Mutagenic Mapping of the Na-K-Cl Cotransporter for Domains Involved in Ion Transport and Bumetanide Binding

The human and shark Na-K-Cl cotransporters (NKCCs) are 74% identical in amino acid sequence yet they display marked differences in apparent affinities for the ions and bumetanide. In this study, we have used chimeras and point mutations to determine which transmembrane domains (tm's) are responsible for the differences in ion transport and in inhibitor binding kinetics. When expressed in HEK-293 cells, all the mutants carry out bumetanide-sensitive 86Rb influx. The kinetic behavior of these constructs demonstrates that the first seven tm's contain all of the residues conferring affinity differences. In conjunction with our previous finding that tm 2 plays an important role in cation transport, the present observations implicate the fourth and seventh tm helices in anion transport. Thus, it appears that tm's 2, 4, and 7 contain the essential affinity-modifying residues accounting for the human–shark differences with regard to cation and anion transport. Point mutations have narrowed the list of candidates to 13 residues within the three tm's. The affinity for bumetanide was found to be affected by residues in the same tm 2–7 region, and also by residues in tm's 11 and 12. Unlike for the ions, changes in bumetanide affinity were nonlinear and difficult to interpret: the Ki(bumetanide) of a number of the constructs was outside the range of sNKCC1 and hNKCC1 Kis.

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Modelling chemical and biological reactions during unsaturated flow through sandy soils

Soil Research ◽

10.1071/sr09145 ◽

2010 ◽

Vol 48 (3) ◽

pp. 221

Author(s):

B. Ludwig ◽

S. Herrmann ◽

K. Michel

Keyword(s):

Ion Transport ◽

Unsaturated Flow ◽

Sandy Soils ◽

Cation Transport ◽

Anion Transport ◽

Calibration Model ◽

Model Variant ◽

Reliable Assessment ◽

Model Predictions ◽

Flow Through

Predicting chemical and biological reactions during unsaturated flow through soils is complex, and the accuracy of model predictions is open to question. Objectives were to test how accurately the transport of nutrients (Ca2+, Mg2+, K+, NH4+, and NO3–) in soils can be predicted when differing amounts of nutrients or acid are added. Undisturbed columns of 2 sandy surface soils from arable sites in Darmstadt, Germany, and Sohar, Oman, were irrigated for 360 and 376 days with 3 mm/day at 10°C. Three treatments were carried out: the columns were irrigated with differing fertilising solutions (Fert-1 or Fert-2 treatments) or with HCl. The model PHREEQC2 was used to calculate 1-dimensional transport, inorganic complexation, dissolution or precipitation of CaCO3, multiple cation exchange, and nitrification. We compared 3 model variants: one with no adjustable parameters, a second with optimised nitrification rates and pCO2, and a third with an additional optimisation of the exchange coefficients. In model variant v2 and v3, the Fert-1 treatment was used for calibration. Model variant v1 was of little use in predicting cation transport in soils. The second was more appropriate for both soils, but an optimisation of the exchange coefficients (model variant v3) was required for a more accurate description (Fert-1) and prediction (Fert-2 and HCl) of ion transport. The included proton buffering reactions resulted in a moderately accurate prediction of pH and release of ions. Nitrification in the Fert-1 and Fert-2 experiments considered in the model variants accounted for a range of 0.03–0.04 µmol N/(day.g soil). Overall, our results indicate that calibration experiments are required for a reliable assessment of ion transport in soils during unsaturated flow. The parameter optimisation program PEST in combination with PHREEQC2 best simulated cation and anion transport in sandy soils under unsaturated conditions.

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The Impact of Glu102Lys on the Factor X Function in a Patient with a Doubly Homozygous Factor X Deficiency (Gla14Lys and Glu102Lys)

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613792 ◽

2000 ◽

Vol 83 (02) ◽

pp. 234-238 ◽

Cited By ~ 6

Author(s):

Eva Forberg ◽

Iris Huhmann ◽

Ester Jimenez-Boj ◽

Herbert Watzke

Keyword(s):

Point Mutations ◽

Site Directed Mutagenesis ◽

Normal System ◽

Factor X ◽

Wild Type ◽

Hek 293 ◽

293 Cells ◽

Functional Defect ◽

A Minor ◽

The Impact

SummaryTwo homozygous point mutations were found in a patient with factor X (FX) deficiency; One results in substitution of Lys for Gla+14 and the second causes a Lys substitution for Glu102. The proposita has a severely reduced FX coagulant activity in the extrinsic (<1% of normal) and in the intrinsic (30% of normal) system of coagulation and after activation with Russel’s viper venom (18% of normal). The FX antigen is reduced in this patient to 20% of normal. The substitution of Lys for Glu102 in FX deficiency has been reported previously in a heterozygous state in conjunction with a Lys for Gla+14 substitution and with a Pro for Ser334 substitution. The contribution of the Lys for Glu102 substitution in the observed combined FX defect in these patients was unclear. The mutation causing the Glu102Lys substitution was introduced by site directed mutagenesis into a wild-type FX cDNA, and recombinant protein was expressed in HEK 293 cells. Compared to the wild-type FX cDNA, the mutant construct had a 67% activity upon activation in the extrinsic system, 93% activity upon activation in the intrinsic system and 72% after activation with RVV. The data presented show that the substitution of Lys for Glu102 results in a minor functional defect of the FX molecule.

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Genetic Determinants of Cell Type-Specific Poliovirus Propagation in HEK 293 Cells

Journal of Virology ◽

10.1128/jvi.79.10.6281-6290.2005 ◽

2005 ◽

Vol 79 (10) ◽

pp. 6281-6290 ◽

Cited By ~ 38

Author(s):

Stephanie A. Campbell ◽

Jennifer Lin ◽

Elena Y. Dobrikova ◽

Matthias Gromeier

Keyword(s):

Point Mutations ◽

Structural Features ◽

Genetically Engineered ◽

Nucleotide Substitutions ◽

Entry Site ◽

Stem Loop ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Domain V

ABSTRACT The ability of poliovirus to propagate in neuronal cells can be reduced by introducing appropriate nucleotide substitutions into the viral genome. Specific mutations scattered throughout the poliovirus genome yielded the live attenuated vaccine strains of poliovirus. Neuron-specific propagation deficits of the Sabin strains are partially encrypted within a confined region of the internal ribosomal entry site (IRES), which carries attenuating point mutations in all three serotypes. Recently, high levels of neurovirulence attenuation were achieved with genetically engineered polioviruses containing heterologous IRES elements. This is exemplified with poliovirus recombinants replicating under control of a human rhinovirus type 2 (HRV2) IRES element. We have carried out experiments delineating the genetic basis for neuronal IRES function. Neuronal dysfunction of the HRV2 IRES is determined mainly by IRES stem-loop domain V, the locus for attenuating point mutations within the Sabin strains. Neuronal incompetence associated with HRV2 IRES domain V is substantially more pronounced than that observed with the attenuating IRES point mutation of the Sabin serotype 1 vaccine strain. Mix-and-match recombination of polio and HRV2 IRES domain V suggests that the attenuation phenotype correlates with overall structural features rather than primary sequence. Our experiments have identified HEK 293 cells as a novel system for the study of neuron-specific replication phenotypes of poliovirus. This cell line, originally derived from embryonic human kidney, has recently been described to display neuronal characteristics. We report propagation properties in HEK 293 cells for poliovirus recombinants with attenuated neurovirulence in experimental animals that corroborate this observation.

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Transport activity of chimaeric AE2-AE3 chloride/bicarbonate anion exchange proteins

Biochemical Journal ◽

10.1042/bj20030007 ◽

2003 ◽

Vol 371 (3) ◽

pp. 687-696 ◽

Cited By ~ 21

Author(s):

Jocelyne FUJINAGA ◽

Frederick B. LOISELLE ◽

Joseph R. CASEY

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Cytoplasmic Domain ◽

Anion Transport ◽

Transport Activity ◽

Surface Processing ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Anion Transport Activity

Chloride/bicarbonate anion exchangers (AEs), found in the plasma membrane of most mammalian cells, are involved in pH regulation and bicarbonate metabolism. Although AE2 and AE3 are highly similar in sequence, AE2-transport activity was 10-fold higher than AE3 (41 versus 4 mM · min−1 respectively), when expressed by transient transfection of HEK-293 cells. AE2–AE3 chimaeras were constructed to define the region responsible for differences in transport activity. The level of AE2 expression was approx. 30% higher than that of AE3. Processing to the cell surface, studied by chemical labelling and confocal microscopy, showed that AE2 is processed to the cell surface approx. 8-fold more efficiently than AE3. The efficiency of cell-surface processing was dependent on the cytoplasmic domain, since the AE2 domain conferred efficient processing upon the AE3 membrane domain, with a predominant role for amino acids 322–677 of AE2. AE2 that was expressed in HEK-293 cells was glycosylated, but little of AE3 was. However, AE2 expressed in the presence of the glycosylation inhibitor, tunicamycin, was not glycosylated, yet retained 85 ± 8% of anion-transport activity. Therefore glycosylation has little, if any, role in the cell-surface processing or activity of AE2 or AE3. We conclude that the low anion-transport activity of AE3 in HEK-293 cells is due to low level processing to the plasma membrane, possibly owing to protein interactions with the AE3 cytoplasmic domain.

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Multifunctional ion transport properties of human SLC4A11: comparison of the SLC4A11-B and SLC4A11-C variants

AJP Cell Physiology ◽

10.1152/ajpcell.00233.2016 ◽

2016 ◽

Vol 311 (5) ◽

pp. C820-C830 ◽

Cited By ~ 25

Author(s):

Liyo Kao ◽

Rustam Azimov ◽

Xuesi M. Shao ◽

Ricardo F. Frausto ◽

Natalia Abuladze ◽

...

Keyword(s):

Ion Transport ◽

Transport Properties ◽

Late Onset ◽

Sensorineural Deafness ◽

Corneal Dystrophy ◽

Hek 293 ◽

Coupled Mode ◽

293 Cells ◽

Inward Currents ◽

Dystrophic Disease

Congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (CHED with progressive sensorineural deafness), and potentially a subset of individuals with late-onset Fuchs' endothelial corneal dystrophy are caused by mutations in the SLC4A11 gene that results in corneal endothelial cell abnormalities. Originally classified as a borate transporter, the function of SLC4A11 as a transport protein remains poorly understood. Elucidating the transport function(s) of SLC4A11 is needed to better understand how its loss results in the aforementioned posterior corneal dystrophic disease processes. Quantitative PCR experiments demonstrated that, of the three known human NH2-terminal variants, SLC4A11-C is the major transcript expressed in human corneal endothelium. We studied the expression pattern of the three variants in mammalian HEK-293 cells and demonstrated that the SLC4A11-B and SLC4A11-C variants are plasma membrane proteins, whereas SLC4A11-A is localized intracellularly. SLC4A11-B and SLC4A11-C were shown to be multifunctional ion transporters capable of transporting H+ equivalents in both a Na+-independent and Na+-coupled mode. In both transport modes, SLC4A11-C H+ flux was significantly greater than SLC4A11-B. In the presence of ammonia, SLC4A11-B and SLC4A11-C generated inward currents that were comparable in magnitude. Chimera SLC4A11-C-NH2-terminus-SLC4A11-B experiments demonstrated that the SLC4A11-C NH2-terminus functions as an autoactivating domain, enhancing Na+-independent and Na+-coupled H+ flux without significantly affecting the electrogenic NH3-H( n)+ cotransport mode. All three modes of transport were significantly impaired in the presence of the CHED causing p.R109H (SLC4A11-C numbering) mutation. These complex ion transport properties need to be addressed in the context of corneal endothelial disease processes caused by mutations in SLC4A11.

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Inhibition of Na+-K+-2Cl−cotransport by mercury

AJP Cell Physiology ◽

10.1152/ajpcell.1999.277.4.c684 ◽

1999 ◽

Vol 277 (4) ◽

pp. C684-C692 ◽

Cited By ~ 42

Author(s):

Steven C. Jacoby ◽

Edith Gagnon ◽

Luc Caron ◽

John Chang ◽

Paul Isenring

Keyword(s):

Ion Transport ◽

Binding Sites ◽

Essential Role ◽

Species Differences ◽

Transmembrane Domain ◽

Transport Proteins ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells

Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH−) groups. The inorganic form (Hg2+) is toxic to epithelial tissues and interacts with various transport proteins including the Na+ pump and Cl− channels. In this study, we determined whether the Na+-K+-Cl−cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues, is also affected by mercurial substrates. To characterize the interaction, we measured the effect of Hg2+ on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg2+inhibits Na+-K+-Cl−cotransport, with inhibitor constant ( K i) values of 25 μM for the shark carrier (sNKCC1) and 43 μM for the human carrier. In further studies, we took advantage of species differences in Hg2+ affinity to identify residues involved in the interaction. An analysis of human-shark chimeras and of an sNKCC1 mutant (Cys-697→Leu) reveals that transmembrane domain 11 plays an essential role in Hg2+binding. We also show that modification of additional SH− groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.

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Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

International Journal of Molecular Sciences ◽

10.3390/ijms22094637 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4637

Author(s):

Daniel Barth ◽

Andreas Lückhoff ◽

Frank J. P. Kühn

Keyword(s):

Amino Acid Residues ◽

Hek 293 ◽

Complete Inactivation ◽

293 Cells ◽

Activation Mechanisms ◽

Specific Regulation ◽

Species Specific ◽

Inside Out ◽

Positively Charged

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

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Preliminary Study on Pasta Samples Characterized in Antioxidant Compounds and Their Biological Activity on Kidney Cells

Nutrients ◽

10.3390/nu13041131 ◽

2021 ◽

Vol 13 (4) ◽

pp. 1131 ◽

Cited By ~ 1

Author(s):

Federico Di Marco ◽

Francesco Trevisani ◽

Pamela Vignolini ◽

Silvia Urciuoli ◽

Andrea Salonia ◽

...

Keyword(s):

Indoleacetic Acid ◽

Antioxidant Properties ◽

Antioxidant Compounds ◽

Kidney Cells ◽

Hek 293 ◽

293 Cells ◽

High Concentrations ◽

Β Carotene ◽

Egg Pasta

Pasta is one of the basic foods of the Mediterranean diet and for this reason it was chosen for this study to evaluate its antioxidant properties. Three types of pasta were selected: buckwheat, rye and egg pasta. Qualitative–quantitative characterization analyses were carried out by HPLC-DAD to identify antioxidant compounds. The data showed the presence of carotenoids such as lutein and polyphenols such as indoleacetic acid, (carotenoids from 0.08 to 0.16 mg/100 g, polyphenols from 3.7 to 7.4 mg/100 g). To assess the effect of the detected metabolites, in vitro experimentation was carried out on kidney cells models: HEK-293 and MDCK. Standards of β-carotene, indoleacetic acid and caffeic acid, hydroalcoholic and carotenoid-enriched extracts from samples of pasta were tested in presence of antioxidant agent to determine viability variations. β-carotene and indoleacetic acid standards exerted a protective effect on HEK-293 cells while no effect was detected on MDCK. The concentrations tested are likely in the range of those reached in body after the consumption of a standard pasta meal. Carotenoid-enriched extracts and hydroalcoholic extracts showed different effects, observing rescues for rye pasta hydroalcoholic extract and buckwheat pasta carotenoid-enriched extract, while egg pasta showed milder dose depending effects assuming pro-oxidant behavior at high concentrations. The preliminary results suggest behaviors to be traced back to the whole phytocomplexes respect to single molecules and need further investigations.

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Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts

Molecules ◽

10.3390/molecules26040973 ◽

2021 ◽

Vol 26 (4) ◽

pp. 973

Author(s):

Tilen Koklič ◽

Alenka Hrovat ◽

Ramon Guixà-González ◽

Ismael Rodríguez-Espigares ◽

Damaris Navio ◽

...

Keyword(s):

Lipid Rafts ◽

Order Parameter ◽

Spin Probe ◽

Gonadotropin Releasing Hormone ◽

Paramagnetic Resonance ◽

Hek 293 ◽

293 Cells ◽

Gonadotropin Releasing Hormone Receptor ◽

Electron Paramagnetic

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-β-cyclodextrin (methyl-β-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

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