Balázs Sarkadi

An inventory of the human ABC proteins.

Currently 30 human ABC proteins are represented by full sequences in various databases, and this paper provides a brief overview of these proteins. ABC proteins are composed of transmembrane domains (TMDs), and nucleotide binding domains (NBDs, or ATP-binding cassettes, ABSs). The arrangement of these domains, together with available membrane topology models of the family members, are presented. Based on their sequence similarity scores, the members of the human ABC protein family can be grouped into eight subfamilies. At present the MDR/TAP, the ALD, the MRP/CFTR, the ABC1, the White, the RNAseL inhibitor, the ANSA, and the GCN20 subfamilies are identified. Mutations of many human ABC proteins are known to be causative in inherited diseases, and a short description of the molecular pathology of these ABC gene-related genetic diseases is also provided.

The Role of ABC Transporters in Drug Resistance, Metabolism and Toxicity

ATP Binding Cassette (ABC) transporters form a special family of membrane proteins, characterized by homologous ATP-binding, and large, multispanning transmembrane domains. Several members of this family are primary active transporters, which significantly modulate the absorption, metabolism, cellular effectivity and toxicity of pharmacological agents. This review provides a general overview of the human ABC transporters, their expression, localization and basic mechanism of action. Then we shortly deal with the human ABC transporters as targets of therapeutic interventions in medicine, including cancer drug resistance, lipid and other metabolic disorders, and even gene therapy applications. We place a special emphasis on the three major groups of ABC transporters involved in cancer multidrug resistance (MDR). These are the classical P-glycoprotein (MDR1, ABCB1), the multidrug resistance associated proteins (MRPs, in the ABCC subfamily), and the ABCG2 protein, an ABC half-transporter. All these proteins catalyze an ATP-dependent active transport of chemically unrelated compounds, including anticancer drugs. MDR1 (P-glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds. Based on the physiological expression and role of these transporters, we provide examples for their role in Absorption-Distribution-Metabolism-Excretion (ADME) and toxicology, and describe several basic assays which can be applied for screening drug interactions with ABC transporters in the course of drug research and development.

The role of ABC transporters in drug absorption, distribution, metabolism, excretion and toxicity (ADME–Tox)

ATP binding cassette (ABC) drug transporters play an important role in cancer drug resistance, protection against xenobiotics, and in general in the passage of drugs through cellular and tissue barriers. This review explores how human ABC transporters modulate the pharmacological effects of various drugs, and how this predictable ADME-TOX modulation can be used during the process of drug discovery and development. We provide a description of the relevant human ABC drug transporters and review the models and assay systems that can be applied for the analysis of their expected drug interactions. The use of the in vitro, in vivo, in silico models, their combination, and the emerging clinical information are evaluated with respect to their potential application in early drug screening.

Activation of ion transport pathways by changes in cell volume.

Swelling-activated K+ and Cl- channels, which mediate RVD, are found in most cell types. Prominent exceptions to this rule include red cells, which together with some types of epithelia, utilize electroneutral [K(+)-Cl-] cotransport for down-regulation of volume. Shrinkage-activated Na+/H+ exchange and [Na(+)-K(+)-2 Cl-] cotransport mediate RVI in many cell types, although the activation of these systems may require special conditions, such as previous RVD. Swelling-activated K+/H+ exchange and Ca2+/Na+ exchange seem to be restricted to certain species of red cells. Swelling-activated calcium channels, although not carrying sufficient ion flux to contribute to volume changes may play an important role in the activation of transport pathways. In this review of volume-activated ion transport pathways we have concentrated on regulatory phenomena. We have listed known secondary messenger pathways that modulate volume-activated transporters, although the evidence that volume signals are transduced via these systems is preliminary. We have focused on several mechanisms that might function as volume sensors. In our view, the most important candidates for this role are the structures which detect deformation or stretching of the membrane and the skeletal filaments attached to it, and the extraordinary effects that small changes in concentration of cytoplasmic macromolecules may exert on the activities of cytoplasmic and membrane enzymes (macromolecular crowding). It is noteworthy that volume-activated ion transporters are intercalated into the cellular signaling network as receptors, messengers and effectors. Stretch-activated ion channels may serve as receptors for cell volume itself. Cell swelling or shrinkage may serve a messenger function in the communication between opposing surfaces of epithelia, or in the regulation of metabolic pathways in the liver. Finally, these transporters may act as effector systems when they perform regulatory volume increase or decrease. This review discusses several examples in which relatively simple methods of examining volume regulation led to the discovery of transporters ultimately found to play key roles in the transmission of information within the cell. So, why volume? Because its functionally important, its relatively cheap (if you happened to have everything else, you only need some distilled water or concentrated salt solution), and since it involves many disciplines of experimental biology, its fun to do.

Calcein accumulation as a fluorometric functional assay of the multidrug transporter

Acetoxymethyl ester (AM) derivatives of various fluorescent indicators (fura-2, fluo-3, indo-1, BCECF, calcein) are actively extruded by the multidrug transporter (MDR1, P-glycoprotein-Homolya, L. et al. (1993) J. Biol. Chem. 268, 21493-21496). In the present paper we show that the measurement of the accumulation of a fluorescent cell viability marker, calcein, can be effectively used as a rapid and sensitive fluorometric and flow cytometric assay for studying P-glycoprotein function. The rate of calcein accumulation in human MDR1-expressing cells is significantly lower than in the control cells, while various drug-resistance reversing agents (verapamil, vinblastine, oligomycin, cyclosporin A and UIC2 monoclonal antibody) greatly increase calcein trapping only in the MDR1-expressing cells. Since calcein-AM is not fluorescent and free calcein is not a substrate of the multidrug transporter, the assay is readily applicable for rapid kinetic studies of the MDR1 function. Calcein has a high fluorescence intensity in the visible range, thus changes in calcein uptake can be easily visualised and MDR1-expressing and control cells separated by conventional flow cytometry.

Functional Multidrug Resistance Protein (MRP1) Lacking the N-terminal Transmembrane Domain

The human multidrug resistance protein (MRP1) causes drug resistance by extruding drugs from tumor cells. In addition to an MDR-like core, MRP1 contains an N-terminal membrane-bound region (TMD0) connected to the core by a cytoplasmic linker (L0). We have studied truncated MRP1 versions containing either the MDR-like core alone or the core plus linker L0, produced in the baculovirus-insect (Sf9) cell system. Their function was examined in isolated membrane vesicles. Full-length MRP1 showed ATP-dependent, vanadate-sensitive accumulation of leukotriene C4 and N-ethylmaleimide glutathione. In addition, leukotriene C4-stimulated, vanadate-dependent nucleotide occlusion was detected. The MDR-like core was virtually inactive. Co-expression of the core with the N-terminal region including L0 fully restored MRP1 function. Unexpectedly, a truncated MRP1 mutant lacking the entire TMD0 region but still containing L0 behaved like wild-type MRP1 in vesicle uptake and nucleotide trapping experiments. We also expressed the MRP1 constructs in polarized canine kidney derived MDCKII cells. Like wild-type MRP1, the MRP1 protein without the TMD0 region was routed to the lateral plasma membrane and transported dinitrophenyl glutathione and daunorubicin. The TMD0L0 and the MRP1 minus TMD0L0 remained in an intracellular compartment. Taken together, these experiments strongly suggest that the TMD0 region is neither required for the transport function of MRP1 nor for its proper routing to the plasma membrane.

ABCG2 – a transporter for all seasons

The human ABCG2 (ABCP/MXR/BCRP) protein is a recently recognized ABC half‐transporter, which forms homodimers in the plasma membrane and actively extrudes a wide variety of chemically unrelated compounds from the cells. This protein protects our cells and tissues against various xenobiotics, with a crucial role in the intestine, liver, placenta, and the blood–brain barrier. Moreover, ABCG2 seems to have a key function in stem cell protection/regulation, and also in hypoxic defense mechanisms. Widely occurring single nucleotide polymorphisms in ABCG2 may affect absorption and distribution, altering the effectiveness and toxicity of drugs in large populations. At the clinics, overexpression of ABCG2 in tumor cells confers cancer multidrug resistance to a variety of newly developed anticancer agents. On the other hand, specific substrate mutants of ABCG2 are advocated for use as selectable markers in stem‐cell based gene therapy.

Membrane topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters

A group of ATP‐binding cassette (ABC) transporters, including the yeast cadmium transporter (YCF1), the mammalian multidrug resistance‐associated protein (MRP), the multispecific organic anion transporter and its congener (MOAT and EBCR), as well as the sulfonylurea receptor (SUR), group into a subfamily by sequence comparison. We suggest that these MRP‐related proteins are also characterized by a special, common membrane topology pattern. The most studied ABC transporters, the cystic fibrosis transmembrane conductance regulator (CFTR) and the multidrug resistance (MDR) proteins, were shown to contain a tandem repeat of six transmembrane helices, each set followed by an ATP‐binding domain. According to the present study, in contrast to various membrane topology predictions proposed for the different MRP‐related proteins, they all seem to have a CFTR/MDR‐like core structure, and an additional, large, N‐terminal hydrophobic region. This latter domain is predicted to contain 4–6 (most probably 5) transmembrane helices, and is occasionally glycosylated on the cell surface. Since all the MRP‐related transporters were shown to interact with anionic compounds, the N‐terminal membrane‐bound domain may have a key role in these interactions.

MDR3 P-glycoprotein, a Phosphatidylcholine Translocase, Transports Several Cytotoxic Drugs and Directly Interacts with Drugs as Judged by Interference with Nucleotide Trapping

The human MDR3 gene is a member of the multidrug resistance (MDR) gene family. The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine. The MDR1 P-glycoprotein related transports cytotoxic drugs. Its overexpression can make cells resistant to a variety of drugs. Attempts to show that MDR3 P-glycoprotein can cause MDR have been unsuccessful thus far. Here, we report an increased directional transport of several MDR1 P-glycoprotein substrates, such as digoxin, paclitaxel, and vinblastine, through polarized monolayers of MDR3-transfected cells. Transport of other good MDR1 P-glycoprotein substrates, including cyclosporin A and dexamethasone, was not detectably increased. MDR3 P-glycoprotein-dependent transport of a short-chain phosphatidylcholine analog and drugs was inhibited by several MDR reversal agents and other drugs, indicating an interaction between these compounds and MDR3 P-gp. Insect cell membranes from Sf9 cells overexpressing MDR3 showed specific MgATP binding and a vanadate-dependent,N-ethylmaleimide-sensitive nucleotide trapping activity, visualized by covalent binding with [α-32P]8-azido-ATP. Nucleotide trapping was (nearly) abolished by paclitaxel, vinblastine, and the MDR reversal agents verapamil, cyclosporin A, and PSC 833. We conclude that MDR3 P-glycoprotein can bind and transport a subset of MDR1 P-glycoprotein substrates. The rate of MDR3 P-glycoprotein-mediated transport is low for most drugs, explaining why this protein is not detectably involved in multidrug resistance. It remains possible, however, that drug binding to MDR3 P-glycoprotein could adversely affect phospholipid or toxin secretion under conditions of stress (e.g. in pregnant heterozygotes with oneMDR3 null allele).

Loss of ATP-dependent transport activity in pseudoxanthoma elasticum-associated mutants of human ABCC6 (MRP6).

Mutations in the ABCC6 (MRP6) gene cause pseudoxanthoma elasticum (PXE), a rare heritable disorder resulting in the calcification of elastic fibers. In the present study a cDNA encoding a full-length normal variant of ABCC6 was amplified from a human kidney cDNA library, and the protein was expressed in Sf9 insect cells. In isolated membranes ATP binding as well as ATP-dependent active transport by ABCC6 was demonstrated. We found that glutathione conjugates, including leukotriene C4 and N-ethylmaleimideS-glutathione (NEM-GS), were actively transported by human ABCC6. Organic anions (probenecid, benzbromarone, indomethacin), known to interfere with glutathione conjugate transport of human ABCC1 and ABCC2, inhibited the ABCC6-mediated NEM-GS transport in a specific manner, indicating that ABCC6 has a unique substrate specificity. We have also expressed three missense mutant forms of ABCC6, which have recently been shown to cause PXE. MgATP binding was normal in these proteins; ATP-dependent NEM-GS or leukotriene C4 transport, however, was abolished. Our data indicate that human ABCC6 is a primary active transporter for organic anions. In the three ABCC6 mutant forms examined, the loss of transport activity suggests that these mutations result in a PXE phenotype through a direct influence on the transport activity of this ABC transporter.