Ruiz A et al. (2018), Human Mutations in SLC2A9 (Glut9) Affect Transp...

XB-ART-55076

Front Physiol 2018 Jan 01;9:476. doi: 10.3389/fphys.2018.00476.

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Human Mutations in SLC2A9 (Glut9) Affect Transport Capacity for Urate.

Ruiz A , Gautschi I , Schild L , Bonny O .

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SLC2A9 or Glut9 is a voltage sensitive urate transporter, mainly expressed in the kidneys, the liver, and the intestine. Human Glut9 loss-of-function mutations were identified in familial hypouricemia, and several single nucleotide polymorphisms (SNPs) were associated with lower serum urate, further indicating that Glut9 is a major determinant of serum uric acid level. To get insights in Glut9 transport characteristics, we systematically analyzed the function of known human Glut9 mutants using 14C-urate uptake assay and two-electrode voltage clamp (TEVC) in the Xenopus laevis oocyte expression system. Surface expression was assessed by immunostaining and biotinylation. We found decreased urate transport by flux studies for most of the variants. No variant was permissive for glucose transport. We could further differentiate two behaviors among the mutants: those harboring poor overall and cell-surface expression leading to low activity and those fully expressed at the cell surface, but presenting decreased activity. We studied the latter by TEVC and observed, in depolarized conditions, decreased inward currents measured in presence of 400 μM urate, partially reversed in 1 mM urate. In addition, we showed that C210F displays lower transport ability. By contrast, N333S showed decreased urate transport activity and urate affinity, suggesting that it may belong to the urate binding pocket. Systematic analysis of Glut9 mutants confirms Glut9 as putative target for the treatment of hyperuricemia and brings new insights in Glut9 structure - function.

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Species referenced: Xenopus laevis
Genes referenced: slc2a1 slc2a9 slc2a9l
GO keywords: urate transmembrane transporter activity [+]

???displayArticle.omims??? HYPOURICEMIA, RENAL, 2; RHUC2

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	FIGURE 1. Glut9-mediated [14C] urate uptake in Xenopus laevis oocytes. (A) [14C] urate uptake was measured after 45 min incubation time in X. laevis oocytes injected with human or mouse Glut9a, Glut9b or with water (H2O). Total concentration of urate in the uptake solution is 400 μM. (B,C) Glut9 transports [14C] urate, but not [14C] D-Glucose. (D) The introduction of a triple flagged tag (Glut9 3xF) did not affect urate transport by Glut9. Statistical analysis were performed using a Kruskal–Wallis test followed by a correction with Dunnett’s test (p < 0.05). Each bar represents the mean and standard deviation of 3 independent batches of oocytes, with 10 oocytes per batch and condition. N.i, not injected; NS, not significant.
	FIGURE 2. Transport studies on human Glut9 loss-of-function mutations. (A) [14C] urate uptake assay was systematically performed on published Glut9 loss-of-function mutants and revealed decreased activity for all of them (i.e., L75R, T125R, R198C, C210F, G216R, N333S, and R380W) except for mutants R171C and P412R that do not reach significance. (B) None of these mutants displayed D-glucose transport activity when tested for [14C] D-glucose uptake. (C) Immunohistochemistry with an anti-Flag antibody on cryosections of oocytes injected with WT-Glut9 or mutant showed conserved expression at the cell surface for R171C, R198C, C210F, N333S, R380W, P412R and a decreased cell surface expression for L75R, T125M, and G216R. Representative pictures of three experiments from different oocyte batches are shown. (D) Cell surface assessment by biotinylation assay on oocytes injected either with Glut9 WT or the designated Glut9 mutants. One representative experiment out of 4 is shown. (E) Densitometric analysis revealed no difference in the ratio between biotinylation and total lysates for each Glut9 mutant when compared to WT Glut9. (F) Summary of cell surface expression (biotinylation) and transport activity (urate uptake) of all tested mutants reported to Glut9 WT. R198C, C210F, N333S, R380W, and P412R (in black) are the Glut9 mutants displaying preserved cell surface expression but decreased transport activity. L75R T125M, R171C, and G216R (in gray) are less expressed than WT and except for R171C, have less activity. Statistical analysis was performed using a Kruskal–Wallis test followed by a correction with Dunnett’s test (p < 0.05). Each experiment was performed on 3 independent batches of oocytes with 10 oocytes per batch and condition for uptake assay and 20 oocytes per condition for the biotinylation assay.
	FIGURE 3. Transport studies on Glut9 SNPs associated with low SUA. [14C]urate (A) and [14C]glucose (B) uptake in oocytes injected with Glut9 SNPs. (C) Immunohistochemistry with an anti-Flag antibody of cryosections of oocytes injected with WT-Glut9 and SNPs V253I, R265H, and P350L reveals a decreased expression at the cell surface for V253I. Protein expression was also verified by biotinylation and densitometric analysis (D,E). (F) Comparison of expression level and activity of WT-Glut9 or SNPs. Statistical analysis was performed using a Kruskal–Wallis test followed by a correction with Dunnett’s test (p < 0.05) with 3 batches of oocytes with 10 oocytes per batch and per condition for the fluxes and 20 oocytes for biotinylation.
	FIGURE 4. Electrogenicity of human Glut9 upon urate exposure. Representative trace current of H2O-(A) or Glut9-(B) injected oocytes in presence of 400 μM urate. The dashed line represents the current at baseline (0 mV).
	FIGURE 5. Electrophysiological studies of WT-Glut9. I/V curves for H2O-injected and WT-Glut9-injected oocytes exposed to 400 μM (A) or 1 mM (B) of urate. Three batches of oocytes were used with 5 oocytes per batch. Data are means ±SD. Asterisks denote p < 0.05 between Glut9- and H2O-injected oocytes.
	FIGURE 6. Electrophysiological studies of mutants Glut9. I/V curves for the following Glut9 mutants measured at 400 μM and 1 mM urate were built: R198C (A), C210F (B), N333S (C), R380W (D), and P412R (E). For the ease of comparison, the I/V curves for Glut9-WT and for water-injected-oocytes are represented on each panel. Three batches of oocytes were used with 5 oocytes per batch. Asterisks denote p < 0.05 by unpaired t-test between WT-injected and mutant-injected oocytes.
	FIGURE 7. Concentration-response analysis for P412R. [14C]urate uptake was measured in H2O, WT, or P412R-injected oocytes at increasing concentrations of urate: 0.02, 0.20, 0.40, and 1 mM. Ten oocytes per batch on 3 batches of oocytes were used. A Dunnett’s multiple comparison test was used to compare P412R to WT and water (∗p < 0.05).
	FIGURE 8. Electrophysiological studies of Glut9 SNPs. I/V curves built upon exposure to 400 μM or 1 mM of urate for: Glut9 WT and water-injected oocytes (A) or Glut9 SNPs injected-oocytes: V253I (B), R265H (C), and P350L (D). For the ease of comparison, the I/V curves for Glut9-WT and for water-injected-oocytes are represented on each panel. Three different batches of oocytes were used with 5 oocytes per batch. ∗Represents p < 0.05 compared to WT-injected oocytes.
	FIGURE 9. Urate kinetics of WT-Glut9. Glut9-injected oocytes were sequentially exposed to increasing urate concentrations (0.01, 0.1, 0.4, 1, 3, and 5 mM) and current was recorded by TEVC, clamped at 0 mV. Each measurement was repeated on atleast 5 oocytes per batch on atleast 3 batches. Detail analysis of Vmax and Km are reported in Table 3.
	FIGURE 10. Predicted secondary structure of Glut9. Representation of human Glut9 mutations and SNPs associated with variations of serum uric acid. Transmembrane domains are indicated by gray boxes. Extracellular side is upper and intracellular side is lower. The corresponding five main residues involved in glucose binding in hGLUT1 have been highlighted in yellow as indication.
	FIGURE 11. 3D homology model for Glut9, based on human Glut1 crystal structure. (A) Lateral view of Glut9-WT. (B,C) Represent views from the cytoplasmic side into the direction of the putative pore of the transporter. The different helices are illustrated and the residues involved in human familial hypouricemia are illustrated in red, as WT amino acids on (B) and as mutated amino acids on (C).

References [+] :

Anzai, Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans. 2008, Pubmed, Xenbase