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

Sulfur, most abundantly found in the environment as sulfate (SO42-), 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 SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.

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. We have developed a comprehensive method, which we have termed "wicking", that greatly simplifies many experimental aspects of the lipid bilayer system. Wicking allows one to manually insert ion channels into planar lipid bilayers in a matter of seconds, without the use of a magnetic stir bar or the addition of other chemicals to monitor or promote the fusion of proteoliposomes. We used the wicking method in conjunction with a standard membrane capacitance test and a simple method of proteoliposome preparation that generates a heterogeneous mixture of vesicle sizes. To determine the robustness of this technique, we selected two ion channels that have been well characterized in the literature: CLIC1 and α-hemolysin. When reconstituted using the wicking technique, CLIC1 showed biophysical characteristics congruent with published reports from other groups; and α-hemolysin demonstrated Type A and B events when threading single stranded DNA through the pore. We conclude that the wicking method gives the investigator a high degree of control over many aspects of the lipid bilayer system, while greatly reducing the time required for channel reconstitution.

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-ß-D-ribofuranosyl) isoxanthopterin (3MI), or the common fluorophore 5-carboxyfluorescein (5-FAM). We use a multiphoton microscopic technique to monitor nucleic acid uptake LLC-PK1, a pig renal tubular cell line that is known to have multiple uptake pathways. We find that the two fluorophores enter the cells at different rates, suggesting that choice of fluorescent moiety influences the uptake pathway used by a cell. Finally, we reconstituted an LLC-PK1 membrane channel that is selective for nucleic acids in planar lipid bilayers, and tested the ability of the labeled nucleic acids to permeate the channel. We find that 3MI, and not 5-FAM labeled oligonucleotides can traverse the plasma membrane through the channel. These results have implications for future studies aimed at delivering pteridine moieties to cells and for tracking nucleic acid transport into tissues.

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 ßγ 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. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gßγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.

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. 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 Kir6.2Delta36 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.

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. The regulatory subunit has been identified as cytosolic malate dehydrogenase (cMDH). cMDH is described in the literature as a soluble protein that is not associated with plasma membrane. Yet a role for cMDH as the regulatory subunit of NACh requires that it be present at the plasma membrane. To resolve this conflict, studies were initiated to determine whether cMDH could be found at the plasma membrane. Before performing localization studies, a suitable model system that expressed NACh was identified. A channel was identified in LLC-PK(1) cells, a line derived from pig proximal tubule, that is selective for nucleic acid and has a conductance of approximately 10 pS. It exhibits dose-dependent blockade by heparan sulfate or L-malate. These characteristics are similar to what has been reported for NACh from rat kidney and indicate that NACh is present in LLC-PK(1) cells. LLC-PK(1) cells were therefore used as a model system for immunolocalization of cMDH. Both immunofluorescence and immunoelectron microscopy demonstrated cMDH at the plasma membrane of LLC-PK(1) cells. This finding supports prior functional data that describe a role for cMDH as the regulatory subunit of NACh.

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. The channel consists of a pore-forming subunit that is blocked by heparan sulfate and a regulatory subunit that is blocked by L-malate (7). The current studies were performed to compare the characteristics of the nucleic acid-conducting channel in native BBM with the characteristics that have been determined for the complex reconstituted from purified proteins. BBM were purified by differential centrifugation and reconstituted in lipid bilayers. Current was not observed until oligodeoxynucleotide (ODN) was added. Conductance was 9.1 +/- 0.9 pS; rectification and voltage dependence were not observed. Reversal potential (E(rev)) shifted to +14 +/- 0.1 mV by a 10-fold gradient for ODN but was not altered when gradients were created for any other ion. Open probability increased significantly with an increase in Ca(2+) on the trans chamber of the bilayer apparatus. Changes in cis Ca(2+) were without effect. Addition of L-malate to the cis chamber or heparan sulfate to the trans chamber significantly reduced the open probability of the channel. These data demonstrate that the nucleic acid channel in BBM is electrophysiologically and pharmacologically identical to that previously reported for purified protein and demonstrate that a nucleic acid-conducting channel is a component of renal BBM.

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. The other urate transport proteins, organic anion transporters 1 and 3, are also members of the organic anion transporter family. These proteins have been localized to the basolateral membrane of proximal tubular cells: organic anion transporter 1 is an electroneutral organic anion exchanger; the mechanism of urate transport on organic anion transporter 3 remains to be determined. SUMMARY: The molecular definition and localization of four urate transport proteins provides a basis for developing a molecular model of the bi-directional transport of urate in renal proximal tubules. It seems likely that the urate-anion exchanger is responsible for luminal reabsorption while the urate transporter/channel permits secretion of urate from the cell into the lumen. Since organic anion transporters 1 and 3 reside in the basolateral membrane, one or both may be relevant in the reabsorptive flux of urate into the peritubular capillary as well as in the cellular uptake of urate from the peritubular space, the first step in the process of urate secretion. Knowledge of the molecular basis of urate transport should provide greater insights into states of altered transport as well as assist in development of drugs to modify urate flux.

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. Lactose significantly increased hUAT open probability but only when added to the channel's extracellular side. This effect on open probability was mimicked by glucose, but not ribose, suggesting a role for extracellular glucose in regulating hUAT channel activity. These functional observations support a four-transmembrane-domain structural model of hUAT, as previously predicted from the primary structure of UAT. hUAT and UAT, however, are not functionally identical: hUAT has a significantly lower single-channel conductance and open probability is voltage independent. These differences suggest that evolutionary changes in specific amino acids in these highly homologous proteins are functionally relevant in defining these biophysical properties.

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). Reconstitution of p45 in planar lipid bilayers resulted in formation of a channel which gated in the absence of nucleic acid and which was more selective for anions (including oligonucleotide) than cations. This channel exhibited transitions from one level of current to another (or to the closed state); however the incidence of transitions was rare. Channel activity was not observed when p36 was reconstituted alone. Reconstitution of p36 with p45 restored nucleic acid dependence and selectivity to the channel. Protein sequence analysis identified p36 as cytosolic malate dehydrogenase (cMDH). Experiments were performed to prove that cMDH is a regulatory subunit of NACh. Selective activity was observed when p45 was reconstituted with pig heart cMDH but not with mitochondrial MDH. Both the enzyme substrate l-malate and antiserum raised against cMDH block NACh activity. These data demonstrate that a nucleic acid conducting channel is a complex of at least two proteins, p45 and cMDH. Furthermore, these data establish that cMDH confers nucleic acid selectivity of the channel.

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. In addition, we review channel functions predicted by our molecular model that resulted in the novel finding that the urate channel activity is regulated by sugars and adenosine. Finally, the presence and possible functions of at least 4 isoforms of UAT and a closely related gene hUAT2 are discussed.

Specific cytochemical localization of sodium-potassium dependent adenosine-triphosphatase (Na -K -atp-ASE) activity in cortical renal tubules of rat kidney

Utilizid para-nitrophenil-phosphate as an unspecific substrate cytochemical methods have described for and electron microscopi localizarion of Na -K -ATP-ase activity (ATP-ase-A). However, when applied to renal tubules, these methods did not clearly reveal the quantitative and quantitative distribution of the enzime. In an attempt to improve this method, we evaluated shorter fixation times, using freh paraformaldehyde after immersion of the renal tissue in an isosmotic sucrose tris buffer solution and utilized ATP as the specific substrate for the ATP-ase. The results indicated good preservation of the tissue, and under these conditions ATP was able to penetrate of the cells allowing a reliable identification at the ultrastructural level, of the ATP-ase-A, associated with subcellular structures for with the enzymatic reaction was positive. The activity observed suggest that the basolateral plasma membranes of proximal and distal renal tubules are the major sites of sites of Na -K -ATP-ase localization. However, there was also significant hydrolitic activity detectived on the surface (brush border) of proximal convoluted tubules, that disappeared with the elimination of the Na and K from the incubation medium. In both cases enzymatic activities were insensitive to 10 mM Ouabain in the medium