Sodium-coupled electrogenic transport of pyroglutamate (5-oxoproline) via SLC5A8, a monocarboxylate transporter

Pyroglutamate, also known as 5-oxoproline, is a structural analog of proline. This amino acid derivative is a byproduct of glutathione metabolism, and is reabsorbed efficiently in kidney by Na+-coupled transport mechanisms. Previous studies have focused on potential participation of amino acid transport systems in renal reabsorption of this compound. Here we show that it is not the amino acid transport systems but instead the Na+-coupled monocarboxylate transporter SLC5A8 that plays a predominant role in this reabsorptive process. Expression of cloned human and mouse SLC5A8 in mammalian cells induces Na+-dependent transport of pyroglutamate that is inhibitable by various SLC5A8 substrates. SLC5A8-mediated transport of pyroglutamate is saturable with a Michaelis constant of 0.36 ± 0.04 mM. Na+-activation of the transport process exhibits sigmoidal kinetics with a Hill coefficient of 1.8 ± 0.4, indicating involvement of more than one Na+ in the activation process. Expression of SLC5A8 in Xenopuslaevis oocytes induces Na+-dependent inward currents in the presence of pyroglutamate under voltage-clamp conditions. The concentration of pyroglutamate necessary for induction of half-maximal current is 0.19 ± 0.01 mM. The Na+-activation kinetics is sigmoidal with a Hill coefficient of 2.3 ± 0.2. Ibuprofen, a blocker of SLC5A8, suppressed pyroglutamate-induced currents in SLC5A8-expressing oocytes; the concentration of the blocker necessary for causing half-maximal inhibition is 14 ± 1 μM. The involvement of SLC5A8 can be demonstrated in rabbit renal brush border membrane vesicles by showing that the Na+-dependent uptake of pyroglutamate in these vesicles is inhibitable by known substrates of SLC5A8. The Na+ gradient-driven pyroglutamate uptake was stimulated by an inside-negative K+ diffusion potential induced by valinomycin, showing that the uptake process is electrogenic.