ABC transporter

ATP-binding cassette transporters (ABC-transporter) are members of a superfamily that is one of the largest, and most ancient families with representatives in all extant phyla from prokaryotes to humans. ^[4]. These are transmembrane proteins that function in the transport of a wide variety of substrates across extra- and intracellular membranes, including metabolic products, lipids and sterols, and drugs. Proteins are classified as ABC transporters based on the sequence and organization of their ATP-binding domain(s), also known as nucleotide-binding folds (NBFs). ABC transporters are involved in tumour resistance, cystic fibrosis, bacterial multidrug resistance, and a range of other inherited human diseases.

Function

ABC-transporters utilize the energy of ATP hydrolysis to transport various substrates across cellular membranes. Within bacteria, ABC-transporters mainly pump essential compounds such as sugars, vitamins, and metal ions into the cell. Within eukaryotes, ABC-transporters mainly transport molecules to the outside of the plasma membrane or into membrane-bound organelles such as the endoplasmic reticulum, mitochondria, etc. More recently, ABC-transporters have been shown to exist within the placenta, indicating they could play a protective role for the developing fetus against xenobiotics.¹

Structure

Typical ABC-transporters will contain two transmembrane domains (TMs), each of which consists of α-helices, which cross the phospholipid bilayer multiple times. These helices form between six to eleven (usually six) membrane-spanning regions. These transmembrane domains provide the specificity for the substrate, and prevent unwanted molecules from using the transporter. In between the TMs is a ligand binding-domain, which is on the extracellular side of the protein for importers and on the cytoplasmic side for exporters.

All ABC proteins also contain either one or two ATP-binding domain(s), (nucleotide-binding folds (NBFs)) and are located on the cytoplasm side of the membrane. These folds are divided into parts or motifs, called Walker A and Walker B, which are separated by approximately 90-120 amino acids. In addition, there is a third short and highly-conserved motif (called LSGGQ motif, C motif, or "signature" motif) located after the Walker B motif. Unlike the Walker A and Walker B motifs, which are found in other proteins that hydrolyze ATP, the signature motif is unique to ABC transporters. Other conserved motifs include the Q-loop, the his-loop, the pro-loop, and the D-loop. These folds form the cassettes, which the protein family is named after. The transmembrane domains and nucleotide-binding folds are often arranged in the order NH3+-TM-NBF-TM-NBF-COO-.

Many ABC transporters may be classified as half transporters or full transporters. Full transporters consist of the typical two TMs and two NBFs. Half transporters consist of only one TM and one NBF and must combine with another half transporter to gain functionality. Half transporters can thus form homodimers if two identical ABC transporters join, and heterodimers if two unlike ABC transporters join. Although these are the most common domain organisations, others exist. For instance, many bacterial importers are composed of four polypeptides. Every possible organisation for these domains has been observed (Higgins, 1992).

Method of operation

The mechanism of ABC transporters has not been fully identified. It is known that, when an ATP molecule binds to each cassette of an ABC-transporter, it induces a conformational change in which the NBFs interact more closely. This is communicated to the TMs, which shift to re-orientate the translocation pathway. This action is called the power stroke. In this conformation, the substrate binding is lower affinity, and so the substrates can be released.

Particular areas of controversy include the roles of ATP binding, ATP hydrolysis, and the subsequent dissociation of the ADP and Pi. Also unclear is how communication between the NBFs and the TMs takes place.

Role in multidrug 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 excretion 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 of 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.

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.[5]

Methods to characterize ABC transporter interactions

There are a number of assay types that allow the detection of ABC transporter interactions with endogeounous and xenobiotic compounds ². The complexity of assay range from relatively simple membrane assays ³ like vesicular transport assay, ATPase assay to more complex cell based assays upto intricate in vivo⁴ detection methodologies ⁵.

The vesicular transport assay detects the translocation of molecules by ABC transporters ⁶. 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 [6] 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).

Subfamilies

Human subfamilies

There are 48 known ABC transporters present in humans, which are classified into seven families by the Human Genome Organization.

Prokaryotic subfamilies

Images

Many structures of water-soluble domains of ABC proteins have been produced in recent years. ^[9]

References

^ Gedeon C, Behravan J, Koren G, Piquette-Miller M (2006). "Transport of glyburide by placental ABC transporters: implications in fetal drug exposure". Placenta 27 (11-12): 1096–102. doi:10.1016/j.placenta.2005.11.012. PMID 16460798.
^ Glavinas H, Krajcsi P, Cserepes J, Sarkadi B.The role of ABC transporters in drug resistance, metabolism and toxicity.Curr Drug Deliv. 2004 Jan;1(1):27-42.[1]
^ Glavinas H, Méhn D, Jani M, Oosterhuis B, Herédi-Szabó K, Krajcsi P.Utilization of membrane vesicle preparations to study drug-ABC transporter interactions. Expert Opin Drug Metab Toxicol. 2008 Jun;4(6):721-32. [2]
^ Jeffrey P, Summerfield SG.Challenges for blood-brain barrier (BBB) screening. Xenobiotica. 2007 Oct-Nov;37(10-11):1135-51.
^ This entire volume is dedicated to various methods used: Methods in enzymology Volume 292, 1998 [3]
^ Horio M, Gottesman MM, Pastan I.ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells.Proc Natl Acad Sci U S A. 1988 May;85(10):3580-4.

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