Functional Analysis and Molecular Modeling of a Cloned Urate Transporter/Channel

Abstract

Recombinant protein, designated UAT, prepared from a cloned rat renal cDNA library functions as a selective voltage-sensitive urate transporter/channel when fused with lipid bilayers. Since we previously suggested that UAT may represent the mammalian electrogenic urate transporter, UAT has been functionally characterized in the presence and absence of potential channel blockers, several of which are known to block mammalian electrogenic urate transport. Two substrates, oxonate (a competitive uricase inhibitor) and pyrazinoate, that inhibit renal electrogenic urate transport also block UAT activity. Of note, oxonate selectively blocks from the cytoplasmic side of the channel while pyrazinoate only blocks from the channel's extracellular face. Like oxonate, anti-uricase (an electrogenic transport inhibitor) also selectively blocks channel activity from the cytoplasmic side. Adenosine blocks from the extracellular side exclusively while xanthine blocks from both sides. These effects are consistent with newly identified regions of homology to uricase and the adenosine A1/A3 receptor in UAT and localize these homologous regions to the cytoplasmic and extracellular faces of UAT, respectively. Additionally, computer analyses identified four putative alpha-helical transmembrane domains, two beta sheets, and blocks of homology to the E and B loops of aquaporin-1 within UAT. The experimental observations substantiate our proposal that UAT is the molecular representation of the renal electrogenic urate transporter and, in conjunction with computer algorithms, suggest a possible molecular structure for this unique channel.

Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter

“…Ion channel activity was assayed in a lipid bilayer system as described previously (20,21). Purified hUAT protein was sonicated with a 1:1 mixture of bovine phosphatidylserine (PS) and phosphatidylethanolamine (PE) (Avanti Polar Lipids Inc., Alabaster, Alabama, USA), suspended in 220 mM Cs 2 SO 4 , 10 mM HEPES-NaOH, pH 7.4, and sonicated to form proteoliposomes.…”

“…In our previously published model for rUAT based on computer-assisted modeling and experimental data in lipid bilayer studies, it was proposed that rUAT contains four transmembrane domains, with intracellular amino and carboxy termini (21). Based on the high degree of homology between rUAT and hUAT, we tested the hypothesis that hUAT would have a similar topology.…”

“…Based on the homology between hUAT and rUAT and our preliminary topologic analysis of hUAT (Figures 4 and 5), it seems likely that the molecular model proposed for rUAT (21) also applies to hUAT. Using this model (Figure 13), it is of interest that insertion of exon 5 introduces a second region of homology to uricase (the presumed urate-binding domain in hUAT) just upstream of the previously identified block of amino acids in rUAT with homology to uricase (21). Deletion of exon 10 eliminates the entire second β-galactoside-binding site in an extracellular domain of the protein (21).…”

“…Using this model (Figure 13), it is of interest that insertion of exon 5 introduces a second region of homology to uricase (the presumed urate-binding domain in hUAT) just upstream of the previously identified block of amino acids in rUAT with homology to uricase (21). Deletion of exon 10 eliminates the entire second β-galactoside-binding site in an extracellular domain of the protein (21). The presence of a second urate-binding site could modify the affinity of hUAT for urate and thereby modify channel function.…”

Is There a Pathogenetic Role for Uric Acid in Hypertension and Cardiovascular and Renal Disease?

“…After filtration, uric acid undergoes both reabsorption and secretion in the proximal tubule, and this process is mediated by a urate/anion exchanger and a voltagesensitive urate channel. 3,4 Organic anions such as lactate decrease urate secretion by competing for urate through the organic anion transporter, whereas several substances, including probenacid and benziodarone, have opposite effects. 5 Hyperuricemia is usually defined as Ͼ6.5 or 7.0 mg/dL in men and Ͼ6.0 mg/dL in women.…”