ABC transporter

thumb|Lipid [[flippase MsbA]]

thumb|[[Molybdate-transporting ATPase|Molybdate transporter AB2C2 complex, open state]]

The ABC transporters, ATP synthase (ATP)-binding cassette transporters are a transport system superfamily that is one of the largest and possibly one of the oldest gene families. It is represented in all extant phyla, from prokaryotes to humans. ABC transporters belong to translocases.

ABC transporters often consist of multiple subunits, one or two of which are transmembrane proteins and one or two of which are membrane-associated AAA ATPases. The ATPase subunits utilize the energy of adenosine triphosphate (ATP) binding and hydrolysis to provide the energy needed for the translocation of substrates across membranes, either for uptake or for export of the substrate.

Most of the uptake systems also have an extracytoplasmic receptor, a solute binding protein. Some homologous ATPases function in non-transport-related processes such as translation of RNA and DNA repair. ABC transporters are considered to be an ABC superfamily based on the similarities of the sequence and organization of their ATP-binding cassette (ABC) domains, even though the integral membrane proteins appear to have evolved independently several times, and thus comprise different protein families. Like the ABC exporters, it is possible that the integral membrane proteins of ABC uptake systems also evolved at least three times independently, based on their high resolution three-dimensional structures. ABC uptake porters take up a large variety of nutrients, biosynthetic precursors, trace metals and vitamins, while exporters transport lipids, sterols, drugs, and a large variety of primary and secondary metabolites. Some of these exporters in humans are involved in tumor resistance, cystic fibrosis and a range of other inherited human diseases. High level expression of the genes encoding some of these exporters in both prokaryotic and eukaryotic organisms (including human) result in the development of resistance to multiple drugs such as antibiotics and anti-cancer agents.

Hundreds of ABC transporters have been characterized from both prokaryotes and eukaryotes. ABC genes are essential for many processes in the cell, and mutations in human genes cause or contribute to several human genetic diseases. Forty eight ABC genes have been reported in humans. Among these, many have been characterized and shown to be causally related to diseases present in humans such as cystic fibrosis, adrenoleukodystrophy, Stargardt disease, drug-resistant tumors, Dubin–Johnson syndrome, Byler's disease, progressive familiar intrahepatic cholestasis, X-linked sideroblastic anemia, ataxia, and persistent and hyperinsulimenic hypoglycemia.

Function

ABC transporters utilize the energy of ATP binding and hydrolysis to transport various substrates across cellular membranes. They are divided into three main functional categories. In prokaryotes, importers mediate the uptake of nutrients into the cell. The substrates that can be transported include ions, amino acids, peptides, sugars, and other molecules that are mostly hydrophilic. The membrane-spanning region of the ABC transporter protects hydrophilic substrates from the lipids of the membrane bilayer thus providing a pathway across the cell membrane. Eukaryotes do not possess any importers. Exporters or effluxers, which are present both in prokaryotes and eukaryotes, function as pumps that extrude toxins and drugs out of the cell. In gram-negative bacteria, exporters transport lipids and some polysaccharides from the cytoplasm to the periplasm. The third subgroup of ABC proteins do not function as transporters, but are rather involved in translation and DNA repair processes. Pathogens use siderophores, such as Enterobactin, to scavenge iron that is in complex with high-affinity iron-binding proteins or erythrocytes. These are high-affinity iron-chelating molecules that are secreted by bacteria and reabsorb iron into iron-siderophore complexes. The chvE-gguAB gene in Agrobacterium tumefaciens encodes glucose and galactose importers that are also associated with virulence. Transporters are extremely vital in cell survival such that they function as protein systems that counteract any undesirable change occurring in the cell. For instance, a potential lethal increase in osmotic strength is counterbalanced by activation of osmosensing ABC transporters that mediate uptake of solutes. Other than functioning in transport, some bacterial ABC proteins are also involved in the regulation of several physiological processes. They also play important roles in biosynthetic pathways, including extracellular polysaccharide biosynthesis and cytochrome biogenesis.

Eukaryotic

Although most eukaryotic ABC transporters are effluxers, some are not directly involved in transporting substrates. In the cystic fibrosis transmembrane regulator (CFTR) and in the sulfonylurea receptor (SUR), ATP hydrolysis is associated with the regulation of opening and closing of ion channels carried by the ABC protein itself or other proteins. Such diseases include Mendelian diseases and complex genetic disorders such as cystic fibrosis, adrenoleukodystrophy, Stargardt disease, Tangier disease, immune deficiencies, progressive familial intrahepatic cholestasis, Dubin–Johnson syndrome, Pseudoxanthoma elasticum, persistent hyperinsulinemic hypoglycemia of infancy due to focal adenomatous hyperplasia, X-linked sideroblastosis and anemia, age-related macular degeneration, familial hypoapoproteinemia, Retinitis pigmentosum, cone rod dystrophy, and others. possibly related to the outward transport of the lipid platelet activating factor (PAF). It has also been reported that ABCB1 mediates transport of cortisol and dexamethasone, but not of progesterone in ABCB1 transfected cells. MDR1 can also transport cholesterol, short-chain and long-chain analogs of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (SM), and glucosylceramide (GlcCer). Multispecific transport of diverse endogenous lipids through the MDR1 transporter can possibly affect the transbilayer distribution of lipids, in particular of species normally predominant on the inner plasma membrane leaflet such as PS and PE. Evidence has shown that placental expression of the ABC-transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are increased in preterm compared to term placentae, with P-gp expression further increased in preterm pregnancies with chorioamnionitis. To a lesser extent, increasing maternal BMI also associated with increased placental ABC-transporter expression, but only at preterm. These domains consist of two trans-membrane (T) domains and two cytosolic (A) domains. The two T domains alternate between an inward and outward facing orientation, and the alternation is powered by the hydrolysis of adenosine triphosphate or ATP. ATP binds to the A subunits and it is then hydrolyzed to power the alternation, but the exact process by which this happens is not known. The four domains can be present in four separate polypeptides, which occur mostly in bacteria, or present in one or two multi-domain polypeptides. In most exporters, the N-terminal transmembrane domain and the C-terminal ABC domains are fused as a single polypeptide chain, arranged as TMD-NBD-TMD-NBD. An example is the E. coli hemolysin exporter HlyB. Importers have an inverted organization, that is, NBD-TMD-NBD-TMD, where the ABC domain is N-terminal whereas the TMD is C-terminal, such as in the E. coli MacB protein responsible for macrolide resistance. importer involved in the uptake of vitamin B12. Most exporters, such as in the multidrug exporter Sav1866 from Staphylococcus aureus, are made up of a homodimer consisting of two half transporters or monomers of a TMD fused to a nucleotide-binding domain (NBD). A full transporter is often required to gain functionality. Some ABC transporters have additional elements that contribute to the regulatory function of this class of proteins. In particular, importers have a high-affinity binding protein (BP) that specifically associates with the substrate in the periplasm for delivery to the appropriate ABC transporter. Exporters do not have the binding protein but have an intracellular domain (ICD) that joins the membrane-spanning helices and the ABC domain. The ICD is believed to be responsible for communication between the TMD and NBD. This diagnostic fold can also be found in the MalF and MalG TM subunits of MalFGK2 and the Met transporter MetI. In the MetI transporter, a minimal set of 5 transmembrane helices constitute this fold while an additional helix is present for both ModB and MalG. The common organization of the fold is the "up-down" topology of the TM2-5 helices that lines the translocation pathway and the TM1 helix wrapped around the outer, membrane-facing surface and contacts the other TM helices.

The type II ABC importer fold is observed in the twenty TM helix-domain of BtuCD a homologous transporter from Haemophilus influenzae. In BtuCD, the packing of the helices is complex. The noticeable pattern is that the TM2 helix is positioned through the center of the subunit where it is surrounded in close proximity by the other helices. Meanwhile, the TM5 and TM10 helices are positioned in the TMD interface. The membrane spanning region of ABC exporters is organized into two "wings" that are composed of helices TM1 and TM2 from one subunit and TM3-6 of the other, in a domain-swapped arrangement. A prominent pattern is that helices TM1-3 are related to TM4-6 by an approximate twofold rotation around an axis in the plane of the membrane. It is generally observed that the ATP bound state is associated with the most extensive interface between ABC domains, whereas the structures of nucleotide-free transporters exhibit conformations with greater separations between the ABC domains. GlcV, MJ1267, E. coli MalK (E.c.MalK), T. litoralis MalK (TlMalK), and exporters such as TAP, HlyB, MJ0796, Sav1866, In these transporters, ATP is bound to the ABC domain. Two molecules of ATP are positioned at the interface of the dimer, sandwiched between the Walker A motif of one subunit and the LSGGQ motif of the other. and reported in structures of MJ0796, the NBD subunit of the LolD transporter from Methanococcus jannaschii

Nucleotide binding is required to ensure the electrostatic and/or structural integrity of the active site and contribute to the formation of an active NBD dimer. Binding of ATP is stabilized by the following interactions: (1) ring-stacking interaction of a conserved aromatic residue preceding the Walker A motif and the adenosine ring of ATP, (2) hydrogen-bonds between a conserved lysine residue in the Walker A motif and the oxygen atoms of the β- and γ-phosphates of ATP and coordination of these phosphates and some residues in the Walker A motif with Mg2+ ion, In addition, a residue that suggests the tight coupling of ATP binding and dimerization, is the conserved histidine in the H-loop. This histidine contacts residues across the dimer interface in the Walker A motif and the D loop, a conserved sequence following the Walker B motif.

The enzymatic hydrolysis of ATP requires proper binding of the phosphates and positioning of the γ-phosphate to the attacking water. and coordinate with Mg2+. ABC importers and exporters have a common mechanism for transporting substrates. They are similar in their structures. The model that describes the conformational changes associated with the binding of the substrate is the alternating-access model. In this model, the substrate binding site alternates between outward- and inward-facing conformations. The relative binding affinities of the two conformations for the substrate largely determines the net direction of transport. For importers, since translocation is directed from the periplasm to the cytoplasm, the outward-facing conformation has higher binding affinity for the substrate. In contrast, the substrate binding affinity in exporters is greater in the inward-facing conformation.

The general mechanism for the transport cycle of ABC transporters has not been fully elucidated, but substantial structural and biochemical data has accumulated to support a model in which ATP binding and hydrolysis is coupled to conformational changes in the transporter. The resting state of all ABC transporters has the NBDs in an open dimer configuration, with low affinity for ATP. This open conformation possesses a chamber accessible to the interior of the transporter. The transport cycle is initiated by binding of substrate to the high-affinity site on the TMDs, which induces conformational changes in the NBDs and enhances the binding of ATP. Two molecules of ATP bind, cooperatively, to form the closed dimer configuration. The closed NBD dimer induces a conformational change in the TMDs such that the TMD opens, forming a chamber with an opening opposite to that of the initial state. The affinity of the substrate to the TMD is reduced, thereby releasing the substrate. Hydrolysis of ATP follows and then sequential release of Pi and then ADP restores the transporter to its basal configuration. Although a common mechanism has been suggested, the order of substrate binding, nucleotide binding and hydrolysis, and conformational changes, as well as interactions between the domains is still debated.

Several groups studying ABC transporters have differing assumptions on the driving force of transporter function. It is generally assumed that ATP hydrolysis provides the principal energy input or "power stroke" for transport and that the NBDs operate alternately and are possibly involved in different steps in the transport cycle. However, recent structural and biochemical data shows that ATP binding, rather than ATP hydrolysis, provides the "power stroke". It may also be that since ATP binding triggers NBD dimerization, the formation of the dimer may represent the "power stroke." In addition, some transporters have NBDs that do not have similar abilities in binding and hydrolyzing ATP and that the interface of the NBD dimer consists of two ATP binding pockets suggests a concurrent function of the two NBDs in the transport cycle. Also, ATP binding induces substantial conformational changes in the TMDs. Spectroscopic, protease accessibility and crosslinking studies have shown that ATP binding to the NBDs induces conformational changes in multidrug resistance-associated protein-1 (MRP1), HisPMQ, LmrA, and Pgp. Two dimensional crystal structures of AMP-PNP-bound Pgp showed that the major conformational change during the transport cycle occurs upon ATP binding and that subsequent ATP hydrolysis introduces more limited changes. Rotation and tilting of transmembrane α-helices may both contribute to these conformational changes. Other studies have focused on confirming that ATP binding induces NBD closed dimer formation. Biochemical studies of intact transport complexes suggest that the conformational changes in the NBDs are relatively small. In the absence of ATP, the NBDs may be relatively flexible, but they do not involve a major reorientation of the NBDs with respect to the other domains. ATP binding induces a rigid body rotation of the two ABC subdomains with respect to each other, which allows the proper alignment of the nucleotide in the active site and interaction with the designated motifs. There is strong biochemical evidence that binding of two ATP molecules can be cooperative, that is, ATP must bind to the two active site pockets before the NBDs can dimerize and form the closed, catalytically active conformation.

In gram-negative organisms, ABC transporters mediate secretion of protein substrates across inner and outer membranes simultaneously without passing through the periplasm. This type of secretion is referred to as type I secretion, which involves three components that function in concert: an ABC exporter, a membrane fusion protein (MFP), and an outer membrane factor (OMF). An example is the secretion of hemolysin (HlyA) from E. coli where the inner membrane ABC transporter HlyB interacts with an inner membrane fusion protein HlyD and an outer membrane facilitator TolC. TolC allows hemolysin to be transported across the two membranes, bypassing the periplasm. P-glycoprotein is the best-studied efflux pump and as such has offered important insights into the mechanism of bacterial pumps. Multidrug resistant (MDR) homologs, also known as P-glycoproteins, represent the largest subfamily in plants with 22 members and the second largest overall ABC subfamily. The B subfamily of plant ABC transporters (ABCBs) are characterized by their localization to the plasma membrane. Plant ABCB transporters are characterized by heterologously expressing them in Escherichia coli, Saccharomyces cerevisiae, Schizosaccharomyces pombe (fission yeast), and HeLa cells to determine substrate specificity.

Plant ABCB transporters have shown to transport the phytohormone indole-3-acetic acid ( IAA), also known as auxin, the essential regulator for plant growth and development. The directional polar transport of auxin mediates plant environmental responses through processes such as phototropism and gravitropism. Two of the best studied auxin transporters, ABCB1 and ABCB19, have been characterized to be primary auxin exporters

Sav1866

The first high-resolution structure reported for an ABC exporter was that of Sav1866 (3.A.1.106.2) from Staphylococcus aureus. Sav1866 is a homolog of multidrug ABC transporters. It shows significant sequence similarity to human ABC transporters of subfamily B that includes MDR1 and TAP1/TAP2. The ATPase activity of Sav1866 is known to be stimulated by cancer drugs such as doxorubicin, vinblastine and others, which suggests similar substrate specificity to P-glycoprotein and therefore a possible common mechanism of substrate translocation. Sav1866 is a homodimer of half transporters, and each subunit contains an N-terminal TMD with six helices and a C-terminal NBD. The NBDs are similar in structure to those of other ABC transporters, in which the two ATP binding sites are formed at the dimer interface between the Walker A motif of one NBD and the LSGGQ motif of the other. The ADP-bound structure of Sav1866 shows the NBDs in a closed dimer and the TM helices split into two "wings" oriented towards the periplasm, forming the outward-facing conformation. Each wing consists of helices TM1-2 from one subunit and TM3-6 from the other subunit. It contains long intracellular loops (ICLs or ICD) connecting the TMDs that extend beyond the lipid bilayer into the cytoplasm and interacts with the 8=D. Whereas the importers contain a short coupling helix that contact a single NBD, Sav1866 has two intracellular coupling helices, one (ICL1) contacting the NBDs of both subunits and the other (ICL2) interacting with only the opposite NBD subunit. MsbA from E. coli is 36% identical to the NH2-terminal half of human MDR1, suggesting a common mechanism for transport of amphiphatic and hydrophobic substrates. The MsbA gene encodes a half transporter that contains a transmembrane domain (TMD) fused with a nucleotide-binding domain (NBD). It is assembled as a homodimer with a total molecular mass of 129.2 kD. MsbA contains 6 TMDs on the periplasmic side, an NBD located on the cytoplasmic side of the cell membrane, and an intracellular domain (ICD), bridging the TMD and NBD. This conserved helix extending from the TMD segments into or near the active site of the NBD is largely responsible for crosstalk between TMD and NBD. In particular, ICD1 serves as a conserved pivot about which the NBD can rotate, therefore allowing the NBD to disassociate and dimerize during ATP binding and hydrolysis.

thumb|Structures of MsbA depicting the three conformational states: open apo (), closed apo (), and nucleotide-bound ()

Previously published (and now retracted) X-ray structures of MsbA were inconsistent with the bacterial homolog Sav1866. The structures were reexamined and found to have an error in the assignment of the hand resulting to incorrect models of MsbA. Recently, the errors have been rectified and new structures have been reported. and EPR spectroscopy studies. The relatively large chamber allows it to accommodate large head groups such as that present in lipid A. Significant conformational changes are required to move the large sugar head groups across the membrane. The difference between the two nucleotide-free (apo) structures is the ≈30° pivot of TM4/TM5 helices relative to the TM3/TM6 helices. In the closed apo state (from V. cholerae MsbA), the NBDs are aligned and although closer, have not formed an ATP sandwich, and the P loops of opposing monomers are positioned next to one another. In comparison to the open conformation, the dimer interface of the TMDs in the closed, inward-facing conformation has extensive contacts. For both apo conformations of MsbA, the chamber opening is facing inward. The structure of MsbA-AMP-PNP (5'-adenylyl-β-γ-imidodiphosphate), obtained from S. typhimurium, is similar to Sav1866. The NBDs in this nucleotide-bound, outward-facing conformation, come together to form a canonical ATP dimer sandwich, that is, the nucleotide is situated in between the P-loop and LSGGQ motif. The conformational transition from MsbA-closed-apo to MsbA-AMP-PNP involves two steps, which are more likely concerted: a ≈10° pivot of TM4/TM5 helices towards TM3/TM6, bringing the NBDs closer but not into alignment followed by tilting of TM4/TM5 helices ≈20° out of plane. The twisting motion results in the separation of TM3/TM6 helices away from TM1/TM2 leading to a change from an inward- to an outward- facing conformation. Thus, changes in both the orientation and spacing of the NBDs dramatically rearrange the packing of transmembrane helices and effectively switch access to the chamber from the inner to the outer leaflet of the membrane. Recent work has resulted in the discovery of MsbA inhibitors.

Mechanism of transport for exporters

thumb|left|Proposed mechanism of transport for ABC exporters. This model was based on structural and biochemical studies on MsbA.

ABC exporters have a transport mechanism that is consistent with both the alternating-access model and ATP-switch model. In the apo states of exporters, the conformation is inward-facing and the TMDs and NBDs are relatively far apart to accommodate amphiphilic or hydrophobic substrates. For MsbA, in particular, the size of the chamber is large enough to accommodate the sugar groups from lipopolysaccharides (LPS). As has been suggested by several groups, binding of substrate initiates the transport cycle. The "power stroke", that is, ATP binding that induces NBD dimerization and formation of the ATP sandwich, drives the conformational changes in the TMDs. In MsbA, the sugar head groups are sequestered within the chamber during the "power stroke". The cavity is lined with charged and polar residues that are likely solvated creating an energetically unfavorable environment for hydrophobic substrates and energetically favorable for polar moieties in amphiphilic compounds or sugar groups from LPS. Since the lipid cannot be stable for a long time in the chamber environment, lipid A and other hydrophobic molecules may "flip" into an energetically more favorable position within the outer membrane leaflet. The "flipping" may also be driven by the rigid-body shearing of the TMDs while the hydrophobic tails of the LPS are dragged through the lipid bilayer. Repacking of the helices switches the conformation into an outward-facing state. ATP hydrolysis may widen the periplasmic opening and push the substrate towards the outer leaflet of the lipid bilayer. Hydrolysis of the second ATP molecule and release of Pi separates the NBDs followed by restoration of the resting state, opening the chamber towards the cytoplasm for another cycle.

Role in multi drug resistance

ABC transporters are known to play a crucial role in the development of multidrug resistance (MDR). In MDR, patients that are on medication eventually develop resistance not only to the drug they are taking but also to several different types of drugs. This is caused by several factors, one of which is increased expulsion of the drug from the cell by ABC transporters. For example, the ABCB1 protein (P-glycoprotein) functions in pumping tumor suppression drugs out of the cell. Pgp also called MDR1, ABCB1, is the prototype of ABC transporters and also the most extensively-studied gene. Pgp is known to transport organic cationic or neutral compounds. A few ABCC family members, also known as MRP, have also been demonstrated to confer MDR to organic anion compounds. The most-studied member in ABCG family is ABCG2, also known as BCRP (breast cancer resistance protein) confer resistance to most Topoisomerase I or II inhibitors such as topotecan, irinotecan, and doxorubicin.

It is unclear exactly how these proteins can translocate such a wide variety of drugs, however, one model (the hydrophobic vacuum cleaner model) states that, in P-glycoprotein, the drugs are bound indiscriminately from the lipid phase based on their hydrophobicity.

The Discovery of the first eukaryotic ABC transporter protein came from studies on tumor cells and cultured cells that exhibited resistance to several drugs with unrelated chemical structures. These cells were shown to express elevated levels of multidrug-resistance (MDR) transport protein which was originally called P-glycoprotein (P-gp), but it is also referred to as multidrug resistance protein 1 (MDR1) or ABCB1. This protein uses ATP hydrolysis, just like the other ABC transporters, to export a large variety of drugs from the cytosol to the extracellular medium. In multidrug-resistant cells, the MDR1 gene is frequently amplified. This results in a large overproduction of the MDR1 protein.

The substrates of mammalian ABCB1 are primarily planar, lipid-soluble molecules with one or more positive charges. All of these substrates compete with one another for transport, suggesting that they bind to the same or overlapping sites on the protein. Many of the drugs that are transported out by ABCB1 are small, nonpolar drugs that diffuse across the extracellular medium into the cytosol, where they block various cellular functions. Drugs such as colchicine and vinblastine, which block assembly of microtubules, freely cross the membrane into the cytosol, but the export of these drugs by ABCB1 reduces their concentration in the cell. Therefore, it takes a higher concentration of the drugs is required to kill the cells that express ABCB1 than those that do not express the gene.

To solve the problems associated with multidrug-resistance by MDR1, different types of drugs can be used or the ABC transporters themselves must be inhibited. For other types of drugs to work, they must bypass the resistance mechanism, which is the ABC transporter. To do this other anticancer drugs can be utilized such as alkylating drugs (cyclophosphamide), antimetabolites (5-fluorouracil), and the anthracycline modified drugs (annamycin and doxorubicin-peptide). These drugs would not function as a substrate of ABC transporters, and would thus not be transported. The other option is to use a combination of ABC inhibitory drugs and anticancer drugs at the same time. This would reverse the resistance to the anticancer drugs so that they could function as intended. The substrates that reverse the resistance to anticancer drugs are called chemosensitizers.

Physiological role

In addition to conferring MDR in tumor cells, ABC transporters are also expressed in the membranes of healthy cells, where they facilitate the transport of various endogenous substances, as well as of substances foreign to the body. For instance, ABC transporters such as Pgp, the MRPs and BCRP limit the absorption of many drugs from the intestine, and pump drugs from the liver cells to the bile as a means of removing foreign substances from the body. A large number of drugs are either transported by ABC transporters themselves or affect the transport of other drugs. The latter scenario can lead to drug-drug interactions, sometimes resulting in altered effects of the drugs.

Methods to characterize ABC transporter interactions

There are a number of assay types that allow the detection of ABC transporter interactions with endogenous and xenobiotic compounds. The complexity of assay range from relatively simple membrane assays. like vesicular transport assay, ATPase assay to more complex cell based assays up to intricate in vivo detection methodologies.

Membrane assays

The vesicular transport assay detects the translocation of molecules by ABC transporters. Membranes prepared under suitable conditions contain inside-out oriented vesicles with the ATP binding site and substrate binding site of the transporter facing the buffer outside. Substrates of the transporter are taken up into the vesicles in an ATP dependent manner. Rapid filtration using glass fiber filters or nitrocellulose membranes are used to separate the vesicles from the incubation solution and the test compound trapped inside the vesicles is retained on the filter. The quantity of the transported unlabelled molecules is determined by HPLC, LC/MS, LC/MS/MS. Alternatively, the compounds are radiolabeled, fluorescent or have a fluorescent tag so that the radioactivity or fluorescence retained on the filter can be quantified.

Various types of membranes from different sources (e.g. insect cells, transfected or selected mammalian cell lines) are used in vesicular transport studies. Membranes are commercially available or can be prepared from various cells or even tissues e.g. liver canalicular membranes. This assay type has the advantage of measuring the actual disposition of the substrate across the cell membrane. Its disadvantage is that compounds with medium-to-high passive permeability are not retained inside the vesicles making direct transport measurements with this class of compounds difficult to perform.

The vesicular transport assay can be performed in an "indirect" setting, where interacting test drugs modulate the transport rate of a reporter compound. This assay type is particularly suitable for the detection of possible drug-drug interactions and drug-endogenous substrate interactions. It is not sensitive to the passive permeability of the compounds and therefore detects all interacting compounds. Yet, it does not provide information on whether the compound tested is an inhibitor of the transporter, or a substrate of the transporter inhibiting its function in a competitive fashion. A typical example of an indirect vesicular transport assay is the detection of the inhibition of taurocholate transport by ABCB11 (BSEP).

Whole cell based assays

Efflux transporter-expressing cells actively pump substrates out of the cell, which results in a lower rate of substrate accumulation, lower intracellular concentration at steady state, or a faster rate of substrate elimination from cells loaded with the substrate. Transported radioactive substrates or labeled fluorescent dyes can be directly measured, or in an indirect set up, the modulation of the accumulation of a probe substrate (e.g. fluorescent dyes like rhodamine 123, or calcein) can be determined in the presence of a test drug.

ABCA

The ABCA subfamily is composed of 12 full transporters split into two subgroups. The first subgroup consists of seven genes that map to six different chromosomes. These are ABCA1, ABCA2, ABCA3, and ABCA4, ABCA7, ABCA12, and ABCA13. The other subgroup consists of ABCA5 and ABCA6 and ABCA8, ABCA9 and ABCA10. A8-10.

All of subgroup 2 is organized into a head to tail cluster of chromosomes on chromosome 17q24. Genes in this second subgroup are distinguished from ABCA1-like genes by having 37-38 exons as opposed to the 50 exons in ABCA1.

The ABCA1 subgroup is implicated in the development of genetic diseases. In the recessive Tangier's disease, the ABCA1 protein is mutated. Also, the ABCA4 maps to a region of chromosome 1p21 that contains the gene for Stargardt's disease. This gene is found to be highly expressed in rod photoreceptors and is mutated in Stargardt's disease, recessive retinitis pigmentism, and the majority of recessive cone-rod dystrophy. They are known to be involved in ion transport, toxin secretion, and signal transduction.

Three families of ABC exporters are defined by their evolutionary origins. Most uptake systems (all except 3.A.1.21) are of the ABC2 type, divided into type I and type II by the way they handle nucleotides. A special subfamily of ABC2 importers called ECF use a separate subunit for substrate recognition.

ABC1 ():

3.A.1.106 The Lipid Exporter (LipidE) Family
3.A.1.108 The β-Glucan Exporter (GlucanE) Family
3.A.1.109 The Protein-1 Exporter (Prot1E) Family
3.A.1.110 The Protein-2 Exporter (Prot2E) Family
3.A.1.111 The Peptide-1 Exporter (Pep1E) Family
3.A.1.112 The Peptide-2 Exporter (Pep2E) Family
3.A.1.113 The Peptide-3 Exporter (Pep3E) Family
3.A.1.117 The Drug Exporter-2 (DrugE2) Family
3.A.1.118 The Microcin J25 Exporter (McjD) Family
3.A.1.119 The Drug/Siderophore Exporter-3 (DrugE3) Family
3.A.1.123 The Peptide-4 Exporter (Pep4E) Family
3.A.1.127 The AmfS Peptide Exporter (AmfS-E) Family
3.A.1.129 The CydDC Cysteine Exporter (CydDC-E) Family
3.A.1.135 The Drug Exporter-4 (DrugE4) Family
3.A.1.139 The UDP-Glucose Exporter (U-GlcE) Family (UPF0014 Family)
3.A.1.201 The Multidrug Resistance Exporter (MDR) Family (ABCB)
3.A.1.202 The Cystic Fibrosis Transmembrane Conductance Exporter (CFTR) Family (ABCC)
3.A.1.203 The Peroxysomal Fatty Acyl CoA Transporter (P-FAT) Family (ABCD)
3.A.1.206 The a-Factor Sex Pheromone Exporter (STE) Family (ABCB)
3.A.1.208 The Drug Conjugate Transporter (DCT) Family (ABCC) (Dębska et al., 2011)
3.A.1.209 The MHC Peptide Transporter (TAP) Family (ABCB)
3.A.1.210 The Heavy Metal Transporter (HMT) Family (ABCB)
3.A.1.212 The Mitochondrial Peptide Exporter (MPE) Family (ABCB)
3.A.1.21 The Siderophore-Fe3+ Uptake Transporter (SIUT) Family

ABC2 ( [partial]):

3.A.1.101 The Capsular Polysaccharide Exporter (CPSE) Family
3.A.1.102 The Lipooligosaccharide Exporter (LOSE) Family
3.A.1.103 The Lipopolysaccharide Exporter (LPSE) Family
3.A.1.104 The Teichoic Acid Exporter (TAE) Family
3.A.1.105 The Drug Exporter-1 (DrugE1) Family
3.A.1.107 The Putative Heme Exporter (HemeE) Family
3.A.1.115 The Na+ Exporter (NatE) Family
3.A.1.116 The Microcin B17 Exporter (McbE) Family
3.A.1.124 The 3-component Peptide-5 Exporter (Pep5E) Family
3.A.1.126 The β-Exotoxin I Exporter (βETE) Family
3.A.1.128 The SkfA Peptide Exporter (SkfA-E) Family
3.A.1.130 The Multidrug/Hemolysin Exporter (MHE) Family
3.A.1.131 The Bacitracin Resistance (Bcr) Family
3.A.1.132 The Gliding Motility ABC Transporter (Gld) Family
3.A.1.133 The Peptide-6 Exporter (Pep6E) Family
3.A.1.138 The Unknown ABC-2-type (ABC2-1) Family
3.A.1.141 The Ethyl Viologen Exporter (EVE) Family (DUF990 Family; )
3.A.1.142 The Glycolipid Flippase (G.L.Flippase) Family
3.A.1.143 The Exoprotein Secretion System (EcsAB(C))
3.A.1.144: Functionally Uncharacterized ABC2-1 (ABC2-1) Family
3.A.1.145: Peptidase Fused Functionally Uncharacterized ABC2-2 (ABC2-2) Family
3.A.1.146: The actinorhodin (ACT) and undecylprodigiosin (RED) exporter (ARE) family
3.A.1.147: Functionally Uncharacterized ABC2-2 (ABC2-2) Family
3.A.1.148: Functionally Uncharacterized ABC2-3 (ABC2-3) Family
3.A.1.149: Functionally Uncharacterized ABC2-4 (ABC2-4) Family
3.A.1.150: Functionally Uncharacterized ABC2-5 (ABC2-5) Family
3.A.1.151: Functionally Uncharacterized ABC2-6 (ABC2-6) Family
3.A.1.152: The lipopolysaccharide export (LptBFG) Family ()
3.A.1.204 The Eye Pigment Precursor Transporter (EPP) Family (ABCG)
3.A.1.205 The Pleiotropic Drug Resistance (PDR) Family (ABCG)
3.A.1.211 The Cholesterol/Phospholipid/Retinal (CPR) Flippase Family (ABCA)
9.B.74 The Phage Infection Protein (PIP) Family
all uptake systems (3.A.1.1 - 3.A.1.34 except 3.A.1.21)
3.A.1.1 Carbohydrate Uptake Transporter-1 (CUT1)
3.A.1.2 Carbohydrate Uptake Transporter-2 (CUT2)
3.A.1.3 Polar Amino Acid Uptake Transporter (PAAT)
3.A.1.4 Hydrophobic Amino Acid Uptake Transporter (HAAT)
3.A.1.5 Peptide/Opine/Nickel Uptake Transporter (PepT)
3.A.1.6 Sulfate/Tungstate Uptake Transporter (SulT)
3.A.1.7 Phosphate Uptake Transporter (PhoT)
3.A.1.8 Molybdate Uptake Transporter (MolT)
3.A.1.9 Phosphonate Uptake Transporter (PhnT)
3.A.1.10 Ferric Iron Uptake Transporter (FeT)
3.A.1.11 Polyamine/Opine/Phosphonate Uptake Transporter (POPT)
3.A.1.12 Quaternary Amine Uptake Transporter (QAT)
3.A.1.13 Vitamin B12 Uptake Transporter (B12T)
3.A.1.14 Iron Chelate Uptake Transporter (FeCT)
3.A.1.15 Manganese/Zinc/Iron Chelate Uptake Transporter (MZT)
3.A.1.16 Nitrate/Nitrite/Cyanate Uptake Transporter (NitT)
3.A.1.17 Taurine Uptake Transporter (TauT)
3.A.1.19 Thiamin Uptake Transporter (ThiT)
3.A.1.20 Brachyspira Iron Transporter (BIT)
3.A.1.21 Siderophore-Fe3+ Uptake Transporter (SIUT)
3.A.1.24 The Methionine Uptake Transporter (MUT) Family (Similar to 3.A.1.3 and 3.A.1.12)
3.A.1.27 The γ-Hexachlorocyclohexane (HCH) Family (Similar to 3.A.1.24 and 3.A.1.12)
3.A.1.34 The Tryptophan (TrpXYZ) Family
ECF uptake systems
3.A.1.18 The Cobalt Uptake Transporter (CoT) Family
3.A.1.22 The Nickel Uptake Transporter (NiT) Family
3.A.1.23 The Nickel/Cobalt Uptake Transporter (NiCoT) Family
3.A.1.25 The Biotin Uptake Transporter (BioMNY) Family
3.A.1.26 The Putative Thiamine Uptake Transporter (ThiW) Family
3.A.1.28 The Queuosine (Queuosine) Family
3.A.1.29 The Methionine Precursor (Met-P) Family
3.A.1.30 The Thiamin Precursor (Thi-P) Family
3.A.1.31 The Unknown-ABC1 (U-ABC1) Family
3.A.1.32 The Cobalamin Precursor (B12-P) Family
3.A.1.33 The Methylthioadenosine (MTA) Family

ABC3 ():

3.A.1.114 The Probable Glycolipid Exporter (DevE) Family
3.A.1.122 The Macrolide Exporter (MacB) Family
3.A.1.125 The Lipoprotein Translocase (LPT) Family
3.A.1.134 The Peptide-7 Exporter (Pep7E) Family
3.A.1.136 The Uncharacterized ABC-3-type (U-ABC3-1) Family
3.A.1.137 The Uncharacterized ABC-3-type (U-ABC3-2) Family
3.A.1.140 The FtsX/FtsE Septation (FtsX/FtsE) Family
3.A.1.207 The Eukaryotic ABC3 (E-ABC3) Family

Images

Many structures of water-soluble domains of ABC proteins have been produced in recent years.