ABC Transporter Assays
Background of ABC Transporters
ATP-binding cassette (ABC) transporters are a superfamily of ubiquitous membrane proteins, translocating ions, lipids, peptides, and steroids across membranes powered by ATPs. Two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs) are required for the ABC transporter's normal functioning. ABC transporters participate in a vast array of pathological and physiological processes.
Fig.1 Cryo-EM structure of human ABCA4 in the apo state. (Xie, 2021)
Distributions and Functions of ABC Transporters
ABC transporters are generally present in membranes of cells, which regulate the ABC transporters’ functions of translocation, DNA repair, and chromosome maintenance. They are related to many inherited and metabolic diseases, including Stargardt disease, Alzheimer’s disease, Dubin–Johnson syndrome, adrenoleukodystrophy, X-linked sideroblastic, cystic fibrosis, hypoglycemia, Byler's disease, ataxia, etc. In addition, overexpression of ABC transporters can result in chemotherapeutic drug resistance.
Mechanisms of ABC Transporters
ABC transporters are a family of active transporters powered by ATP binding and/or hydrolysis on NBDs to migrate substrates across cell membranes through conformational alteration in the TMDs. The ABC transporter inhibitors are capable of optimization of the ABC transporter-caused drug resistance.
Published Data
| Paper Title | Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays |
| Journal | The FASEB Journal |
| Published | 2012 |
| Abstract | The largest family of transmembrane proteins is encoded by the ATP-binding cassette (ABC) genes. ABC transporters translocate a wide range of substrates across membranes. As the physiological function of ABC transporters remains incompletely understood, the study aimed to develop a novel method to study the substrate spectrum of ABC transporters. They put the transporter-containing inside-out membrane vesicles in body fluids extracted from mouse urine and analyzed the vesicle content by liquid chromatography/mass spectrometry (LC/MS)-based metabolomics. This approach has allowed them to obtain a comprehensive and relatively unbiased inventory of physiological substrates transported by ABCC2, which is a protein present at sites of uptake and elimination. |
| Result |
They used vesicular transport assays and found new substrates of ABCC2 in mice urine, including glucuronides of plant-derived xenobiotics, a class of compounds to which humans are exposed on a daily basis. In addition, the results have indicated that the excretion of these compounds in vivo depends on ABCC2. When compared to wild-type mice, the urinary excretion of several glucuronides was increased up to 20-fold in Abcc2-/- mice. In summary, the transportomics method described by the study has broad applicability, which is not restricted to urine but can be applied to other ATP-dependent transport proteins as well.
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Fig.2 Schematic representation of the use of vesicular transport assays to identify unknown ABCC2 substrates in body fluids. (Krumpochova, 2012)
References
- Xie, T.; et al. Structural basis of substrate recognition and translocation by human ABCA4. Nat Commun. 2021, 12: 3853-3853.
- Krumpochova, P.; et al. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays. The FASEB Journal. 2012, 26(2): 738-747.
