Urea Channel Research Articles

Elucidating the pH gating mechanism of Helicobacter pyloris urea channel UreI.

Over 50% of the world population suffer from chronic gastric infection with Helicobacter pyloris which is linked to peptic ulcer disease and stomach cancer. Yet, the efficacy of the common therapy including antibiotics is decreasing. An alternative drug target could be H. pyloris pH gatedinner-membrane urea channel UreI, which is pivotal for the survival of the pathogen in the acidic environment of the human stomach. However, despite in vivo studies and high-resolution structures in the open and closed state, HpUreIs gating mechanism is still elusive. Using yeast complementation assays, we tested (i) homologues urea channels, (ii) point mutations of charged residues also on the cytoplasmic side, and (iii) variants carrying changes at the periplasmic side at the N- and C-terminus as well as in periplasmic loop 1 (PL1). The functionality and pH gating behaviour of these constructs are compared to the wild type protein in the physiological relevant pH range from 4.0 to 7.0. Our in vivo studies are guided and accompanied by an extensive sequence- and structure-based analysis of homologues urea channels. The results question the hypothesis of PL1 and PL2 constituting the main pH-sensor of HpUreI but confirm the importance of the N- and C-terminus in the pH gating mechanism. For example, additional amino acids at the C-terminus provoke a urea impermeable HpUreI, emphasizing its importance for the stability of the open state. Furthermore, the urea channels from Helicobacter hepaticus and Streptococcus salivarius, lacking such extensive periplasmic loops, show similar pH dependence as HpUreI. Overall, yeast complementation assays are an cost-effective tool for testing qualitative differences in protein variant activity for a broad range of solutes.

The aquaporin 8 (AQP8) membrane channel gene is present in the elasmobranch dogfish (Squalus acanthias) genome and is expressed in brain but not in gill, kidney or intestine.

The transport mechanisms for water, ammonia and urea in elasmobranch gill, kidney and gastrointestinal tract remain to be fully elucidated. Aquaporin 8 (AQP8) is a known water, ammonia and urea channel that is expressed in the kidney and respiratory and gastrointestinal tracts of mammals and teleost fish. However, at the initiation of this study in late 2019, there was no copy of an elasmobranch aquaporin 8 gene identified in the genebank even for closely related holocephalon species such as elephant fish (Callorhinchus milii) or for the elasmobranch little skate (Leucoraja erinacea). A transcriptomic study in spiny dogfish (Squalus acanthias) also failed to identify a copy. Hence this study has remedied this and identified the AQP8 cDNA sequence using degenerate PCR. Agarose electrophoresis of degenerate PCR reactions from dogfish tissues showed a strong band from brain cDNA and faint bands of a similar size in gill and liver. 5' and 3' RACE was used to complete the AQP8 cDNA sequence. Primers were then designed for further PCR reactions to determine the distribution of AQP8 mRNA expression in dogfish tissues. This showed that AQP8 is only expressed in dogfish brain and AQP8 therefore clearly can play no role in water, ammonia and urea transport in the gill, kidney or gastrointestinal tract. The role of AQP8 in dogfish brain remains to be determined.

Expression and Transport Characteristics of Urea Channel Protein B in Rat Bladder Cance

Expression and Transport Characteristics of Urea Channel Protein B in Rat Bladder Cance

PH-dependent gating mechanism of the Helicobacter pylori urea channel revealed by cryo-EM.

The urea channel of Helicobacter pylori (HpUreI) is an ideal drug target for preventing gastric cancer but incomplete understanding of its gating mechanism has hampered development of inhibitors for the eradication of H. pylori. Here, we present the cryo-EM structures of HpUreI in closed and open conformations, both at a resolution of 2.7 Å. Our hexameric structures of this small membrane protein (~21 kDa/protomer) resolve its periplasmic loops and carboxyl terminus that close and open the channel, and define a gating mechanism that is pH dependent and requires cooperativity between protomers in the hexamer. Gating is further associated with well-resolved changes in the channel-lining residues that modify the shape and length of the urea pore. Site-specific mutations in the periplasmic domain and urea pore identified key residues important for channel function. Drugs blocking the urea pore based on our structures should lead to a new strategy for H. pylori eradication.

Deep Proteomic Quantification of Inner Medullary Collecting Duct Protein Phosphorylation: Response to Vasopressin

Progress in protein mass spectrometry instrumentation and labeling methods have led to dramatic increases in the depth of phospho‐peptide identification and quantification. Here we used tandem mass tag (TMT) labeling and LC‐MS/MS for deep identification of phospho‐proteomic responses to the vasopressin analog dDAVP (1 nM for 30 minutes) in inner medullary collecting duct (IMCD) suspensions from rats. We analyzed three pairs of samples (dDAVP vs. vehicle) on different days. The analysis yielded 16 GB of spectral data mapping to 11,658 phospho‐peptides in 1758 distinct proteins. Vehicle vs. vehicle comparisons allowed assessment of the variability of the method and permitted dual criteria to be applied for stringent identification of abundance changes (based on t‐statistic and z‐score). Among all phospho‐peptides quantified, only 93 were increased in abundance, and only 49 were decreased, indicating very selective regulation within the broad range of phospho‐peptides quantified. A substantial number of the increases were in aquaporin‐2 and the UT‐A urea channel, including a previously unidentified phosphorylation site at Ser918 of UT‐A1 (sequence RAS*MITK) that was more than doubled in abundance. Abundance changes in aquaporin‐2 peptides confirmed prior findings. Of interest, phosphorylation changes in Raf1 (increased phosphorylation at Ser259, a kinase inhibitory site) and A‐Raf (decreased phosphorylation of Ser255, a kinase activating site) provide an explanation for findings that vasopressin decreases ERK activation and AQP2 phosphorylation at Ser261 in the collecting duct. Finally, vasopressin significantly increased phosphorylation of protein kinase D1 (Prkd1) at Ser255 and of phosphatidylinositol 4‐kinase beta at Ser551. Both proteins play central roles in regulated exocytosis of ENaC, and we speculate that they could play similar roles in vasopressin‐stimulated exocytotic apical insertion of aquaporin‐2 and UT‐A1.Support or Funding InformationV.D. was a member of the 2017 Biomedical Engineering Student Internship Program at the National Institutes of Health.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting the Helicobacter pylori urea channel and urease

Targeting the <i>Helicobacter pylori</i> urea channel and urease

Identification of UT-A1- and AQP2-interacting proteins in rat inner medullary collecting duct.

The urea channel UT-A1 and the water channel aquaporin-2 (AQP2) mediate vasopressin-regulated transport in the renal inner medullary collecting duct (IMCD). To identify the proteins that interact with UT-A1 and AQP2 in native rat IMCD cells, we carried out chemical cross-linking followed by detergent solubilization, immunoprecipitation, and LC-MS/MS analysis of the immunoprecipitated material. The analyses revealed 133 UT-A1-interacting proteins and 139 AQP2-interacting proteins, each identified in multiple replicates. Fifty-three proteins that were present in both the UT-A1 and the AQP2 interactomes can be considered as mediators of housekeeping interactions, likely common to all plasma membrane proteins. Among proteins unique to the UT-A1 list were those involved in posttranslational modifications: phosphorylation (protein kinases Cdc42bpb, Phkb, Camk2d, and Mtor), ubiquitylation/deubiquitylation (Uba1, Usp9x), and neddylation (Nae1 and Uba3). Among the proteins unique to the AQP2 list were several Rab proteins (Rab1a, Rab2a, Rab5b, Rab5c, Rab7a, Rab11a, Rab11b, Rab14, Rab17) involved in membrane trafficking. UT-A1 was found to interact with UT-A3, although quantitative proteomics revealed that most UT-A1 molecules in the cell are not bound to UT-A3. In vitro incubation of UT-A1 peptides with the protein kinases identified in the UT-A1 interactome revealed that all except Mtor were capable of phosphorylating known sites in UT-A1. Overall, the UT-A1 and AQP2 interactomes provide a snapshot of a dynamic process in which UT-A1 and AQP2 are produced in the rough endoplasmic reticulum, processed through the Golgi apparatus, delivered to endosomes that move into and out of the plasma membrane, and are regulated in the plasma membrane.

Genetic Diversity as Consequence of a Microaerobic and Neutrophilic Lifestyle.

As a neutrophilic bacterium, Helicobacter pylori is growth deficient under extreme acidic conditions. The gastric pathogen is equipped with an acid survival kit, regulating urease activity by a pH-gated urea channel, opening below pH 6.5. After overcoming acid stress, the bacterium’s multiplication site is situated at the gastric mucosa with near neutral pH. The pathogen exhibits exceptional genetic variability, mainly due to its capability of natural transformation, termed competence. Using single cell analysis, we show here that competence is highly regulated in H. pylori. DNA uptake complex activity was reversibly shut down below pH 6.5. pH values above 6.5 opened a competence window, in which competence development was triggered by the combination of pH increase and oxidative stress. In contrast, addition of sublethal concentrations of the DNA-damaging agents ciprofloxacin or mitomycin C did not trigger competence development under our conditions. An oxygen-sensitive mutant lacking superoxide dismutase (sodB) displayed a higher competent fraction of cells than the wild type under comparable conditions. In addition, the sodB mutant was dependent on adenine for growth in broth and turned into non-cultivable coccoid forms in its absence, indicating that adenine had radical quenching capacity. Quantification of periplasmically located DNA in competent wild type cells revealed outstanding median imported DNA amounts of around 350 kb per cell within 10 min of import, with maximally a chromosomal equivalent (1.6 Mb) in individual cells, far exceeding previous amounts detected in other Gram-negative bacteria. We conclude that the pathogen’s high genetic diversity is a consequence of its enormous DNA uptake capacity, triggered by intrinsic and extrinsic oxidative stress once a neutral pH at the site of chronic host colonization allows competence development.

Structure, function, and inhibitors of the acid-gatedHelicobacter pyloriurea channel, an essential component for acid survival and chronic infection

Structure, function, and inhibitors of the acid-gated<i>Helicobacter pylori</i>urea channel, an essential component for acid survival and chronic infection

Colloidal bismuth subcitrate impedes proton entry into Helicobacter pylori and increases the efficacy of growth-dependent antibiotics.

Successful eradication of Helicobacter pylori is becoming more difficult, mainly due to emerging antibiotic resistance. Treatment regimens containing bismuth have increased efficacy, but the mechanism is unknown. Helicobacter pylori is a neutralophile adapted to survive the acidic gastric environment via acid acclimation, but demonstrates more robust growth at neutral pH. Many antibiotics used to treat H. pylori rely on bacterial growth. To investigate the mechanism of increased efficacy of bismuth-containing H. pylori treatment regimens. RNAseq and qPCR, urease activity in permeabilised and intact bacteria, internal pH and membrane potential were measured with and without colloidal bismuth subcitrate (CBS). Bacterial survival was assessed with CBS and/or ampicillin. Genes involved with metabolism and growth were upregulated in the presence of CBS at acidic pH. Urease activity of permeabilised H. pylori at pH 7.4 and 4.5 decreased in the presence of CBS, but intact urease activity decreased only at acidic pH. The fall in cytoplasmic pH with external acidification was diminished by CBS. The increase in membrane potential in response to urea addition at acidic medium pH was unaffected by CBS. The impact of CBS and ampicillin on H. pylori survival was greater than either agent alone. Bismuth is not acting directly on urease or the urea channel. Colloidal bismuth subcitrate impedes proton entry into the bacteria, leading to a decrease in the expected fall in cytoplasmic pH. With cytoplasmic pH remaining within range for increased metabolic activity of a neutralophile, the efficacy of growth-dependent antibiotics is augmented.

Structure, Function, and Inhibitors of the Acid‐Gated Helicobacter pylori urea Channel, an Essential Component for Acid Survival and Chronic Infection

Half the world's population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated innermembrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease produces NH3 and CO2, neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea. Follow-up microsecond-scale unrestrained molecular dynamics studies now provide a detailed mechanism of urea and water transport by HpUreI. Strugatsky, D., McNulty, R.M., Munson, K., Chen, C.-K., Soltis, S.M., Sachs, G., Luecke H. “Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori” Nature 493, 255–258 (2013). Luecke, H. & Sachs, G. “Helicobacter pylori's Achilles' Heel” (2013) Immuno-Gastroenterology 2, 76. McNulty, R., Ulmschneider, J.P., Luecke, H., Ulmschneider, M.B. ”Mechanisms of molecular transport through the urea channel of Helicobacter pylori” (2013) Nature Communications 4, article number 2900.

Computation and simulation of the structural characteristics of the kidney urea transporter and behaviors of urea transport.

Urea transporters are a family of membrane proteins that transport urea molecules across cell membranes and play important roles in a variety of physiological processes. Although the crystal structure of bacterial urea channel dvUT has been solved, there lacks an understanding of the dynamics of urea transport in dvUT. In this study, by using molecular dynamics simulations, Monte Carlo methods, and the adaptive biasing force approach, we built the equilibrium structure of dvUT, calculated the variation in the free energy of urea, determined the urea-binding sites of dvUT, gained insight into the microscopic process of urea transport, and studied the water permeability in dvUT including the analysis of a water chain in the pore. The strategy used in this work can be applied to studying transport behaviors of other membrane proteins.

Morphology and crystalline structure of inclusion compounds formed between poly(ethylene glycol) and urea

The poly(ethylene glycol) (PEG, with M w 2000)-urea inclusion compound (IC) crystallized at high temperature region showed two typical orientations, flat-on and edge-on crystals. 2D-XRD and polarized FTIR spectroscopy revealed that the PEG chains within urea channels were perpendicular to the substrate in flat-on oriented crystals, while PEG chain axes were parallel to the substrate and lay along the growth direction in the edge-on crystals. FTIR absorption bands of PEG in the ICs are sensitive to orientation of the crystals. A scheme of PEG chain packing in the urea IC channel was proposed, which could explain the orientation of the crystal nucleus causing the two types of morphologies. Furthermore, functioning of PEG2000 chain end with analine had significantly influence on the morphology and orientation of the inclusion compound crystals, due to the defects caused by large terminal groups included in the urea channel.

Identification of protein kinases that phosphorylate the urea channel protein UT‐A1 (1137.11)

Vasopressin regulates the urea channel protein UT‐A1 by phosphorylation at Ser84 and Ser486. What kinases are responsible for these phosphorylation events remains an open question. To identify protein kinases that interact with UT‐A1 in IMCD, we employed crosslinking with BSOCOES, UT‐A1 immunoprecipitation, SDS‐PAGE separation, and quantitative mass spectrometry of trypsin digests of gel pieces. The peptides passing stringent spectral quality thresholds were quantified (label‐free) to identify those with high signal:noise (UTA1 antibody/preimmune IgG&gt;4). This identified four protein kinases bound to the UT‐A1 (Phosphorylase Kinase [PhK], Calmodulin‐Dependent Kinase 2δ□[CAMK2], CDC42‐Binding Protein Kinase β□[CDC42BPK] and mTOR). In vitro phosphorylation assays were carried out by incubating these kinases or Protein Kinase A (purified, active recombinant) with synthetic peptides containing the known phosphorylation sites in UT‐A1. Phosphorylation was determined by immunoblotting using phospho‐specific antibodies. Four of the five tested kinases phosphorylated Ser486 (PKA &gt; CDC42BPK &gt; PhK &gt; CAMK2). Kinetic studies of Ser486 phosphorylation (1 min‐24 hr) also showed PKA &gt; CDC42BPK &gt; PhK. In contrast, only CDC42BPK phosphorylated Ser84‐UT‐A1. mTOR did not phosphorylate either site. These results provide important data for modeling vasopressin signaling pathways.Grant Funding Source: Supported by NHLBI

Challenging the state of the art in protein structure prediction: Highlights of experimental target structures for the 10th Critical Assessment of Techniques for Protein Structure Prediction Experiment CASP10.

For the last two decades, CASP has assessed the state of the art in techniques for protein structure prediction and identified areas which required further development. CASP would not have been possible without the prediction targets provided by the experimental structural biology community. In the latest experiment, CASP10, more than 100 structures were suggested as prediction targets, some of which appeared to be extraordinarily difficult for modeling. In this article, authors of some of the most challenging targets discuss which specific scientific question motivated the experimental structure determination of the target protein, which structural features were especially interesting from a structural or functional perspective, and to what extent these features were correctly reproduced in the predictions submitted to CASP10. Specifically, the following targets will be presented: the acid-gated urea channel, a difficult to predict transmembrane protein from the important human pathogen Helicobacter pylori; the structure of human interleukin (IL)-34, a recently discovered helical cytokine; the structure of a functionally uncharacterized enzyme OrfY from Thermoproteus tenax formed by a gene duplication and a novel fold; an ORFan domain of mimivirus sulfhydryl oxidase R596; the fiber protein gene product 17 from bacteriophage T7; the bacteriophage CBA-120 tailspike protein; a virus coat protein from metagenomic samples of the marine environment; and finally, an unprecedented class of structure prediction targets based on engineered disulfide-rich small proteins.

Mechanisms of Molecular Transport through the Proton-Gated Urea Channel of Helicobacter Pylori

Helicobacter pylori survival in acidic environments relies on cytoplasmic hydrolysis of gastric urea into ammonia and carbon dioxide, which buffer the pathogen's periplasm. Urea uptake is greatly enhanced and regulated by HpUreI, a proton-gated inner-membrane channel protein essential for gastric survival of H. pylori. The crystal structure of HpUreI describes a static snapshot of the channel with two constriction sites near the center of the bilayer that are too narrow to allow passage of urea or even water. Here we report the atomic resolution urea transport mechanism, revealed by unrestrained microsecond equilibrium molecular dynamics simulations of the hexameric channel assembly. Two consecutive constrictions open to allow conduction of urea, which is guided through the channel by an interplay of conserved residues that determine proton rejection and solute selectivity. Remarkably, HpUreI conducts water at rates equivalent to aquaporins, which might be essential for efficient transport of urea at small concentration gradients.

Mechanisms of molecular transport through the urea channel of Helicobacter pylori

Helicobacter pylori survival in acidic environments relies on cytoplasmic hydrolysis of gastric urea into ammonia and carbon dioxide, which buffer the pathogen’s periplasm. Urea uptake is greatly enhanced and regulated by HpUreI, a proton-gated inner membrane channel protein essential for gastric survival of H. pylori. The crystal structure of HpUreI describes a static snapshot of the channel with two constriction sites near the center of the bilayer that are too narrow to allow passage of urea or even water. Here we describe the urea transport mechanism at atomic resolution, revealed by unrestrained microsecond equilibrium molecular dynamics simulations of the hexameric channel assembly. Two consecutive constrictions open to allow conduction of urea, which is guided through the channel by interplay between conserved residues that determine proton rejection and solute selectivity. Remarkably, HpUreI conducts water at rates equivalent to aquaporins, which might be essential for efficient transport of urea at small concentration gradients.

Urea channel inhibitors: a new functional class of aquaretics

Using large-scale small molecule screening techniques, Li et al. have identified a compound that inhibits the UT-B urea channel. They propose that this or similar compounds could be used as aquaretic agents to increase water excretion without changes in electrolyte excretion. Such compounds would potentially be useful in treatment of hyponatremic disorders. Here we review the physiological basis for the action of urea channel inhibitors in the kidney and assess their clinical potential.

The Role of ExbD in Periplasmic pH Homeostasis in Helicobacter pylori

Helicobacter pylori, a neutralophile, colonizes the acidic environment of the human stomach by employing acid acclimation mechanisms that regulate periplasmic and cytoplasmic pH. The regulation of urease activity is central to acid acclimation. Inactive urease apoenzyme, UreA/B, requires nickel for activation. Accessory proteins UreE, F, G, and H are required for nickel insertion into apoenzyme. The ExbB/ExbD/TonB complex transfers energy from the inner to outer membrane, providing the driving force for nickel uptake. Therefore, the aim of this study was to determine the contribution of ExbD to pH homeostasis. A nonpolar exbD knockout was constructed and survival, growth, urease activity, and membrane potential were determined in comparison with wildtype. Survival of the ΔexbD strain was significantly reduced at pH 3.0. Urease activity as a function of pH and UreI activation was similar to the wildtype strain, showing normal function of the proton-gated urea channel, UreI. The increase in total urease activity over time in acid seen in the wildtype strain was abolished in the ΔexbD strain, but recovered in the presence of supraphysiologic nickel concentrations, demonstrating that the effect of the ΔexbD mutant is due to loss of a necessary constant supply of nickel. In acid, ΔexbD also decreased its ability to maintain membrane potential and periplasmic buffering in the presence of urea. ExbD is essential for maintenance of periplasmic buffering and membrane potential by transferring energy required for nickel uptake, making it a potential nonantibiotic target for H. pylori eradication.

Mass spectrometry‐based pseudo‐western blots

Modern systems biology utilizes protein mass spectrometry (MS) to quantify thousands of proteins in a given tissue. The alternative to MS, western blotting, is low‐throughput, but gives quantitative information as a function of protein molecular weight, providing critical information about protein modifications. Here, we describe an approach that combines the advantages of both: “pseudo‐western blots” (pWBs). Protein extracts are separated by standard SDS‐PAGE, and each lane is sliced into 40 successive blocks followed by in‐gel trypsin digestion. Individual samples are analyzed by label‐free quantitative LC‐MS/MS (Velos‐Orbitrap). The resulting data were then translated into a western blot‐like format (abundance vs. slice number) using a heat map generator. To establish proof‐of‐principle, we analyzed kidney inner medullas from rats subjected to high‐ or low‐water intake for 3 days. The data were assembled into hundreds of pWBs. pWBs of the vasopressin‐regulated water channel aquaporin‐2 (Aqp2) and a urea channel (Slc14a2) matched corresponding antibody‐based western blots and reproduced changes. Our results show the feasibility of the pWB approach for systems biology applications. (G.L. supported by NIBIB Biomedical Engineering Student Internship Program, 2012)