Mutational analysis of histidine residues in human organic anion transporter 4 (hOAT4).
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.
Department of Pharmaceutics, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA.
Fanfan
F
Zhou
Zui
Z
Pan
Jianjie
J
Ma
Guofeng
G
You
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000187:drug effects / Q000235:genetics; D000818:Animals; D019556:COS Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis / Q000379:methods*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D029362:Organic Anion Transporters, Sodium-Independent / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology
D029362:Organic Anion Transporters, Sodium-Independent; C427207:SLC22A11 protein, human; D006639:Histidine; D004047:Diethyl Pyrocarbonate; D000409:Alanine