Abstract
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.
Highlights
Uric acid (UA) is a weak acid mostly found as urate in the serum at physiological pH
We have previously shown that mouse Glut9 transports urate and we have suggested that the simplest model of transport fitting our observations was a uniporter (Bibert et al, 2009)
We first assessed human and mouse Glut9 isoforms a and b, by [14C] urate uptake into X. laevis oocytes and showed that they display the same transport rate (Figure 1A).We further verified that urate transport was specific for Glut9 by comparing urate and glucose uptake using Glut2 as control (Figures 1B,C)
Summary
Uric acid (UA) is a weak acid mostly found as urate in the serum at physiological pH. Due to successive mutations in the gene coding for uricase (Wu et al, 1992), humans lost the ability to degrade UA and have higher SUA concentrations by about thirty times compared to other species (Keilin, 1959). If this might have led to evolutionary advantages, it contributes to significant morbidity, including gout, tophi and kidney stones. Most of urate transport takes place in the proximal tubule and results in net urate reabsorption of 90–95% of the filtered load. SLC2A9 ( called Glut or URATv1) proved to be central to urate reabsorption, as indicated by several lines of evidence
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