The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding

Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding.     

Photoaffinity labelling of Ca2+ channels with [3H]azidopine

A 1,4-dihydroypyridine arylazide photoaffinity ligand, [3H]azidopine (50.6 Ci/mmol), has been synthesized. [3H]Azidopine binds reversibly with a Kd of 350 pM to guinea-pig skeletal muscle membranes in the absence of ultraviolet light. The reversible [3H]azidopine binding is inhibited steroselectively by 1,4-dihydropyridines, phenylalkylamine Ca2+ channel blockers and La3+. Covalent incorporation into membrane proteins after photolysis was investigated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. [3H]Azidopine is photoincorporated specifically into a protein of Mr approximately 145 000. The covalent labelling of the Mr approximately 145 000 band is inhibited stereoselectively by drugs and cations which block the reversible [3H]azidopine binding. It is suggested that [3H]azidopine is photoincorporated into a subunit of the putative Ca2+ channel.     

Photoaffinity labeling of the purified skeletal muscle calcium antagonist receptor by a novel benzothiazepine, [3H]azidobutyryl diltiazem

Previous photoaffinity-labeling studies with [3H]azidopine, (+) [3H]PN200-110, and [3H]LU 49888 have demonstrated that 1,4-dihydropyridines (nifedipine-like drugs) and phenylalkylamines (verapamil-like drugs) bind exclusively to the 165-kDa alpha 1 subunit of skeletal muscle calcium channels. However, it has not been conclusively determined whether benzothiazepines (diltiazem-like drugs), which represent the third group of calcium antagonists, also bind to the alpha 1 subunit. Here we report data obtained with a newly developed benzothiazepine photoaffinity probe, [3H]azidobutyryl diltiazem. This drug competes with diltiazem for the benzothiazepine-binding site and, in purified calcium channel preparations, specifically labels the 165-kDa polypeptide which does not change its electrophoretic mobility upon disulfide reduction. These data show that benzothiazepines, just like 1,4-dihydropyridines and phenylalkylamines, bind to the alpha 1 subunit of the skeletal muscle calcium channels.     

Characterization of drug transport,ATP hydrolysis,and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation

The overexpression of the human ATP-binding cassette half-transporter, ABCG2 (placenta-specific ABC transporter, mitoxantrone resistance-associated protein, breast cancer resistance protein), causes multidrug resistance in tumor cells. An altered drug resistance profile and substrate recognition were suggested for wild-type ABCG2 and its mutant variants (R482G and R482T); the mutations were found in drug-selected tumor cells. In order to characterize the different human ABCG2 transporters without possible endogenous dimerization partners, we expressed these proteins and a catalytic center mutant (K86M) in Sf9 insect cells. Transport activity was followed in intact cells, whereas the ATP binding and hydrolytic properties of ABCG2 were studied in isolated cell membranes. We found that the K86M mutant had no transport or ATP hydrolytic activity, although its ATP binding was retained. The wild-type ABCG2 and its variants, R482G and R482T, showed characteristically different drug and dye transport activities; mitoxantrone and Hoechst 33342 were transported by all transporters, whereas rhodamine 123 was only pumped by the R482G and R482T mutants. In each case, ABCG2-dependent transport was blocked by the specific inhibitor, fumitremorgin C. A relatively high basal ABCG2-ATPase, inhibited by fumitremorgin C, was observed in all active proteins, but specific drug stimulation could only be observed in the case of R482G and R482T mutants. We found that ABCG2 is capable of a vanadate-dependent adenine nucleotide trapping. Nucleotide trapping was stimulated by the transported compounds in the R482G and R482T variants but not in the wild-type ABCG2. These experiments document the applicability of the Sf9 expression system for parallel, quantitative examination of the specific transport and ATP hydrolytic properties of different ABCG2 proteins and demonstrate significant differences in their substrate interactions.     

Disulfiram is a potent modulator of multidrug transporter Cdr1p of Candida albicans

To find novel drugs for effective antifungal therapy in candidiasis, we examined disulfiram, a drug used for the treatment of alcoholism, for its role as a potential modulator of Candida multidrug transporter Cdr1p. We show that disulfiram inhibits the oligomycin-sensitive ATPase activity of Cdr1p and 2.5mM dithiothreitol reverses this inhibition. Disulfiram inhibited the binding of photoaffinity analogs of both ATP ([alpha-(32)P]8-azidoATP; IC(50)=0.76 microM) and drug-substrates ([(3)H]azidopine and [(125)I]iodoarylazidoprazosin; IC(50) approximately 12 microM) to Cdr1p in a concentration-dependent manner, suggesting that it can interact with both ATP and substrate-binding site(s) of Cdr1p. Furthermore, a non-toxic concentration of disulfiram (1 microM) increased the sensitivity of Cdr1p expressing Saccharomyces cerevisiae cells to antifungal agents (fluconazole, miconazole, nystatin, and cycloheximide). Collectively these results demonstrate that disulfiram reverses Cdr1p-mediated drug resistance by interaction with both ATP and substrate-binding sites of the transporter and may be useful for antifungal therapy.     

Identification of a novel 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide in calcium channel preparations

A 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide has been identified by photoaffinity labeling of purified rabbit and guinea pig skeletal muscle calcium channel preparations. The arylazide ligands (-)-[3H]azidopine and (-)-5-[(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4,5- trimethoxyphenyl)-2-isopropylvaleronitrile [( N-methyl-3H]LU 49888) were used to label 1,4-dihydropyridine- and phenylalkylamine-binding sites, respectively. A single, 155 to 170-kDa polypeptide was specifically labeled by both ligands in rabbit and guinea pig preparations provided that the skeletal muscle membranes used for purification were derived from fresh and not previously frozen and thawed tissue. The photoaffinity labeled polypeptide (termed here alpha 1) is different from the previously described alpha subunit in that it has the identical electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels irrespective of pretreatment either with N-ethylmaleimide or with dithiothreitol. The use of transverse tubular membranes isolated from previously frozen and thawed skeletal muscle results in a purified calcium channel preparation devoid of the alpha 1 subunit. In these preparations proteolytic degradation products of alpha 1 are labeled with both (-)-[3H]azidopine and [N-methyl-3H]LU 49888. Another large molecular weight polypeptide (termed here alpha 2) was also present in every purified calcium channel preparation studied. alpha 2 is distinct from alpha 1 in that reduction with dithiothreitol changes its apparent mass from 160-190 to 130-150 kDa. The alpha 2 subunit is not photoaffinity labeled either with (-)-[3H]azidopine or [N-methyl-3H]LU 49888. These data suggest that two distinct high molecular weight polypeptides (termed alpha 1 and alpha 2) are putative subunits of skeletal muscle calcium channels. Only the alpha 1 subunit contains both 1,4-dihydropyridine and phenylalkylamine receptors. alpha 2 is the same as the previously described alpha subunit (Curtis, B. M., and Catterall, W. A. (1984) Biochemistry 23, 2113-2118), but is neither a 1,4-dihydropyridine- nor a phenylalkylamine-binding protein.     

Photoaffinity labeling of the organic cation/H+ exchanger in renal brush border membrane vesicles.

The brush border membrane of the proximal tubule contains two efflux pathways for organic cations from the cell to the tubular fluid: a P-glycoprotein and an organic cation/H+ exchanger. There is evidence that they transport many of the same substrates. Their structural relatedness is unknown and is the subject of this report. The experimental approach was to identify the exchanger with photoaffinity labeling reagents. The rationale was that if the P-glycoprotein and the organic cation/H+ exchanger transport many of the same substrates, then they might be photoaffinity labeled by the same reagents. [125I]Iodoarylazidoprazosin and [3H]azidopine are two reagents, which have been used, to photoaffinity label the P-glycoprotein. We found that several polypeptides were photolabeled in a time- and concentration-dependent manner. The photoincorporation into only two of these polypeptides (41 and 28 kDa) was blocked extensively by the presence of known substrates for the exchanger. The photoaffinity labeling of only the 41-kDa polypeptide was affected by treatment with the chemical reagents, N-ethylmaleimide and dithiothreitol, which are known to affect the exchanger reaction. The findings are consistent with the interpretation that a 41-kDa polypeptide is, or is a component of, the exchanger.     

Diazipine, a novel photoaffinity probe for dihydropyridine receptors of calcium channels.

A new 1,4-dihydropyridine photoaffinity ligand, [3H]diazipine, has been assessed by binding and photolabeling, and compared with a currently used [3H]azidopine. [3H]Diazipine reversibly binds to skeletal muscle Ca2+ channels with a similar affinity to [3H]azidopine, but [3H]diazipine labels the channel two times more efficiently and no release of the incorported amount is observed after dithiothreitol treatment.     

Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [³H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [³H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [¹²⁵I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-gp, MRP1, and MXR were determined by MTT assay. THC significantly increased the sensitivity of vinblastine, mitoxantrone and etoposide in drug resistance KB-V-1, MCF7AdrVp3000 and MRP1-HEK 293 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Taken together, this study clearly showed that THC inhibits the efflux function of P-gp, MXR and MRP1 and it is able to extend the MDR reversing activity of curcuminoids in vivo.     

The role of hydrogen bond acceptor groups in the interaction of substrates with Pdr5p, a major yeast drug transporter

The yeast ABC (ATP-binding cassette protein) multidrug transporter Pdr5p transports a broad spectrum of xenobiotic compounds, including antifungal and antitumor agents. Previously, we demonstrated that substrate size is an important factor in substrate-transporter interaction and that Pdr5p has at least three substrate-binding sites. In this study, we use a combination of whole cell transport assays and photoaffinity labeling of Pdr5p with [(125)I]iodoarylazidoprazosin in purified plasma membrane vesicles to study the behavior of two series of novel substrates: trityl (triphenylmethyl) and carbazole derivatives. The results indicate that site 2, defined initially by tritylimidazole efflux, requires at least a single hydrogen bond acceptor group (electron pair donor). In contrast, complete inhibition of rhodamine 6G efflux and [(125)I]iodoarylazidoprazosin binding at site 1 requires substrates with three electronegative groups. Carbazole and trityl substrates with two groups show saturating, incomplete inhibition at this site. This type of inhibition is frequently observed in bacterial multidrug-binding proteins that use a pocket with multiple binding sites. The presence of multiple sites with different requirements for substrate-Pdr5p interaction may explain the broad specificity of xenobiotic compounds transported by this protein.     

Functional characterization of Candida albicans ABC transporter Cdr1p

In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and ΔF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (ΔF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized ΔF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).     

Photoaffinity probes for the alpha 1-adrenergic receptor and the calcium channel bind to a common domain in P-glycoprotein

P-glycoprotein is a 130-180-kDa integral membrane protein that is overproduced in multidrug-resistant cells. The protein appears to act as an energy-dependent drug efflux pump that has broad specificity for structurally diverse hydrophobic antitumor drugs. Many agents, such as the calcium channel blocker verapamil, reverse multidrug resistance and also interact with P-glycoprotein. The goal of this work was to determine if a common binding site participates in the transport of antitumor drugs and/or the reversal of drug resistance. This was done by comparing the peptide maps of P-glycoprotein (encoded by mdr1b) after it was labeled with a photoactive calcium channel blocker, [3H]azidopine, and a newly identified photoaffinity analog for P-glycoprotein 2-[4-(4-azido-3-[125I]iodobenzoyl) piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline [( 125I]iodoaryl azidoprazosin). [125I] Iodoaryl azidoprazosin, which classically has been used to identify the alpha 1-adrenergic receptor, bound to P-glycoprotein and was preferentially competed by vinblastine greater than actinomycin D greater than doxorubicin greater than colchicine. Peptide maps derived from P-glycoprotein labeled with [3H]azidopine or [125I]iodoaryl azidoprazosin were identical. After maximal digestion under conditions for Cleveland mapping, a single major 6-kDa fragment was obtained after digestion with V8 protease, whereas two major fragments, 6.5 and 5.5 kDa, were detected after digestion with chymotrypsin. The 6.0-kDa V8 fragment and the 6.5-kDa chymotrypsin fragment were both found when P-glycoprotein encoded by mdr1a and mdr1b was compared. Despite its specific interaction with P-glycoprotein, neither iodoaryl azidoprazosin nor prazosin markedly reversed resistance compared with verapamil or azidopine. Further, multidrug-resistant cells were 900-fold resistant to vinblastine but only 5-fold resistant to prazosin. These data demonstrate that structurally diverse reversal and/or antitumor agents are likely to have differential affinity for a small common domain of P-glycoprotein.     

Linsitinib (OSI-906) antagonizes ATP-binding cassette subfamily G member 2 and subfamily C member 10-mediated drug resistance

In this study we investigated the effect of linsitinib on the reversal of multidrug resistance (MDR) mediated by the overexpression of the ATP-binding cassette (ABC) subfamily members ABCB1, ABCG2, ABCC1 and ABCC10. Our results indicate for the first time that linsitinib significantly potentiate the effect of anti-neoplastic drugs mitoxantrone (MX) and SN-38 in ABCG2-overexpressing cells; paclitaxel, docetaxel and vinblastine in ABCC10-overexpressing cells. Linsitinib moderately enhanced the cytotoxicity of vincristine in cell lines overexpressing ABCB1, whereas it did not alter the cytotoxicity of substrates of ABCC1. Furthermore, linsitinib significantly increased the intracellular accumulation and decreased the efflux of [³H]-MX in ABCG2-overexpressing cells and [³H]-paclitaxel in ABCC10-overexpressing cells. However, linsitinib, at a concentration that reversed MDR, did not significantly alter the expression levels of either the ABCG2 or ABCC10 transporter proteins. Furthermore, linsitinib did not significantly alter the intracellular localization of ABCG2 or ABCC10. Moreover, linsitinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner. Overall, our study suggests that linsitinib attenuates ABCG2- and ABCC10-mediated MDR by directly inhibiting their function as opposed to altering ABCG2 or ABCC10 protein expression.     

Azidopine noncompetitively interacts with vinblastine and cyclosporin A binding to P-glycoprotein in multidrug resistant cells.

It is believed that P-glycoprotein (P-gp) is an energy-dependent drug efflux pump responsible for decreased drug accumulation in multidrug resistant (MDR) cells. In this study, we investigated whether azidopine, a photoactive dihydropyridine calcium channel blocker, is transported by P-gp in MDR Chinese hamster lung cells, DC-3F/VCRd-5L, and whether its binding site(s) on P-gp are distinct from those of Vinca alkaloids and cyclosporins. The efflux of azidopine from MDR cells was energy-dependent and inhibited by the cytotoxic agent vinblastine (VBL). Cyclosporin A (CsA), a modulator of MDR, also increased azidopine accumulation in MDR cells by decreasing the energy-dependent efflux of azidopine. P-gp in these cells was the only protein specifically bound to [3H]azidopine in photoaffinity experiments. The specific photoaffinity labeling of P-gp by [3H]azidopine was inhibited by CsA, SDZ 33-243, nonradioactive azidopine, and VBL with median concentrations (IC50) of 0.5, 0.62, 1.7, and 25 microM, respectively. The equilibrium binding of azidopine to plasma membranes of MDR variant DC-3F/VCRd-5L cells showed a single class of specific binding sites having a dissociation constant of 1.20 microM and a maximum binding capacity of 4.47 nmol/mg of protein. Kinetic analysis indicated that the inhibitory effect of VBL and CsA on azidopine binding to plasma membranes of MDR cells was noncompetitive, indicating that azidopine binds to P-gp at a binding site(s) different from the binding site(s) of these drugs.     

Iodomycin and iodipine, a structural analogue of azidopine, bind to a common domain in hamster P-glycoprotein.

Both the overexpression of P-glycoprotein and the broad range of substrates of this ATP-binding cassette (ABC) transporter induce the phenomenon of multidrug resistance, one major cause of the failure of cancer chemotherapy in humans. This study reports that [125I]iodipine, a structural analogue of the 1,4-dihydropyridine azidopine, shares a common binding site with iodomycin, a Bolton-Hunter derivative of the anthracycline daunomycin. This binding site is different from that described for iodoarylazidoprazosin, which is presumed to share a common binding site with azidopine. Edman sequencing revealed that [125I]iodipine had photolabelled the same peptide as iodomycin and spans the primary sequence of hamster isoform pgp1 from amino acid 230 to amino acid 312.     

The Dopamine Transporter Is Absent in Parkinsonian Putamen and Reduced in the Caudate Nucleus

The neuronal dopamine transporter/uptake site can be covalently labeled with the photoaffinity probe 1-(2-[bis-(4-fluorophenyl) methoxy]ethyl)-4-[2-(4-azido-3-[125I]iodophenyl)ethyl]piperazine [( 125I]FAPP) and visualized following sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Upon photolysis, [125I]FAPP specifically incorporated into a polypeptide of apparent Mr = 62,000 in membranes from both the putamen and the caudate nucleus of control, Alzheimer's, schizophrenia, and Huntington's diseased brain, and following complete deglycosylation, migrated as an Mr approximately 48,000 polypeptide. In parkinsonian postmortem putamen, however, there was no detectable photoincorporation of [125I]FAPP into the ligand binding subunit of the dopamine transporter. [125I]FAPP did specifically label the Mr 62,000 polypeptide of parkinsonian caudate, although with efficiencies of 20-50% of control. The asymmetrical loss of the dopamine transporter in Parkinson's diseased striatum was confirmed in reversible receptor binding experiments using [3H]GBR-12935 (3H-labeled 1-[2-(diphenylmethoxy) ethyl]-4-(3-phenylpropyl)piperazine). In parkinsonian putamen, mazindol competitively inhibited the binding of [3H]GBR-12935 with an estimated affinity (Ki approximately 2,000 nM) 10 times lower than in controls (Ki approximately 30 nM), while the affinity of maxindol for [3H]GBR-12935 binding in the caudate was equal to that seen with controls (Ki approximately 50 nM). The proportion of [3H]GBR-12935 binding sites recognized by mazindol with high affinity in Parkinson's diseased caudate was, however, reduced by 50-80%.(ABSTRACT TRUNCATED AT 250 WORDS)     

ABCG2 confers resistance to indolocarbazole compounds by ATP-dependent transport

The ABC half-transporter, ABCG2, is known to confer resistance to chemotherapeutic agents including indolocarbazole derivatives. MCF7 cells were introduced by either wild type ABCG2 (ABCG2-482R) or mutant ABCG2 (-482T), whose amino acid at position 482 is substituted to threonine from arginine, and their cross-resistance pattern was analyzed. Although this amino acid substitution seems to affect cross-resistance patterns, both 482T- and 482R-transfectants showed strong resistance to indolocarbazoles, confirming that ABCG2 confers resistance to them. For further characterization of ABCG2-mediated transport, we investigated indolocarbazole compound A (Fig. 1) excretion in cell-free system. Compound A was actively transported in membrane vesicles prepared from one of the 482T- transfectants and its uptake was supported by hydrolysis of various nucleoside triphosphates. This transport was inhibited completely by the other indolocarbazole compound, but not by mitoxantrone, implying that the binding site of mitoxantrone or the transport mechanisms for mitoxantrone is different from those of indolocarbazoles. These results showed that ABCG2 confers resistance to indolocarbazoles by transporting them in an energy-dependent manner.     

Specific derivatization of the vesicle monoamine transporter with novel carrier-free radioiodinated reserpine and tetrabenazine photoaffinity labels

Two iodophenylazide derivatives of reserpine and one iodophenylazide derivative of tetrabenazine have been synthesized and characterized as photoaffinity labels of the vesicle monoamine transporter (VMAT2). These compounds are 18-O-[3-(3'-iodo-4'-azidophenyl)-propionyl]methyl reserpate (AIPPMER), 18-O-[N-(3'-iodo-4'-azidophenethyl)glycyl]methyl reserpate (IAPEGlyMER), and 2-N-[(3'-iodo-4'-azidophenyl)-propionyl]tetrabenazine (TBZ-AIPP). Inhibition of [3H]dopamine uptake into purified chromaffin granule ghosts showed IC50 values of approximately 37 nM for reserpine, 83 nM for AIPPMER, 200 nM for IAPEGlyMER, and 2.1 microM for TBZ-AIPP. Carrier-free radioiodinated [125I]IAPEGlyMER and [125I]TBZ-AIPP were synthesized and used to photoaffinity label chromaffin granule membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed specific [125I]IAPEGlyMER labeling of a polypeptide that migrated as a broad band (approximately 55-90 kDa), with the majority of the label located between 70 and 80 kDa. The labeling by [125I]IAPEGlyMER was blocked by 100 nM reserpine, 10 microM tetrabenazine, 1 mM serotonin, and 10 mM (-)-norepinephrine and dopamine. Analysis of [125I]TBZ-AIPP-labeled chromaffin granule membranes by SDS-PAGE and autoradiography demonstrated specific labeling of a similar polypeptide, which was blocked by 1 microM reserpine and 10 microM tetrabenazine. Incubation of [125I]TBZ-AIPP-photolabeled chromaffin granule membranes in the presence of the glycosidase N-glycanase shifted the apparent molecular weight of VMAT2 to approximately 51 kDa. These data indicate that [125I]IAPEGlyMER and [125I]TBZ-AIPP are effective photoaffinity labels for VMAT2.     

The role of conserved tryptophan and acidic residues in the human dopamine transporter as characterized by site-directed mutagenesis

The human dopamine (DA) transporter (hDAT) contains multiple tryptophans and acidic residues that are completely or highly conserved among Na(+)/Cl(-)-dependent transporters. We have explored the roles of these residues using non-conservative substitution. Four of 17 mutants (E117Q, W132L, W177L and W184L) lacked plasma membrane immunostaining and were not functional. Both DA uptake and cocaine analog (i.e. 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane, CFT) binding were abolished in W63L and severely damaged in W311L. Four of five aspartate mutations (D68N, D313N, D345N and D436N) shifted the relative selectivity of the hDAT for cocaine analogs and DA by 10-24-fold. In particular, mutation of D345 in the third intracellular loop still allowed considerable [(3)H]DA uptake, but caused undetectable [(3)H]CFT binding. Upon anti-C-terminal-hDAT immunoblotting, D345N appeared as broad bands of 66-97 kDa, but this band could not be photoaffinity labeled with cocaine analog [(125)I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid ([(125)I]RTI-82). Unexpectedly, in this mutant, cocaine-like drugs remained potent inhibitors of [(3)H]DA uptake. CFT solely raised the K(m) of [(3)H]DA uptake in wild-type hDAT, but increased K(m) and decreased V(max) in D345N, suggesting different mechanisms of inhibition. The data taken together indicate that mutation of conserved tryptophans or acidic residues in the hDAT greatly impacts ligand recognition and substrate transport. Additionally, binding of cocaine to the transporter may not be the only way by which cocaine analogs inhibit DA uptake.     

A single amino acid residue contributes to distinct mechanisms of inhibition of the human multidrug transporter by stereoisomers of the dopamine receptor antagonist flupentixol.

Both cis and trans isomers of the dopamine receptor antagonist flupentixol inhibit drug transport and reverse drug resistance mediated by the human multidrug transporter P-glycoprotein (Pgp) with a stereoselective potency. The rate of ATP hydrolysis by Pgp and photoaffinity labeling of Pgp with the substrate analogue [125I]iodoarylazidoprazosin ([125I]IAAP) are modulated by each isomer in an opposite manner, suggesting different mechanisms for the inhibitory effect on drug transport. In this study we demonstrate that substitution of a single phenylalanine residue at position 983 (F983) with alanine (F983A) in putative transmembrane (TM) region 12 selectively affects inhibition of Pgp-mediated drug transport by both isomers of flupentixol. In F983A the stimulatory effect of cis(Z)-flupentixol and the inhibitory effect of trans(E)-flupentixol on ATP hydrolysis and [125I]IAAP labeling were significantly altered. This indicates that F983 contributes to inhibition of drug transport by both isomers of flupentixol and plays an important role in stimulation and inhibition of ATP hydrolysis and [125I]IAAP labeling by cis(Z)- and trans(E)-flupentixol, respectively. The near-wild-type level of drug transport by the F983A Pgp mutant dissociates susceptibility to inhibition by flupentixol from drug translocation, indicating the allosteric nature of the flupentixol interaction. The inhibitory effects of cyclosporin A on drug transport, drug-stimulated ATP hydrolysis, and [125I]IAAP labeling as well as the stimulatory effect of verapamil on ATP hydrolysis by Pgp were minimally affected by substitution of F983, suggesting no global alteration in the structural and functional integrity of the mutant. Taken together, our data suggest that distinct mechanisms of inhibition of Pgp-mediated drug transport by the cis and trans isomers of flupentixol are mediated through a common site of interaction.