Abstract
Organic anion transporter 1 (OAT1) is key for the secretion of organic anions in renal proximal tubules. These organic anions comprise endogenous as well as exogenous compounds including frequently used drugs of various chemical structures. The molecular basis for the polyspecificity of OAT1 is not known. Here we mutated a conserved positively charged arginine residue (Arg(466)) in the 11(th) transmembrane helix of human OAT1. The replacement by the positively charged lysine (R466K) did not impair expression of hOAT1 at the plasma membrane of Xenopus laevis oocytes but decreased the transport of p-aminohippurate (PAH) considerably. Extracellular glutarate inhibited and intracellular glutarate trans-stimulated wild type and mutated OAT1, suggesting for the mutant R466K an unimpaired interaction with dicarboxylates. However, when Arg(466) was replaced by the negatively charged aspartate (R466D), glutarate no longer interacted with the mutant. PAH uptake by wild type hOAT1 was stimulated in the presence of chloride, whereas the R466K mutant was chloride-insensitive. Likewise, the uptake of labeled glutarate or ochratoxin A was chloride-dependent in the wild type but not in R466K. Kinetic experiments revealed that chloride did not alter the apparent K(m) for PAH but influenced V(max) in wild type OAT1-expressing oocytes. In R466K mutants the apparent K(m) for PAH was similar to that of the wild type, but V(max) was not changed by chloride removal. We conclude that Arg(466) influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.
Highlights
Membrane of proximal tubules in the kidneys, Organic anion transporter 1 (OAT1) has been shown to interact with a wide variety of structurally diverse therapeutically important compounds like antivirals, diuretics, and nonsteroidal anti-inflammatory drugs [5]
To narrow down the search, we identified amino acids that were oppositely charged in the organic cation transporters (OCTs) [13]
Chloride removal clearly inhibited glutarate uptake as well as ochratoxin A transport in wt-expressing oocytes. Uptake of both glutarate and ochratoxin A was found to be reduced in the mutant R466K and no longer to be dependent on the presence or absence of chloride. These results suggest that optimal transport of PAH, glutarate, and ochratoxin A requires chloride in wt OAT1 but not in the mutant R466K
Summary
Chemicals—All of the chemicals were of reagent grade. p-[glycyl-2-3H]Aminohippurate ([3H]PAH), 1–5 Ci/mmol, was obtained from PerkinElmer Life Sciences. [1,5-14C]Glutaric acid (55 mCi/mmol), was obtained from MP Biomedicals (Heidelberg, Germany). [3H(G)]Ochratoxin A (7.2 Ci/mmol) was obtained from Moravek Biochemicals Inc. (Brea, CA). Uptake of [3H]PAH, [14C]glutarate, or [3H]ochratoxin A was assayed at room temperature (experimental parameters are described in the figure legends) in ORI. For cis-inhibition experiments 1 mM malonic, glutaric, or adipic acid solutions were prepared in ORI, and pH was adjusted to 7.6. Trans-stimulation experiments were performed by injecting oocytes with 46 nl of 5 mM unlabeled glutaric acid solution in water (pH adjusted to 7.6). They were washed and kept on ice for 15 min, and the uptake of [3H]PAH was assayed over 1 h in ORI. Immunocytochemistry—To study hOAT1 trafficking, oocytes were injected with the cRNA of FLAG-tagged wt or mutant hOAT1 or an equivalent volume of water (mocks). PAH transport; Km is the PAH concentration that resulted in halfmaximal transport; [S] is the concentration of unlabeled PAH in the transport reaction; and C is a constant representing the component of total PAH uptake that was not saturated (over the range of substrate concentrations tested) and presumably reflected the combined influence of diffusive flux, nonspecific binding, and/or incomplete washing
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