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Purpose: PET imaging using [11C]metoclopramide revealed the importance of P-glycoprotein (P-gp, ABCB1) in mediating the brain-to-blood efflux of substrates across the blood–brain barrier (BBB). In this work, the elimination rate constant from the brain (kE,brain), calculated from dynamic PET images without the need for arterial blood sampling, was evaluated as an outcome parameter for the interpretation of [11C]metoclopramide PET data. Procedures: kE,brain parameter was obtained by linear regression of log-transformed brain time-activity curves (TACs). kE,brain values (h−1) obtained under baseline conditions were compared with values obtained after complete P-gp inhibition using tariquidar in rats (n = 4) and baboons (n = 4) or after partial inhibition using cyclosporine A in humans (n = 10). In baboons, the sensitivity of kE,brain to measure complete P-gp inhibition was compared with outcome parameters derived from kinetic modeling using a 1-tissue compartment model (1-TCM). Finally, kE,brain-maps were generated in each species using PMOD software. Results: The linear part of the log-transformed brain TACs occurred from 10 to 30 min after radiotracer injection in rats, from 15 to 60 min in baboons, and from 20 to 60 min in humans. P-gp inhibition significantly decreased kE,brain values by 39 ± 12% in rats (p < 0.01), by 32 ± 6% in baboons (p < 0.001), and by 37 ± 22% in humans (p < 0.001). In baboons, P-gp inhibition consistently decreased the brain-to-plasma efflux rate constant k2 (36 ± 9%, p < 0.01) leading to an increase in the total brain volume of distribution (VT, 101 ± 12%, p < 0.001). In all studied species, brain kE,brain-maps displayed decreased P-gp-mediated efflux across the BBB. Conclusions: kE,brain of [11C]metoclopramide provides a simple outcome parameter to describe P-gp function in the living brain when arterial input function data are unavailable, although less sensitive than VT. kE,brain-maps represent easy to compute parametric images reflecting the effect of P-gp on [11C]metoclopramide elimination from the brain. [ABSTRACT FROM AUTHOR]

The recent elucidation of atomistic structures of Cl− channel CFTR provides opportunities for understanding the molecular basis of cystic fibrosis. Despite having been activated through phosphorylation and provided with ATP ligands, several near-atomistic cryo-EM structures of CFTR are in a closed state, as inferred from the lack of a continuous passage through a hydrophobic bottleneck region located in the extracellular portion of the pore. Here, we present repeated, microsecond-long molecular dynamics simulations of human CFTR solvated in a lipid bilayer and aqueous NaCl. At equilibrium, Cl− ions enter the channel through a lateral intracellular portal and bind to two distinct cationic sites inside the channel pore but do not traverse the narrow, de-wetted bottleneck. Simulations conducted in the presence of a strong hyperpolarizing electric field led to spontaneous Cl− translocation events through the bottleneck region of the channel, suggesting that the protein relaxed to a functionally open state. Conformational changes of small magnitude involving transmembrane helices 1 and 6 preceded ion permeation through diverging exit routes at the extracellular end of the pore. The pore bottleneck undergoes wetting prior to Cl− translocation, suggesting that it acts as a hydrophobic gate. Although permeating Cl− ions remain mostly hydrated, partial dehydration occurs at the binding sites and in the bottleneck. The observed Cl− pathway is largely consistent with the loci of mutations that alter channel conductance, anion binding, and ion selectivity, supporting the model of the open state of CFTR obtained in the present study. [ABSTRACT FROM AUTHOR]

Purpose: Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumors in adults. Patients invariably relapse during or after first-line therapy and the median overall survival is 14.6 months. Such poor clinical response is partly ascribed to the activity of ATP-binding cassette (ABC) transporters. The activity of these proteins, severely reduces the amount of therapeutics that penetrates the tumor cells. We hypothesized that ABC transporter expression could correlate with survival surrogates. In this study, we assessed the expression of four commonly expressed ABC transporters in GBM samples and investigated if mRNA levels could serve as a prognostic biomarker. Methods: Human specimens were analyzed by qPCR to assess ABCB1, ABCC1/3 and ABCG2 expression. Kaplan-Meier and multivariate analyses were then used to evaluate the correlation with overall survival (OS) and progression-free survival (PFS). Results: Our cohort included 22 non-tumoral samples as well as 159 GBM tumor specimens. ABC transporters were significantly more expressed in GBM samples compared to non-tumoral tissue. Moreover ABCC1 and 3 mRNA expression were significantly increased at recurrence. Statistical analyses revealed that increased expression of either ABCC1 or ABCC3 did not confer a poorer prognosis. However, increased ABCC1 mRNA levels did correlate with a significantly shorter PFS. Conclusion: In this manuscript, the analyses we conducted suggest that the expression of the four ABC transporters evaluated would not be suitable prognostic biomarkers. We believe that, when estimating prognosis, the plethora of mechanisms implicated in chemoresistance should be analyzed as a multi-facetted entity rather than isolated units. [ABSTRACT FROM AUTHOR]

Main conclusion: Functional expression of a multidrug resistance-type ABC transporter from Lavandulaangustifolia improved yeast resistance to geraniol, a monoterpene constituent of lavender essential oil.Plant ATP-binding cassette (ABC) transporters are a large family of membrane proteins involved in active and selective transport of structurally diverse compounds. In this study, we functionally evaluated LaABCB1, a multidrug resistance (MDR)-type ABC transporter strongly expressed in the secretory cells of lavender glandular trichomes, where monoterpene essential oil constituents are synthesized and secreted. We used LaABCB1 to complement a yeast knockout mutant in which 16 ABC transporters were deleted. Expression of LaABCB1 enhanced tolerance of yeast mutants to geraniol, a key constituent of essential oils in lavenders and numerous other plants. Our findings suggest a role for the MDR-type ABC transporters in the toxicity tolerance of at least certain essential oil constituents in lavender oil glands. [ABSTRACT FROM AUTHOR]

Elacridar (GF120918) is a highly potent inhibitor of both P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), the main efflux transporters expressed at the blood-brain barrier (BBB). Elacridar shows very low aqueous solubility, which complicates its formulation for i.v. administration. An intravenous infusion protocol would be preferred to achieve high and controlled plasma concentrations of elacridar in large animals, including nonhuman primates. Formulation of elacridar for i.v. infusion was achieved using a co-solvent strategy, resulting in an aqueous dispersion with a final concentration of 5 g L−1 elacridar with tetrahydrofuran (5% w/v) in aqueous D-glucose solution (2.5%, w/v). Particle size (mean = 2.8 ± 0.9 μm) remained stable for 150 min. The preparation was i.v. administered as a continuous infusion (12 mg kg−1 h−1 for 90 min) to three baboons. Arterial and venous plasma pharmacokinetics (PK) of elacridar were monitored using a newly developed and validated HPLC-UV method. Elacridar concentration increased rapidly to reach a plateau at 9.5 μg mL−1 within 20 min after the start of infusion. Elacridar PK in venous plasma did not differ from arterial plasma facing the BBB, indicating the absence of an arteriovenous concentration gradient. Intravenous infusion of elacridar allows for controlled exposure of the BBB and offers a useful tool to assess the impact of ABCB1/ABCG2 on drug disposition to the brain in nonhuman primates, a relevant animal model for the study of transporter function at the BBB. [ABSTRACT FROM AUTHOR]

Premature termination codon (PTC) mutations in the ATP-Binding Cassette, Sub-Family A, Member 7 gene ( ABCA7) have recently been identified as intermediate-to-high penetrant risk factor for late-onset Alzheimer's disease (LOAD). High variability, however, is observed in downstream ABCA7 mRNA and protein expression, disease penetrance, and onset age, indicative of unknown modifying factors. Here, we investigated the prevalence and disease penetrance of ABCA7 PTC mutations in a large early onset AD (EOAD)-control cohort, and examined the effect on transcript level with comprehensive third-generation long-read sequencing. We characterized the ABCA7 coding sequence with next-generation sequencing in 928 EOAD patients and 980 matched control individuals. With MetaSKAT rare variant association analysis, we observed a fivefold enrichment ( p = 0.0004) of PTC mutations in EOAD patients (3%) versus controls (0.6%). Ten novel PTC mutations were only observed in patients, and PTC mutation carriers in general had an increased familial AD load. In addition, we observed nominal risk reducing trends for three common coding variants. Seven PTC mutations were further analyzed using targeted long-read cDNA sequencing on an Oxford Nanopore MinION platform. PTC-containing transcripts for each investigated PTC mutation were observed at varying proportion (5-41% of the total read count), implying incomplete nonsense-mediated mRNA decay (NMD). Furthermore, we distinguished and phased several previously unknown alternative splicing events (up to 30% of transcripts). In conjunction with PTC mutations, several of these novel ABCA7 isoforms have the potential to rescue deleterious PTC effects. In conclusion, ABCA7 PTC mutations play a substantial role in EOAD, warranting genetic screening of ABCA7 in genetically unexplained patients. Long-read cDNA sequencing revealed both varying degrees of NMD and transcript-modifying events, which may influence ABCA7 dosage, disease severity, and may create opportunities for therapeutic interventions in AD. [ABSTRACT FROM AUTHOR]

The human breast cancer resistance protein (BCRP, gene symbol ABCG2) is an ATP-binding cassette (ABC) efflux transporter. It was so named because it was initially cloned from a multidrug-resistant breast cancer cell line where it was found to confer resistance to chemotherapeutic agents such as mitoxantrone and topotecan. Since its discovery in 1998, the substrates of BCRP have been rapidly expanding to include not only therapeutic agents but also physiological substances such as estrone-3-sulfate, 17β-estradiol 17-(β- d-glucuronide) and uric acid. Likewise, at least hundreds of BCRP inhibitors have been identified. Among normal human tissues, BCRP is highly expressed on the apical membranes of the placental syncytiotrophoblasts, the intestinal epithelium, the liver hepatocytes, the endothelial cells of brain microvessels, and the renal proximal tubular cells, contributing to the absorption, distribution, and elimination of drugs and endogenous compounds as well as tissue protection against xenobiotic exposure. As a result, BCRP has now been recognized by the FDA to be one of the key drug transporters involved in clinically relevant drug disposition. We published a highly-accessed review article on BCRP in 2005, and much progress has been made since then. In this review, we provide an update of current knowledge on basic biochemistry and pharmacological functions of BCRP as well as its relevance to drug resistance and drug disposition. [ABSTRACT FROM AUTHOR]

Genetic polymorphisms in drug metabolism and transport genes can influence the pharmacokinetics and pharmacodynamics of chemotherapy drugs. We investigated the role of genes involved in metabolic and transport pathways in response to chemotherapy and clinical outcome of osteosarcoma patients. The association between the eight polymorphisms with response to chemotherapy and clinical outcome of patients was carried out by unconditional logistic regression analysis and Cox proportional hazard models. Of 186 patients, 98 patients showed good response to chemotherapy, 64 died, and 97 showed progression at the end of the study. Patients carrying ABCB1 rs1128503 TT genotype and T allele were more likely to have a good response to chemotherapy. ABCC3 rs4148416 TT genotype and T allele and GSTP1 rs1695 GG genotype and G allele were associated with poor response to chemotherapy. In the Cox proportional hazards model, after adjusting for potential confounding factors, patients carrying ABCB1 rs1128503 TT genotype and T allele were associated with lower risk of progression-free survival (PFS) and overall survival (OS). ABCC3 rs4148416 TT genotype and T allele and GSTP1 rs1695 GG genotype and G allele were correlated with high risk of PFS and OS. The ABCB1 TT and GSTP1 GG genotypes were significantly associated with a shorter OS. In conclusion, variants of ABCB1 rs128503, ABCC3 rs4148416, and GSTP1 rs1695 are associated with response to chemotherapy and PFS and OS of osteosarcoma patients; these gene polymorphisms could help in the design of individualized therapy. [ABSTRACT FROM AUTHOR]

The ATP-binding cassette family is one of the largest groupings of membrane proteins, moving allocrites across lipid membranes, using energy from ATP. In bacteria, they reside in the inner membrane and are involved in both uptake and export. In eukaryotes, these transporters reside in the cell’s internal membranes as well as in the plasma membrane and are unidirectional—out of the cytoplasm. The range of substances that these proteins can transport is huge, which makes them interesting for structure–function studies. Moreover, their abundance in nature has made them targets for structural proteomics consortia. There are eight independent structures for ATP-binding cassette transporters, making this one of the best characterised membrane protein families. Our understanding of the mechanism of transport across membranes and membrane protein structure in general has been enhanced by recent developments for this family. [ABSTRACT FROM AUTHOR]

The 72-kDa breast cancer resistance protein (BCRP) is the second member of the subfamily G of the human ATP binding cassette (ABC) transporter superfamily and thus also designated as ABCG2. Unlike P-glycoprotein and MRP1, which are arranged in 2 repeated halves, BCRP is a half-transporter consisting of only 1 nucleotide binding domain followed by 1 membrane-spanning domain. Current experimental evidence suggests that BCRP may function as a homodimer or homotetramer. Overexpression of BCRP is associated with high levels of resistance to a variety of anticancer agents, including anthracyclines, mitoxantrone, and the camptothecins, by enhancing drug efflux. BCRP expression has been detected in a large number of hematological malignancies and solid tumors, indicating that this transporter may play an important role in clinical drug resistance of cancers. In addition to its role to confer resistance against chemotherapeutic agents, BCRP actively transports structurally diverse organic molecules, conjugated or unconjugated, such as estrone-3-sulfate, 17β-estradiol 17-(β-D-glucuronide), and methotrexate. BCRP is highly expressed in the placental syncytiotrophoblasts, in the apical membrane of the epithelium in the small intestine, in the liver canalicular membrane, and at the luminal surface of the endothelial cells of human brain microvessels. This strategic and substantial tissue localization indicates that BCRP also plays an important role in absorption, distribution, and elimination of drugs that are BCRP substrates. This review summarizes current knowledge of BCRP and its relevance to multidrug resistance and drug disposition. [ABSTRACT FROM AUTHOR]

Multidrug resistance-associated protein 1 (MRP1) is one of the major proteins shown to mediate efflux transport of a broad range of antitumor drugs, glucuronide conjugates, and glutathione, in addition to endogenous substrates. Significant differences in substrate selectivity were reported for murine and human MRP1. As preclinical drug disposition and pharmacokinetics studies are often conducted in rats, we have recently cloned the rat MRP1 (rMRP1) and demonstrated that rMRP1 expressed in transfected cells effluxes calcein, a commonly used fluorescence substrate for human MRP1. To further characterize the rat ortholog of MRP1, we isolated a cell line stably expressing recombinant rMRP1. These cells were tested for their ability to transport calcein and a range of chemotherapeutic drugs. Our results showed that cells expressing rMRP1 consistently efflux calcein at a rate 5-fold greater than control cells. The rMRP1 transfected cells, like their human ortholog, can confer drug resistance to vinca alkaloid (vinblastine and vincristine) and anthracycline drugs (daunorubcin and doxorubicin), and the resistance conferred by the MRP1 can be partially abolished by the MRP-specific inhibitors. The transepithelial permeability due to rMRP1 expression in differentiated Madin-Darby canine kidney cells (MDCK) cells was also investigated. The MRP1 transport activity is directional, as demonstrated by directional vinblastine transport. Collectively, our results demonstrate that the cellular expression of rMRP1, like its human ortholog, could confer resistance to anticancer drugs. [ABSTRACT FROM AUTHOR]

An ABC-type transporter in Escherichia coli that transports both l- and d-methionine, but not other natural amino acids, was identified. This system is the first functionally characterized member of a novel family of bacterial permeases within the ABC superfamily. This family was designated the methionine uptake transporter (MUT) family (TC #3.A.1.23). The proteins that comprise the transporters of this family were analyzed phylogenetically, revealing the probable existence of several sequence-divergent primordial paralogues, no more than two of which have been transmitted to any currently sequenced organism. In addition, MetJ, the pleiotropic methionine repressor protein, was shown to negatively control expression of the operon encoding the ABC-type methionine uptake system. The identification of MetJ binding sites (in gram-negative bacteria) or S-boxes (in gram-positive bacteria) in the promoter regions of several MUT transporter-encoding operons suggests that many MUT family members transport organic sulfur compounds. [ABSTRACT FROM AUTHOR]

Dubin-Johnson syndrome is a recessive inherited disorder with conjugated hyperbilirubinemia caused by a dysfunction of multidrug resistance protein 2 (MRP2) on the canalicular membrane of hepatocytes. A mutational analysis of the MRP2 gene was carried out in three Japanese patients and their family members. In two patients, the homozygous mutations c.1901del67 and c,2272del168 were found. In the third patient, a -24C→T polymorphism and the two mutations c.1901del67 and 2026G→C were detected. The 2026G→C mutation was a novel mutation in exon 16 affecting the conversion of Gly[sup 676] to Arg[sup 676] (G676R) in the MRP2 protein, and was not detected in fifty healthy volunteers. The G676R mutation was located in the Walker A motif of the first nucleotide binding domain in the MRP2 protein, and it was suggested that the mutation induced the dysfunction of the MRP2 protein. It was concluded that the compound heterozygosity of the two mutations of the MRP2 gene in the third patient contributed to the induction of hyperbilirubinemia in this case. [ABSTRACT FROM AUTHOR]

The human ATP-binding cassette (ABC) transporters comprise a large family of membrane transport proteins and play a vital role in many cellular processes. The genes provide functions as diverse as peptide transport, cholesterol and sterol transport, bile acid, retinoid, and iron transport. In addition some ABC genes play a role as regulatory elements. Many ABC genes play a role in human genetic diseases, and several are critical drug transport proteins overexpressed in drug resistant cells. Analysis of the gene products allows the genes to be grouped into seven different subfamilies. [ABSTRACT FROM AUTHOR]

Juliano and Ling initially reported the expression of a 170 kDa glycoprotein in the membrane of Chinese hamster ovarian cells in 1976, and named this glycoprotein P-glycoprotein (P-gp) based on its predicted role of causing “permeability” of the cell membrane. After much research on anthracycline-resistance, this P-gp was finally characterized as a multidrug-resistant protein coded by the mdrl gene. Multidrug resistance associated protein (MRP) was initially cloned from H69AR, a human small cell-lung carcinoma cell line which is resistant to doxorubicin (DXR) but does not express P-gp. MRP also excretes substrates through the cell membrane using energy from ATP catabolism. The substrate of MRP is conjugated with glutathione before active efflux from cell membrane. Recently, membrane transporter proteins were re-categorized as members of “ATP-Binding Cassette transporter” (ABC-transporter) superfamily, as shown at http://www.med.rug.nl/mdl/humanabc. htm and http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html. A total of ABC transporters have been defined, and MDR1 and multidrug resistance associated protein 1 (MRP1) were reclassified as ABCB1 and ABCC1, respectively. Their associated superfamilies include 11 and 13 other protein, in addition to ABCB and ABCC, respectively. Lung resistance-related protein (LRP) is not a member of the superfamily of ABC transporter proteins, because it shows nuclear membrane expression and transports substrate between nucleus and cytoplasm. LRP was initially cloned from a non-small cell lung carcinoma cell line, SW1573/2R120 which is resistant to DXR, vincristine, etoposide and gramicidin D and does not express Pgp. The mechanisms of resistance remains unclear, and why some resistant cell lines express P-gp and others express MRP and/or LRP is likewise unclear. [ABSTRACT FROM AUTHOR]

Inherent or acquired resistance of tumor cells to cytotoxic drugs represents a major limitation to the successful chemotherapeutic treatment of cancer. During the past three decades dramatic progress has been made in the understanding of the molecular basis of this phenomenon. Analyses of drug-selected tumor cells which exhibit simultaneous resistance to structurally unrelated anti-cancer drugs have led to the discovery of the human MDR1 gene product, P-glycoprotein, as one of the mechanisms responsible for multidrug resistance. Overexpression of this 170 kDa N-glycosylated plasma membrane protein in mammalian cells has been associated with ATP-dependent reduced drug accumulation, suggesting that P-glycoprotein may act as an energy-dependent drug efflux pump. P-glycoprotein consists of two highly homologous halves each of which contains a transmembrane domain and an ATP binding fold. This overall architecture is characteristic for members of the ATP-binding cassette or ABC superfamily of transporters. Cell biological, molecular genetic and biochemical approaches have been used for structure-function studies of P-glycoprotein and analysis of its mechanism of action. This review summarizes the current status of knowledge on the domain organization, topology and higher order structure of P-glycoprotein, the location of drug- and ATP binding sites within P-glycoprotein, its ATPase and drug transport activities, its possible functions as an ion channel, ATP channel and lipid transporter, its potential role in cholesterol biosynthesis, and the effects of phosphorylation on P-glycoprotein activity. [ABSTRACT FROM AUTHOR]

The major histocompatibility complexes of mice, rats and humans each contain a pair of related genes, Tap-1 and Tap-2, that encode members of a large superfamily of proteins having similar structure and function. The TAP-1 (previously called HAM1 in the mouse) and TAP-2 (HAM2) proteins each contain 6-8 predicted membrane-spanning α helices, and a cytoplasmic domain containing a putative ATP-binding site. Recent evidence suggests that a functional TAP-1/TAP-2 heterodimer is required for efficient presentation of antigens to CD8+ cytotoxic T cells. This heterodimer resides in the membrane of the endoplasmic reticulum (ER), and probably functions to transport peptides (produced in the cytoplasm) into the ER lumen for binding to MHC class I molecules. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor on C backbone assignment of MJ1267, an ATP-binding cassette in the January 2002 issue of the journal "Biomolecular NMR."