Maintenance of urate homeostasis requires urate efflux from urate-producing cells with subsequent renal and gastrointestinal excretion. The molecular basis for urate transport, however, has not been identified. A novel full-length cDNA encoding a 322-amino acid protein, designated UAT (urate transporter), has been cloned from a rat renal cDNA library by antibody screening. UAT mRNA transcripts that approximate 1.55 kilobases are present, but differentially expressed in various rat tissues. Recombinant UAT protein that was expressed from the cloned cDNA in Escherichia coli and purified via immobilized metal affinity chromatography has been functionally reconstituted as a highly selective urate transporter/channel in planar lipid bilayers. The IgG fraction of the polyclonal antibody that was used to select the UAT clone from the cDNA library, but not nonimmune IgG, blocked urate channel activity. Based on the wide tissue distribution of the mRNA for UAT we propose that UAT provides the molecular basis for urate flux across cell membranes, allowing urate that is formed during purine metabolism to efflux from cells and serving as an electrogenic transporter that plays an important role in renal and gastrointestinal urate excretion.

We have identified a 45-kDa protein purified from rat renal brush border membrane that binds short single-stranded nucleic acid sequences. This activity was purified, reconstituted in proteoliposomes, and then fused with model planar lipid bilayers. In voltage-clamp experiments, the reconstituted 45-kDa protein functioned as a gated channel that allows the passage of nucleic acids. Channel activity was observed immediately after addition of oligonucleotide. Channel activity was not observed in the absence of purified protein or of oligonucleotide or when protein was heat-inactivated prior to forming proteoliposomes. In the presence of symmetrical buffered solution and oligonucleotide, current passed linearly over the range of holding potentials tested. Conductance was 10.4 ؎ 0.4 picosiemens (pS) and reversal potential was 0.2 ؎ 1.7 mV. There was no difference in channel conductance or reversal potential between phosphodiester and phosphorothioate oligonucleotides. Ionsubstitution experiments documented a shift in reversal potential only when a concentration gradient for oligonucleotide was established, indicating that movement of oligonucleotide alone was responsible for current. Movement of oligonucleotide across the bilayer was confirmed by using 32 P-labeled oligonucleotides. Channel open probability decreased significantly in the presence of heparan sulfate. These studies provide evidence for a cell surface channel that conducts nucleic acids.

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.

Kir channels are important in setting the resting membrane potential and modulating membrane excitability. A common feature of Kir2 channels and several other ion channels that has emerged in recent years is that they are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. Yet, the mechanism by which cholesterol affects channel function is not clear. We have recently shown that the sensitivity of Kir2 channels to cholesterol depends on residues in the CD loop of the cytosolic domain of the channels with one of the mutations, L222I, abrogating cholesterol sensitivity of the channels completely. Here we show that in addition to Kir2 channels, members of other Kir subfamilies are also regulated by cholesterol. Interestingly, while similarly to Kir2 channels, several Kir channels, Kir1.1, Kir4.1 and Kir6.2Δ36 were suppressed by an increase in membrane cholesterol, the function of Kir3.4* and Kir7.1 was enhanced following cholesterol enrichment. Furthermore, we show that independent of the impact of cholesterol on channel function, mutating residues in the corresponding positions of the CD loop in Kir2.1 and Kir3.4*, inhibits cholesterol sensitivity of Kir channels, thus extending the critical role of the CD loop beyond Kir2 channels.