog 8-azido-[alpha-32P]ATP as well as under equilibrium conditions using [alpha-32P]ATP, in which a cross-linking step is not involved. Similar results were obtained when 8-azido-[alpha-32P]ATP or [alpha-32P]ATP was used. The reaction intermediates were characterized based on their kinetic properties and the nature (triphosphate/diphosphate) of the trapped nucleotide. Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E.S reaction intermediate of the ATPase reaction. Importantly, the posthydrolysis E.P state continues to have low affinity for substrate, suggesting that conformational changes that form the E.S complex are coupled to the conformational change at the transport substrate site to do mechanical work. Thus, the formation of E.S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrate from inner leaflet to outer leaflet of lipid bilayer. This novel approach applies transition state theory to elucidate the mechanism of P-glycoprotein and other ABC transporters and has wider applications in testing cause-effect hypotheses in coupled systems.

Multidrug resistance protein 4 (MRP4/ABCC4), transports cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents, and prostaglandins. In this study we characterize ATP hydrolysis by human MRP4 expressed in insect cells. MRP4 hydrolyzes ATP (Km, 0.62 mm), which is inhibited by orthovanadate and beryllium fluoride. However, unlike ATPase activity of P-glycoprotein, which is equally sensitive to both inhibitors, MRP4-ATPase is more sensitive to beryllium fluoride than to orthovanadate. 8-Azido[alpha-32P]ATP binds to MRP4 (concentration for half-maximal binding approximately 3 microm) and is displaced by ATP or by its non-hydrolyzable analog AMPPNP (concentrations for half-maximal inhibition of 13.3 and 308 microm). MRP4 substrates, the prostaglandins E1 and E2, stimulate ATP hydrolysis 2- to 3-fold but do not affect the Km for ATP. Several other substrates, azidothymidine, 9-(2-phosphonylmethoxyethyl)adenine, and methotrexate do not stimulate ATP hydrolysis but inhibit prostaglandin E2-stimulated ATP hydrolysis. Although both post-hydrolysis transition states MRP4.8-azido[alpha-32P]ADP.Vi and MRP4.8-azido[alpha-32P]ADP.beryllium fluoride can be generated, nucleotide trapping is approximately 4-fold higher with beryllium fluoride. The divalent cations Mg2+ and Mn2+ support comparable levels of nucleotide binding, hydrolysis, and trapping. However, Co2+ increases 8-azido[alpha-32P]ATP binding and beryllium fluoride-induced 8-azido[alpha-32P]ADP trapping but does not support steady-state ATP hydrolysis. ADP inhibits basal and prostaglandin E2-stimulated ATP hydrolysis (concentrations for half-maximal inhibition 0.19 and 0.25 mm, respectively) and beryllium fluoride-induced 8-azido[alpha-32P]ADP trapping, whereas Pi has no effect up to 20 mm. In aggregate, our results demonstrate that MRP4 exhibits substrate-stimulated ATP hydrolysis, and we propose a kinetic scheme suggesting that ADP release from the post-hydrolysis transition state may be the rate-limiting step during the catalytic cycle.

The mechanism(s) involved in agonist-stimulation of TRPC3 channels is not yet known. Here we demonstrate that TRPC3-N terminus interacts with VAMP2 and alphaSNAP. Further, endogenous and exogenously expressed TRPC3 colocalized and coimmunoprecipitated with SNARE proteins in neuronal and epithelial cells. Imaging of GFP-TRPC3 revealed its localization in the plasma membrane region and in mobile intracellular vesicles. Recovery of TRPC3-GFP fluorescence after photobleaching of the plasma membrane region was decreased by brefeldin-A or BAPTA-AM. Cleavage of VAMP2 with tetanus toxin (TeNT) did not prevent delivery of TRPC3 to the plasma membrane region but reduced its surface expression. TeNT also decreased carbachol and OAG, but not thapsigargin, stimulated Ca2+ influx. Importantly, carbachol, not thapsigargin, increased surface expression of TRPC3 that was attenuated by TeNT and not by BAPTA. In aggregate, these data suggest that VAMP2-dependent exocytosis regulates plasma membrane insertion of TRPC3 channels and contributes to carbachol-stimulation of Ca2+ influx.

The yeast (Saccharomyces cerevisiae) multidrug transporter Pdr5p effluxes a broad range of substrates that are variable in structure and mode of action. Previous work suggested that molecular size and ionization could be important parameters. In this study, we compared the relative sensitivity of isogenic PDR5 and pdr5 strains toward putative substrates that are similar in chemical structure. Three series were used: imidazole-containing compounds, trialkyltin chlorides, and tetraalkyltin compounds. We demonstrate that the Pdr5p transporter is capable of mediating transport of substrates that neither ionize nor have electron pair donors and that are much simpler in structure than those transported by the human MDR1-encoded P-glycoprotein. Furthermore, the size of the substrate is critical and independent of any requirement for hydrophobicity. Substrates have surface volumes greater than 90 A(3) with an optimum response at approximately 200-225 A(3) as determined by molecular modeling. Assays measuring the efflux from cells of [(3)H]chloramphenicol and [(3)H]tritylimidazole were used. A concentration-dependent inhibition of chloramphenicol transport was observed with imidazole derivatives but not with either the organotin compounds or the antitumor agent doxorubicin. In contrast, several of the organotin compounds were potent inhibitors of tritylimidazole efflux, but the Pdr5p substrate tetrapropyltin was ineffective in both assays. This argues for the existence of at least three substrate-binding sites on Pdr5p that differ in behavior from those of the mammalian P-glycoprotein. Evidence also indicates that some substrates are capable of interacting at more than one site. The surprising observation that Pdr5p mediates resistance to tetraalkyltins suggests that one of the sites might use only hydrophobic interactions to bind substrates.

Multidrug resistance (MDR) is a phenomenon that is often associated with decreased intracellular drug accumulation in the tumor cells of a patient, resulting from enhanced drug efflux. It is often related to the overexpression of P-glycoprotein (Pgp) on the surface of tumor cells, thereby reducing drug cytotoxicity. In this study, curcumin was tested for its potential ability to modulate the expression and function of Pgp in the multidrug-resistant human cervical carcinoma cell line KB-V1. Western blot analysis and reverse transcription–polymerase chain reaction (RT–PCR) showed that treatment with 1, 5, and 10 μM curcumin for up to 72 hr was able to significantly lower Pgp expression in KB-V1 cells. Curcumin (1–10 μM) decreased Pgp expression in a concentration-dependent manner and was also found to have the same effect on MDR1 mRNA levels. The effect of curcumin on Pgp function was demonstrated by rhodamine 123 (Rh123) accumulation and efflux in Pgp-expressing KB-V1 cells. Curcumin increased Rh123 accumulation in a concentration-dependent manner (1–55 μM) and inhibited the efflux of Rh123 from these cells, but did not affect the efflux of Rh123 from the wild-type drug-sensitive KB-3-1 cells. Treatment of drug-resistant KB-V1 cells with curcumin increased their sensitivity to vinblastine, which was consistent with an increased intracellular accumulation of Rh123. In addition, curcumin inhibited verapamil-stimulated ATPase activity and the photoaffinity labeling of Pgp with the prazosin analog [ 125 I ]iodoarylazidoprazosin in a concentration-dependent manner, demonstrating that curcumin interacts directly with the transporter. Thus, curcumin seems to be able to modulate the in vitro expression and function of Pgp in multidrug-resistant human KB-V1 cells. In summary, this study describes the duel modulation of MDR1 expression and Pgp function by the phytochemical curcumin, which may be an attractive new agent for the chemosensitization of cancer cells.

Cyclosporin A (CsA) treatment of HEK 293 cells expressing the rat heart RHE-1 (NCX1.1, EMBL accession number ) or the rat brain RBE-2 (NCX1.5, GenBank(TM) accession number ) Na(+)-Ca(2+) exchanger inhibited their transport activity in a concentration-dependent manner. The inhibition was detectable at 2 microm CsA, and exposure of the cells to 20 microm CsA resulted in a decrease of the Na(+)-dependent Ca(2+) uptake to about 20% relative to that of untreated cells. Determination of the surface expression of the exchanger protein revealed a parallel concentration-dependent reduction in the amount of the immunoreactive protein. No reduction was detected in the amount of total immunoreactive exchanger protein in CsA-treated cells relative to untreated ones. Among the different drugs tested, only PSC833, an analog of cyclosporin D, mimicked the effects of CsA. Exposure of the transfected cells to the chemically related cyclosporin D and macrolide drugs (FK506 or rapamycin) had no effect on the transport activity or the surface expression of the Na(+)-Ca(2+) exchanger. Co-expression of the human multidrug transporter P-glycoprotein (of which both drugs are modulators) with the cloned Na(+)-Ca(2+) exchanger revealed that transport activity and surface expression of each transporter in the co-transfected system were similar to those of each transporter alone in both the presence and absence of CsA or PSC833. CsA and PSC833 inhibited the surface expression of the NCX1 protein but did not alter the surface expression of P-glycoprotein. Unlike some P-glycoprotein endoplasmic reticulum-retained mutants (Loo, T. W., and Clarke, D. M. (1997) J. Biol. Chem. 272, 709-712), CsA did not rescue RBE-2/F913-->Stop, an endoplasmic reticulum-retained function-competent mutant of the Na(+)-Ca(2+) exchanger (Kasir, J., Ren, X., Furman, I., and Rahamimoff, H. (1999) J. Biol. Chem. 274, 24873-24880) and did not induce its kinesis to the surface membrane, further demonstrating molecular differences between P-glycoprotein and NCX1 mutants for interaction with CsA.

P-glycoprotein (Pgp), the ATP-binding cassette multidrug transporter, exhibits a drug (substrate)-stimulatable ATPase activity, and vanadate (Vi) inhibits this activity by stably trapping the nucleoside diphosphate in the Pgp.ADP.Vi conformation. We recently demonstrated that Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp in the absence of substrate occurs both in the presence of 8-azido-[alpha-(32)P]ATP (following 8-azido-ATP hydrolysis) or 8-azido-[alpha-(32)P]ADP (without hydrolysis) and, the transition state intermediates generated under either condition are functionally indistinguishable. In this study, we compare the effect of substrates on Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp under both non-hydrolysis and hydrolysis conditions. We demonstrate that whereas substrates stimulate the Vi-induced trapping of 8-azido-[alpha-(32)P]ADP under hydrolysis conditions, they strongly inhibit Vi-induced trapping under non-hydrolysis conditions. This inhibition is concentration-dependent, follows first order kinetics, and is effected by drastically decreasing the affinity of nucleoside diphosphate for Pgp during trapping. However, substrates do not affect the binding of nucleoside diphosphate in the absence of Vi, indicating that the substrate-induced conformation exerts its effect at a step distinct from nucleoside diphosphate-binding. Our results demonstrate that during the catalytic cycle of Pgp, although the transition state, Pgp x ADP x P(i) (Vi), can be generated both via the hydrolysis of ATP or by directly providing ADP to the system, in the presence of substrate the reaction is driven in the forward direction, i.e. hydrolysis of ATP. These data suggest that substrate-stimulated ATP hydrolysis by Pgp is a vectorial process.

A series of five homodimeric polyethylene glycol (PEG) linked homodimers based on the multidrug resistance reversal agent (−)-stipiamide were made and tested for their ability to interact with P-glycoprotein, the protein responsible for multidrug resistance, using ATPase and photoaffinity displacement assays. Key reactions include a new alkoxide-mesylate displacement for the assembly of the PEG linkers and a double Sonogashira coupling reaction.

P-glycoprotein (Pgp) is a plasma membrane protein whose overexpression confers multidrug resistance to tumor cells by extruding amphipathic natural product cytotoxic drugs using the energy of ATP. An elucidation of the catalytic cycle of Pgp would help design rational strategies to combat multidrug resistance and to further our understanding of the mechanism of ATP-binding cassette transporters. We have recently reported (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515–2520) that there are two independent ATP hydrolysis events in a single catalytic cycle of Pgp. In this study we exploit the vanadate (Vi)-induced transition state conformation of Pgp (Pgp·ADP·Vi) to address the question of what are the effects of ATP hydrolysis on the nucleotide-binding site. We find that at the end of the first hydrolysis event there is a drastic decrease in the affinity of nucleotide for Pgp coincident with decreased substrate binding. Release of occluded dinucleotide is adequate for the next hydrolysis event to occur but is not sufficient for the recovery of substrate binding. Whereas the two hydrolysis events have different functional outcomesvis a vis the substrate, they show comparablet for both incorporation and release of nucleotide, and the affinities for [α-32P]8-azido-ATP during Vi-induced trapping are identical. In addition, the incorporation of [α-32P]8-azido-ADP in two ATP sites during both hydrolysis events is also similar. These data demonstrate that during individual hydrolysis events, the ATP sites are recruited in a random manner, and only one site is utilized at any given time because of the conformational change in the catalytic site that drastically reduces the affinity of the second ATP site for nucleotide binding. In aggregate, these findings provide an explanation for the alternate catalysis of ATP hydrolysis and offer a mechanistic framework to elucidate events at both the substrate- and nucleotide-binding sites in the catalytic cycle of Pgp.

P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp. The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 μmol·min·mg−1 with aK m for ATP of 0.33 mm. This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the K m for ATP or 8-azidoATP and K i for ADP, suggesting that substrates do not affect nucleotide binding to Pgp. Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [α-32P]8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction. TheK m for [α-32P]8-azidoATP (20 μm) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [α-32P]8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state. Furthermore, Pi exhibits very low affinity for Pgp (K i∼30 mm for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [α-32P]8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction. We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp.

A benzophenone photoaffinity label 9 based on the polyene natural product (-)-stipiamide has been constructed using a diaminoethane spacer and the radioactive agent [3H]-BZDC (N-succinimidyl p-benzoyl-(2,3-3H)-dehydrocinnamate). Photoaffinity experiments show specific binding to human P-glycoprotein (Pgp) in the presence of cis-flupentixol but not with cyclosporin A.

Intercellular transfer of proteins is a mode of communication between cells that is crucial for certain physiological processes. Chemotherapy is the treatment of choice for approximately 50% of all cancers. However, multidrug resistance mediated by drug-efflux pumps such as P-glycoprotein (Pgp) minimizes the effectiveness of such therapy in a large number of patients. A new study demonstrates the functional intercellular transfer of Pgp. Non-genetic transfer of the multidrug resistance phenotype raises fascinating questions about the mechanism and regulation of cell-surface membrane-protein-mediated spread of traits.

Although the phenomenon of simultaneous resistance to multiple cytotoxic drugs (multidrug resistance) in cancer cells has been discussed for more than two decades, and the human and mouse genes encoding an energy-dependent transporter (the multidrug transporter or P-glycoprotein) responsible for multidrug resistance were cloned 10 years ago, there is still considerable controversy about the mechanism of action of this efflux pump and its true biological function. This review summarizes the current research on the mechanism of action of the multidrug transporter, including the hydrophobic cleaner and altered partitioning models, the possible function of P-glycoprotein as a chloride and/or ATP channel, the role of phosphorylation in its function and fact and speculation about its physiological role.

We had proposed earlier that the anaerobe Oxalobacter formigenes sustains a proton-motive force by exploiting a secondary carrier rather than a primary proton pump. In this view, a carrier protein would catalyze the exchange of extracellular oxalate, a divalent anion, and intracellular formate, the monovalent product of oxalate decarboxylation. Such an electrogenic exchange develops an internally negative membrane potential, and since the decarboxylation reaction consumes an internal proton, the combined activity of the carrier and the soluble decarboxylase would constitute an "indirect" proton pump with a stoichiometry of 1H+ per turnover. This model is now verified by identification and purification of OxlT, the protein responsible for the anion exchange reaction. Membranes of O. formigenes were solubilized at pH 7 with 1.25% octyl glucoside in 20 mM 3-(N-morpholino)propanesulfonic acid/K, in the presence of 0.4% Escherichia coli phospholipids and with 20% glucerol present as the osmolyte stabilant. Rapid methods for reconstitution were developed to monitor the distribution of OxlT during biochemical fractionation, allowing its purification by sequential anion and cation exchange chromatography. OxlT proved to be a single hydrophobic polypeptide, of 38 kDa mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a turnover number estimated as at least 1000/s. The properties of OxlT point to an indirect proton pump as the mechanism by which a proton-motive force arises in O. formigenes, and one may reasonably argue that indirect proton pumps take part in bacterial events such as acetogenesis, malolactate fermentation, and perhaps methanogenesis.

We have characterized the minimal functioning unit of UhpT, the secondary carrier that mediates exchange of phosphate and glucose 6-phosphate in Escherichia coli. Membranes of a UhpT overproducing strain were solubilized with 1.25% octyl beta-D-glucopyranoside, in the presence of 0.1% E. coli phospholipid and with 20% glycerol as the osmolyte stabilant. That soluble UhpT could bind its natural substrates was indicated by the protections afforded by sugar phosphates against thermal inactivation or chemical modification with pyridoxal 5'-phosphate. Moreover, the degree of protection correlated with the strength of interaction between UhpT and the test substrate (2-deoxyglucose 6-phosphate = glucose 6-phosphate greater than galactose 6-phosphate = glucose 1-phosphate much greater than glucose 6-sulfate). Other experiments demonstrated that soluble UhpT existed as a monomer. For example, during both high performance liquid chromatography and conventional gel permeation chromatography, the elution pattern of UhpT activity was measured directly by a rapid reconstitution technique. In both cases, and in the presence and absence of substrate, UhpT activity traveled as a single component of Mr 53,000, corresponding closely to the sequence prediction of 50,600. Finally, reconstitution was studied at protein to lipid ratios low enough to achieve between 0.075 and 1.5 UhpT monomers/proteoliposome. Specific activity was constant throughout this range, a finding consistent with the idea of a functional monomer. Mitochondria and chloroplasts provide the only other anion exchange carriers described at this level of biochemical resolution, and these organelle antiporters function as dimers. By contrast, work summarized here places their bacterial counterpart, UhpT, in the same class as the lactose carrier of E. coli and the glucose carrier of the human erythrocyte, both of which function as monomers. Consideration of this pattern in conjunction with the known hydropathy profiles of these proteins suggests a novel scheme for the classification of all secondary carriers, with implications for both the structure and origin of these transport proteins.

Membrane vesicles of three streptococcal strains (Streptococcus faecalis, Streptococcus lactis, and Streptococcus sanguis) were extracted with octyl-beta-D-glucoside in the presence of Escherichia coli lipid and glycerol. For reconstitution, the detergent extract was mixed with bath-sonicated E. coli lipid, in the presence of octyl-beta-D-glucoside, and proteoliposomes were formed by a 25-fold dilution. ATP-dependent calcium accumulation by proteoliposomes was comparable to that found in parent vesicles. Recovery of this calcium transport activity was dependent on the inclusion of an osmolyte protein stabilant (glycerol, etc.) during solubilization. The properties of ATP-driven calcium transport were studied in the reconstituted system. In proteoliposomes, ATP-linked calcium accumulation was not affected by the protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or by the ionophores, valinomycin and nigericin, in the presence of potassium, or by N,N'-dicyclohexylcarbodiimide, an inhibitor of the F0F1-ATPase. On the other hand, calcium transport was completely blocked by micromolar levels of orthovanadate; half-maximal inhibitions were observed at 0.4, 4, and 4 microM vanadate, for S. faecalis, S. lactis, and S. sanguis, respectively. This marked sensitivity to orthovanadate suggests operation of an E1E2-type ion-motive pump. These data demonstrate that, in a reconstituted system, calcium transport is not linked to an ATP-dependent proton circulation via the F0F1-ATPase, but rather is driven by a calcium-translocating ATPase. Thus, calcium extrusion from the cytosol of enteric, lactic acid, or oral streptococci is mediated by an ATP-linked process analogous to the ion-motive ATPases of eukaryotic membranes.

Membrane vesicles of Streptococcus lactis were used to characterize a novel anion exchange involving phosphate and sugar 6-phosphates. For vesicles loaded with 50 mM phosphate at pH 7, homologous phosphate:phosphate exchange had a maximal rate of 130 nmol/min/mg of protein and a Kt of 0.21 mM external phosphate; among phosphate analogues tested, only arsenate replaced phosphate. Heterologous exchange was studied by 2-deoxyglucose 6-phosphate entry into phosphate-loaded vesicles; this reaction had a maximal velocity of 31 nmol/min/mg of protein and a Kt of 26 microM external substrate. Sugar phosphate moved intact during this exchange, since its entry led to loss of internal 32Pi without transfer of 32P to sugar phosphate. Inhibitions of phosphate exchange suggested that the preferred sugar phosphate substrates were (Kiapp): glucose, 2-deoxyglucose, and mannose 6-phosphates (approximately 20 microM) greater than fructose 6-phosphate (150 microM) greater than glucosamine 6-phosphate (420 microM) greater than alpha-methylglucoside 6-phosphate (740 microM). Stoichiometry for phosphate:2-deoxyglucose 6-phosphate antiport was 2:1 at pH 7, and since initial rates of exchange were unaffected by charge carrying ionophores (gramicidin, valinomycin, a protonophore), this unequal stoichiometry indicated the electroneutral exchange of two monovalent phosphates for a single divalent sugar phosphate.

Studies with Escherichia coli cells showed that the transport systems encoded by glpT (sn-glycerol 3-phosphate transport) and uhpT (hexose phosphate transport) catalyze a reversible 32Pi:Pi exchange. This reaction could be used to monitor the glpT or uhpT activities during reconstitution. Membranes from suitably constructed strains were extracted with octylglucoside in the presence of lipid and glycerol, and proteoliposomes were formed by dilution in 0.1 M KPi (pH 7). Both reconstituted systems mediated a 32Pi:Pi exchange which was blocked by the appropriate heterologous substrate, sn-glycerol 3-phosphate (G3P) or 2-deoxyglucose 6-phosphate (2DG6P), with an apparent Ki near 50 microM. In the absence of an imposed cation-motive gradient, Pi-loaded proteoliposomes also transported the expected physiological substrate; Michaelis constants for the transport of G3P or 2DG6P were near 20 microM. The heterologous exchange showed a maximal velocity of 130 nmol/min/mg protein via the glpT system and 11 nmol/min/mg protein for the uhpT system. This difference was expected because the G3P transport activity had been reconstituted from a strain carrying multiple copies of the glpT gene. Taken together, these results suggest that anion exchange may be the molecular basis for transport by the glpT and uhpT proteins.