Structure-based analysis of CysZ-mediated cellular uptake of sulfate.

Sulfur, most abundantly found in the environment as sulfate (SO), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO at the molecular level is limited. CysZ has been described as a SO permease, but its sequence family is without known structural precedent.

Use of a Pteridine Moiety to Track DNA Uptake in Cells.

Fluorescence labeled oligonucleotides have a long history of being used to monitor nucleic acid transport and uptake. However, it is not known if the fluorescent moiety itself physically limits the number of pathways that can be used by the cell due to steric, hydrophobic, or other chemical characteristics. Here, we report a method for comparing the uptake kinetics of oligonucleotides labeled either with the fluorescent pteridine, 3-methyl-8-(2-deoxy-β-D-ribofuranosyl) isoxanthopterin (3MI), or the common fluorophore 5-carboxyfluorescein (5-FAM).

Wicking: a rapid method for manually inserting ion channels into planar lipid bilayers.

The planar lipid bilayer technique has a distinguished history in electrophysiology but is arguably the most technically difficult and time-consuming method in the field. Behind this is a lack of experimental consistency between laboratories, the challenges associated with painting unilamellar bilayers, and the reconstitution of ion channels into them. While there has be a trend towards automation of this technique, there remain many instances where manual bilayer formation and subsequent membrane protein insertion is both required and advantageous.

Gating of a G protein-sensitive mammalian Kir3.1 prokaryotic Kir channel chimera in planar lipid bilayers.

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood.

Comparative analysis of cholesterol sensitivity of Kir channels: role of the CD loop.

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.

Localization of the nucleic acid channel regulatory subunit, cytosolic malate dehydrogenase.

NACh is a nucleic acid-conducting channel found in apical membrane of rat kidney proximal tubules. It is a heteromultimeric complex consisting of at least two proteins: a 45-kDa pore-forming subunit and a 36-kDa regulatory subunit. The regulatory subunit confers ion selectivity and influences gating kinetics.

Presence of the nucleic acid channel in renal brush-border membranes: allosteric modulation by extracellular calcium.

We have previously described a cell surface channel complex that is highly selective for nucleic acid (6, 7). The channel complex was purified to homogeneity by solubilizing renal brush-border membranes (BBM) with CHAPS and separation by liquid chromatography. It was characterized by reconstitution in planar lipid bilayers.

Galectin 9 is the sugar-regulated urate transporter/channel UAT.

UAT, also designated galectin 9, is a multifunctional protein that can function as a urate channel/transporter, a regulator of thymocyte-epithelial cell interactions, a tumor antigen, an eosinophil chemotactic factor, and a mediator of apoptosis. We review the evidence that UAT is a transmembrane protein that transports urate, describe our molecular model for this protein, and discuss the evidence from epitope tag and lipid bilayer studies that support this model of the transporter. The properties of recombinant UAT are compared with those of urate transport into membrane vesicles derived from proximal tubule cells in rat kidney cortex.

Uric acid transport.

Purpose Of Review: The goal of this article is to review the physiology and describe newly defined molecular mechanisms that are responsible for renal urate transport.

Recent Findings: Four complementary DNAs have recently been cloned whose expressed proteins transport urate. Two of these proteins have been localized to the apical membrane of proximal tubular cells: one, a urate transporter/channel, a galectin, is an electrogenic transporter (an ion channel); the second is a urate-anion electroneutral exchanger, a member of the organic anion transporter family.

Functional analysis and molecular model of the human urate transporter/channel, hUAT.

Recombinant protein, designated hUAT, the human homologue of the rat urate transporter/channel (UAT), functions as a highly selective urate channel in lipid bilayers. Functional analysis indicates that hUAT activity, like UAT, is selectively blocked by oxonate from its cytosolic side, whereas pyrazinoate and adenosine selectively block from the channel's extracellular face. Importantly, hUAT is a galectin, a protein with two beta-galactoside binding domains that bind lactose.

Cytosolic malate dehydrogenase confers selectivity of the nucleic acid-conducting channel.

We have described previously a cell surface channel that is highly selective for nucleic acids. Nucleic acid conductance is 10 pS and the channel is at least 10,000-fold more selective for oligodeoxynucleotides than any anion tested (1). Herein we provide evidence that the nucleic acid-conducting channel (NACh) is a heteromultimeric complex of at least two proteins; a 45-kDa pore-forming subunit (p45) and a 36-kDa regulatory subunit (p36).

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

Elevated serum levels of uric acid have been associated with an increased risk for gout, hypertension, cardiovascular disease, and renal failure. The molecular mechanisms for the diminished excretion of urate in these disorders, however, remain poorly understood. Human galectin 9, which is highly homologous to the rat urate transporter rUAT, has been reported to be a secreted or cytosolic protein.

Functional analysis and molecular modeling of a cloned urate transporter/channel.

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.

Identification and characterization of a cell membrane nucleic acid channel.

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.

Molecular cloning and functional reconstitution of a urate transporter/channel.

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.

Calcium regulation of a cell surface nucleic acid channel.

We have recently purified a 45 kDa protein from rat renal brush border membrane that functions as a macromolecular nucleic acid channel when reconstituted in artificial lipid bilayers. To explore the role of calcium in the regulation of this channel, purified protein was reconstituted in bilayers and calcium concentration was altered while voltage clamp experiments were performed. Open probability of the channel was less than 1% in 0 calcium and increased an average of 19-fold when calcium concentration was increased from 0 to 0.

Reconstitution of hepatic uricase in planar lipid bilayer reveals a functional organic anion channel.

Rat renal proximal tubule cell membranes have been reported to contain uricase-like proteins that function as electrogenic urate transporters. Although uricase, per se, has only been detected within peroxisomes in rat liver (where it functions as an oxidative enzyme) this protein has been shown to function as a urate transport protein when inserted into liposomes. Since both the uricase-like renal protein and hepatic uricase can transport urate, reconstitution studies were performed to further characterize the mechanism by which uricase may function as a transport protein.

Urate uptake in membrane vesicles of rat renal cortex: effect of copper.

[2-14C]Urate uptake was studied in brush border and basolateral membrane vesicles of rat renal cortex. In the absence of copper, urate equilibrated without metabolism of transported urate. Exposure of the vesicles to copper significantly stimulated uptake, and in these vesicles uptake was also stimulated by NaCl or KCl gradients.

Metabolism of glutamine by the intact functioning kidney of the dog. Studies in metabolic acidosis and alkalosis.

The renal conversion of glutamine to glucose and its oxidation to CO(2) were compared in dogs in chronic metabolic acidosis and alkalosis. These studies were performed at normal endogenous levels of glutamine utilizing glutamine-(34)C (uniformly labeled) as a tracer. It was observed in five experiments in acidosis that mean renal extraction of glutamine by one kidney amounted to 27.