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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

The Human Multidrug Resistance Protein 4 (MRP4, ABCC4): Functional Analysis of a Highly Polymorphic Gene

Departments of Biopharmaceutical Sciences (N.A., T.N., L.W.C., L.L, K.M.G., D.L.K) and Pharmaceutical Chemistry (C.C.H, S.J.J., M.K., D.S., T.R.T., T.E.F.), University of California, San Francisco, California

ABCC4 encodes multidrug resistance protein 4 (MRP4), a member of the ATP-binding cassette family of membrane transporters involved in the efflux of endogenous and xenobiotic molecules. The aims of this study were to identify single nucleotide polymorphisms of ABCC4 and to functionally characterize selected nonsynonymous variants. Resequencing was performed in a large ethnically diverse population. Ten nonsynonymous variants were selected for analysis of transport function based on allele frequencies and evolutionary conservation. The reference and variant MRP4 cDNAs were constructed by site-directed mutagenesis and transiently transfected into human embryonic kidney cells (HEK 293T). The function of MRP4 variants was compared by measuring the intracellular accumulation of two antiviral agents, azidothymidine (AZT) and adefovir (PMEA). A total of 98 variants were identified in the coding and flanking intronic regions of ABCC4. Of these, 43 variants are in the coding region, and 22 are nonsynonymous. In a functional screen of ten variants, there was no evidence for a complete loss of function allele. However, two variants (G187W and G487E) showed a significantly reduced function compared to reference with both substrates, as evidenced by higher intracellular accumulation of AZT and PMEA compared to the reference MRP4 (43 and 69% increase in accumulation for G187W compared with the reference MRP4, with AZT and PMEA, respectively). The G187W variant also showed decreased expression following transient transfection of HEK 293T cells. Further studies are required to assess the clinical significance of this altered function and expression and to evaluate substrate specificity of this functional change.

Address correspondence to: Professor Deanna L. Kroetz, Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, 1550 4th Street, Box 2911, San Francisco, California. E-mail: deanna.kroetz{at}ucsf.edu