Fig. 3.

Identification and characterization of mouse Svct1 as a urate transporter. All uptake assays were conducted using transiently Svct1-expressing HEK293 cells 48 h after plasmid transfection in Krebs–Ringer buffer. Experimental conditions: pH 7.4 unless otherwise indicated, or pH 10 (f); [1-¹⁴C]-vitamin C in the transport buffer was 20 μM (c, h); incubation time and [8-¹⁴C]-urate in the transport buffer were 5 min and 10 μM, respectively, unless otherwise indicated. a–c Functional expression of Svct1 in HEK293 cells 48 h after the transfection. (a) Immunoblot detection of Svct1 protein in whole-cell lysates. (b) Intracellular localization of Svct1 detected by confocal microscopy. Magnified images of representative cells denoted by yellow squares are demonstrated. Scale bar: 10 μm. (c) [1-¹⁴C]-Vitamin C transport activities into the cells. d–g Svct1 as a urate transporter. (d) Sodium-dependency in Svct1-mediated urate transport, which was insignificantly affected by an alkaline pH condition (pH 10). (e) Time profile for Svct1-mediated [8-¹⁴C]-urate uptake into HEK293 cells. (f) Concentration dependence in Svct1-mediated [8-¹⁴C]-urate transport. Regarding the estimated Michaelis-Menten constant (K_m) and maximal velocity (V_max), the 95% confidence interval values were provided in parentheses. (g) Concentration-dependent inhibition of Svct1-mediated urate transport by vitamin C. IC₅₀, the half-maximal inhibitory concentration. h Mild inhibitory effect of urate on Svct1-mediated vitamin C transport at physiological concentrations. Values are shown as % of vehicle control (g, h). Data are expressed as the mean ± SD; where vertical bars are not displayed, the SD was contained within the limits of the symbol; n = 6 (f), 3 (the others). ^††P < 0.01; ns, not significantly different between groups (two-sided t-test; c, d); ^**P < 0.01 vs. the other groups for pH 7.4 (Tukey–Kramer multiple-comparison test; d); ^##P < 0.01 vs. vehicle control (Williams’ multiple-comparison test; h)