Multidrug resistance-associated protein 2 (MRP2/ABCC2) is a polyspecific efflux transporter of organic anions expressed in hepatocyte canalicular membranes. MRP2 dysfunction, in Dubin-Johnson syndrome or by off-target inhibition, for example by the uricosuric drug probenecid, elevates circulating bilirubin glucuronide and is a cause of jaundice. Here, we determine the cryo-EM structure of rat Mrp2 (rMrp2) in an autoinhibited state and in complex with probenecid. The autoinhibited state exhibits an unusual conformation for this class of transporter in which the regulatory domain is folded within the transmembrane domain cavity. In vitro phosphorylation, mass spectrometry and transport assays show that phosphorylation of the regulatory domain relieves this autoinhibition and enhances rMrp2 transport activity. The in vitro data is confirmed in human hepatocyte-like cells, in which inhibition of endogenous kinases also reduces human MRP2 transport activity. The drug-bound state reveals two probenecid binding sites that suggest a dynamic interplay with autoinhibition. Mapping of the Dubin-Johnson mutations onto the rodent structure indicates that many may interfere with the transition between conformational states.

The plasma membrane transporter ASCT2 is a well-known Na⁺-dependent obligatory antiporter of neutral amino acids. The crucial role of the residue C467 in the recognition and binding of the ASCT2 substrate glutamine, has been highlighted by structure/function relationship studies. The reconstitution in proteoliposomes of the human ASCT2 produced in P. pastoris is here employed to unveil another role of the C467 residue in the transport reaction. Indeed, the site-directed mutant C467A displayed a novel property of the transporter, i.e., the ability of mediating a low but measurable unidirectional transport of [³H]-glutamine. This reaction conforms to the main features of the ASCT2-mediated transport, namely the Na⁺-dependence, the pH dependence, the stimulation by cholesterol included in the proteoliposome membrane, and the specific inhibition by other common substrates of the reconstituted human ASCT2. Interestingly, the WT protein cannot catalyze the unidirectional transport of [³H]-glutamine, demonstrating an unspecific phenomenon. This difference is in favor of a structural conformational change between a WT and C467A mutant that triggers the appearance of the unidirectional flux; this feature has been investigated by comparing the available 3D structures in two different conformations, and two homology models built on the basis of hEAAT1 and GLT_Ph.

The human SLC7A10 transporter, also known as ASC-1, catalyzes the transport of some neutral amino acids. It is expressed in astrocytes, neurons, and adipose tissues, playing roles in learning, memory processes, and lipid metabolism, thus being involved in neurological and metabolic pathologies. Structure/function studies on this transporter are still in their infancy. In this study, we present a methodology for producing the recombinant human transporter in E. coli. Its transport function was assayed in proteoliposomes following the uptake of radiolabeled L-serine. After the testing of several growth conditions, the hASC-1 transporter was successfully expressed in BL21(DE3) codon plus RIL in the presence of 0.5% glucose and induced with 0.05 mM IPTG. After solubilization with C₁₂E₈ and cholesteryl hemisuccinate and purification by Ni-chelating chromatography, hASC-1 was reconstituted in proteoliposomes. In this experimental system it was able to catalyze an Na⁺-independent homologous antiport of L-serine. A Km for L-serine transport of 0.24 mM was measured. The experimental model developed in this work represents a reproducible system for the transport assay of hASC-1 in the absence of interferences. This tool will be useful to unveil unknown transport properties of hASC-1 and for testing ligands with possible application in human pharmacology.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.