Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, antitumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. hOAT4-mediated transport of the organic anion oestrone sulphate in COS-7 cells was inhibited by the histidine-modifying reagent DEPC (diethyl pyrocarbonate). Therefore the role of histidine residues in the function of hOAT4 was examined by site-directed mutagenesis. All five histidine residues of hOAT4 were converted into alanine, singly or in combination. Single replacement of His-47, or simultaneous replacement of His-47/52/83 or His-47/52/83/305/469 (H-less) led to a 50–80% decrease in transport activity. The decreased transport activity of these mutants was correlated with a decreased amount of cell-surface expression, although the total cell expression of these mutants was similar to that of wild-type hOAT4. These results suggest that mutation at positions 47, 47/52/83 and 47/52/83/305/469 impaired membrane expression rather than function. We also showed that, although most of the histidine mutants of hOAT4 were sensitive to inhibition by DEPC, H469A (His-469→Ala) was completely insensitive to inhibition by this reagent. Therefore modification of His-469 is responsible for the inhibition of hOAT4 by DEPC.
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Research Article| November 09 2004
Mutational analysis of histidine residues in human organic anion transporter 4 (hOAT4)
Guofeng YOU 1
*Department of Pharmaceutics, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, U.S.A.
‡Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, U.S.A.
1To whom correspondence should be addressed (email firstname.lastname@example.org).
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Fanfan ZHOU, Zui PAN, Jianjie MA, Guofeng YOU; Mutational analysis of histidine residues in human organic anion transporter 4 (hOAT4). Biochem J 15 November 2004; 384 (1): 87–92. doi: https://doi.org/10.1042/BJ20040751
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