Loss of ATP-dependent Transport Activity in Pseudoxanthoma Elasticum-associated Mutants of Human ABCC6 (MRP6)

Abstract

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 C 4 and N-ethylmaleimide S-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 C 4 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. Pseudoxanthoma elasticum (PXE)1 is a heritable disorder affecting several different elastic tissues including the skin, the elastic Bruch's membrane of the eye, and the arterial system.

Identification of Two Novel Missense Mutations (p.R1221C and p.R1357W) in the ABCC6 (MRP6) Gene in a Japanese Patient with Pseudoxanthoma Elasticum (PXE)

“…p.R1357W was found in NBD2. In vitro transport studies with three PXE-causing mutants (p.V1298F, p.G1302R and p.G1321S) within NBD2, that had been expressed in Sf9 cells, showed markedly reduced transport activities (12). We would expect a similar effect for the p.R1357W.…”

Mutational analysis of theABCC6 gene and the proximalABCC6 gene promoter in German patients with pseudoxanthoma elasticum (PXE)

“…MRP6 belongs to a family of transmembrane proteins which function as efflux transporters (Dean et al, 2001;Haimeur et al, 2004). The physiological function of MRP6 is still unknown, but recent reports suggest its implication in transmembrane transport of glutathionated metabolites (Belinsky et al, 2002;Ilias et al, 2002).…”

ABCC6 mutations in Italian families affected by pseudoxanthoma elasticum (PXE)

“…The precise localization in human tissues and the physiological role of MRP6 are still unknown (Scheffer et al, 2002). Recent reports suggest its implication in the cell extrusion of glutathionated metabolites (Belinsky et al, 2002;Ilias et al, 2002). To date, more than 60 causative mutations for PXE have been reported (Hu et al, 2003;Chassaing et al, 2004).…”