Functional analysis of ABCA8, a new drug transporter

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Abstract

We examined the transport capacity in Xenopus laevis oocytes of human EST KIAA0822/ABCA8, a member of the ABC superfamily. Substrates of ABCC2/MRP-2 such as [14C]estradiol-β-glucuronide, taurocholate, and LTC4, and of organic anion transporter (OAT), such as para-aminohippuric acid, ochratoxin-A, were significantly accumulated while tetraethylammonium and doxorubicin were not. The transport of [14C]estradiol-β-glucuronide was ATP-dependent and Km and Vmax values of 30.4 μM and 66.9 pmol/h/egg, respectively, were estimated. The transport of [14C]estradiol-β-glucuronide was inhibited by substrates/inhibitors of ABCC2/MRP-2, but not by those of the organic cation transporter and multidrug resistance protein (MDR)-1. KIAA0822/ABCA8 possesses two ATP-binding sites and fourteen transmembrane domains. Northern blot analysis revealed expression in most organs, especially in heart, skeletal muscle, and liver. Thus, ABCA8 is a new member of the xenobiotic transporter ABC-subfamily.

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Methods

Database screening for new member of ABC superfamily. We screened the GenBank database by the BLAST program using several known ABC transporters including the P-glycoprotein as the query. We found a Homo sapiens complete cDNA (AB020629, the mRNA of which is 5677 bp), which is now called ABCA8 by the HUGO (Human Gene Organization) Nomenclature Committee. This cDNA was obtained in pBluescript SK(−) from Kazusa DNA Research Institute (Kisarazu, Japan). The Kyte-Doolittle method predicted that the

Screening cDNA libraries

The complete Homo sapiens cDNA corresponding to the KIAA0822 protein (AB020629, 5677 bp mRNA) fulfilled the criteria of our screening. The cDNA was originally cloned as one of the full-length cDNAs with unknown function in human brain by Ohara and co-workers [8] from Kazusa DNA Research Institute (Kisarazu, Chiba, Japan) and registered in GenBank (with Accession No. AB020629). The gene is now called ABCA8 by the HUGO (Human Gene Organization) Nomenclature Committee. However, the function of this

Discussion

By screening an EST-database, we found KIAA0822 (now called ABCA8) as a possible new member of the ABC-transporter superfamily. This ABCA8 protein was shown to transport several drugs with a specificity substrate close to that of ABCC2/MRP-2 and of oatp, which does not belong to the ABC superfamily. The transport maximum is ATP-dependent. The Km of ABCA8 for the transport of [14C]estradiol-β-glucuronide was surprisingly low and similar to that of MRP-2/ABCC2 in Caco-2 cells [6], transfected

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