Functional characterization of nonsynonymous single nucleotide polymorphisms in the human organic anion transporter 4 (hOAT4).
OBJECTIVE
The human organic anion transporter (hOAT) family of transmembrane carrier proteins mediate the cellular flux of anionic substances, including certain hormones and anti-cancer drugs. hOAT4 is highly expressed at the apical membrane of the renal tubular cell and facilitates drug re-absorption in the kidney. In the present study, the impact of 10 nonsynonymous single nucleotide polymorphisms (SNPs) of hOAT4 on transport function in COS-7 cells was characterized.


METHODS
Transport uptake assay was used to assess the function of the variant transporters. Cell surface biotinylation and western blot analysis were used to investigate the expression characteristics of the transporter proteins. Comparative modelling was used to interpret the influence of nonsynonymous changes in terms of hOAT4 structure.


RESULTS
Four naturally occurring hOAT4 variants (L29P, R48Y, V155G and T392I) exhibited a significant loss of function. Substitution of leucine-29, which is a conserved residue in OATs, with a proline residue, impaired the synthesis or the apparent stability of the transporter and membrane insertion was disrupted in the R48Y variant. In the case of the V155G and T392I variants, impaired function was due to decreased affinity of the transporter for oestrone sulphate and impaired transporter-substrate turnover respectively. The T392I variant was inhibited more extensively than the wild-type transporter by the cationic substrate tetraethyl ammonium.


CONCLUSIONS
Several naturally occurring SNPs encode variant hOAT4s that may impair the renal tubular re-absorption of important drug substrates.
Zhou
Fanfan
F
Pharmacogenomics and Drug Development Laboratory, Faculty of Pharmacy, The University of Sydney, Sydney, NSW, Australia. fzhou@pharm.usyd.edu.au
Zhu
Ling
L
Cui
Pei H
PH
Church
W Bret
WB
Murray
Michael
M
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D029362:Organic Anion Transporters, Sodium-Independent / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide
D029362:Organic Anion Transporters, Sodium-Independent; C427207:SLC22A11 protein, human; D004970:Estrone; C017296:estrone sulfate