Type 1 Sodium-dependent Phosphate Transporter (SLC17A1 Protein) Is a Cl−-dependent Urate Exporter

Masafumi Iharada,Takaaki Miyaji,Takahiro Fujimoto,Miki Hiasa,Naohiko Anzai,Hiroshi Omote,Yoshinori Moriyama

doi:10.1074/jbc.m110.122721

Masafumi Iharada, Takaaki Miyaji + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110.122721

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Abstract

SLC17A1 protein (NPT1) is the first identified member of the SLC17 phosphate transporter family and mediates the transmembrane cotransport of Na(+)/P(i) in oocytes. Although this protein is believed to be a renal polyspecific anion exporter, its transport properties are not well characterized. Here, we show that proteoliposomes containing purified SLC17A1 transport various organic anions such as p-aminohippuric acid and acetylsalicylic acid (aspirin) in an inside positive membrane potential (Deltapsi)-dependent manner. We found that NPT1 also transported urate. The uptake characteristics were similar to that of SLC17 members in its Cl(-) dependence and inhibitor sensitivity. When arginine 138, an essential amino acid residue for members of the SLC17 family such as the vesicular glutamate transporter, was specifically mutated to alanine, the resulting mutant protein was inactive in Deltapsi-dependent anion transport. Heterologously expressed and purified human NPT1 carrying the single nucleotide polymorphism mutation that is associated with increased risk of gout in humans exhibited 32% lower urate transport activity compared with the wild type protein. These results strongly suggested that NPT1 is a Cl(-)-dependent polyspecific anion exporter involved in urate excretion under physiological conditions.

Highlights

SLC17A1 protein (NPT1) is the first identified member of the SLC17 type 1 phosphate transporter family [5]
SLC17A5 protein is a biphasic transporter. It acts as a Hϩ/sialic acid cotransporter and when present in synaptic vesicles, it acts as a vesicular excitatory amino acid transporter (VEAT) [11]
Arg184 of VGLUT2, a charged amino acid residue in the transmembrane regions conserved in all members, which corresponds to Arg138 of NPT1, is essential for their transport activity (5, 10 –12)

Summary

EXPERIMENTAL PROCEDURES

Transport Assay—Proteoliposomes (0.4 ␮g protein/single assay) were suspended in 20 mM MOPS-Tris (pH 7.0), 5 mM magnesium acetate, 4 mM KCl, and 0.15 M sodium acetate and incubated for 3 min at 27 °C. For ClϪ transport, the reaction mixture containing 20 mM MOPS-Tris (pH 7.0), 5 mM magnesium acetate, 10 mM PAH, 0.15 M potassium acetate, 2 ␮M valinomycin, and 10 mM radioactive 36ClϪ (740 MBq/g, American Radiolabeled Chemicals Inc.) was preincubated for 3 min at 27 °C. Proteoliposomes containing NPT1 (0.5 ␮g protein per assay) were added to the mixture to initiate the reaction and incubated for a further 1 min. Proteoliposomes were incubated in the standard assay condition in the presence of 20 ␮M [14C]SCN for 3 min, and the internal concentration of [14C]SCN was quantified as described.

RESULTS

PAH uptake

Gene name

DISCUSSION

Control PAH Salicylate Aspirin Diclofenac Phosphate