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Johannes Fürst

Publications and source records attributed to Johannes Fürst.

15 recordsLinked to original sources
PubMed2006

Fast fluorometric method for measuring pendrin (SLC26A4) Cl-/I- transport activity.

Malfunction of the SLC26A4 protein leads to Pendred syndrome, characterized by sensorineural hearing loss, often associated with mild thyroid dysfunction and goiter. It is generally assumed that SLC26A4 acts as a chloride/anion exchanger, which in the thyroid gland transports iodide, and in the inner ear contributes to the conditioning of the endolymphatic fluid. Here we describe a fast fluorometric method able to be used to functionally scrutinize SLC26A4 and its mutants described in Pendred syndrome. The validation of the method was done by functionally characterizing the chloride/iodide transport of SLC26A4, and a mutant, i.e. SLC26A4(S28R), which we previously described in a patient with sensorineural hearing loss, hypothyroidism and goiter. Using the fluorometric method we describe here we can continuously monitor and quantify the iodide or chloride amounts transported by the cells, and we found that the transport capability of the SLC26A4(S28R) mutant protein is markedly reduced if compared to wild-type SLC26A4.

Biological Transport↗
PubMed2006

Glucose induces anion conductance and cytosol-to-membrane transposition of ICln in INS-1E rat insulinoma cells.

The metabolic coupling of insulin secretion by pancreatic beta cells is mediated by membrane depolarization due to increased glucose-driven ATP production and closure of K(ATP) channels. Alternative pathways may involve the activation of anion channels by cell swelling upon glucose uptake. In INS-1E insulinoma cells superfusion with an isotonic solution containing 20 mM glucose or a 30% hypotonic solution leads to the activation of a chloride conductance with biophysical and pharmacological properties of anion currents activated in many other cell types during regulatory volume decrease (RVD), i.e. outward rectification, inactivation at positive membrane potentials and block by anion channel inhibitors like NPPB, DIDS, 4-hydroxytamoxifen and extracellular ATP. The current is not inhibited by tolbutamide and remains activated for at least 10 min when reducing the extracellular glucose concentration from 20 mM to 5 mM, but inactivates back to control levels when cells are exposed to a 20% hypertonic extracellular solution containing 20 mM glucose. This chloride current can likewise be induced by 20 mM 3-Omethylglucose, which is taken up but not metabolized by the cells, suggesting that cellular sugar uptake is involved in current activation. Fluorescence resonance energy transfer (FRET) experiments show that chloride current activation by 20 mM glucose and glucose-induced cell swelling are accompanied by a significant, transient redistribution of the membrane associated fraction of ICln, a multifunctional 'connector hub' protein involved in cell volume regulation and generation of RVD currents.

3-O-Methylglucose↗
PubMed2006

Functional characterization of wild-type and a mutated form of SLC26A4 identified in a patient with Pendred syndrome.

BACKGROUND: Malfunction of the SLC26A4 protein leads to prelingual deafness often associated with mild thyroid dysfunction and goiter. It is assumed that SLC26A4 acts as a chloride/anion exchanger responsible for the iodide organification in the thyroid gland, and conditioning of the endolymphatic fluid in the inner ear. METHODS: Chloride uptake studies were made using HEK293-Phoenix cells expressing human wild type SLC26A4 (pendrin) and a mutant (SLC26A4(S28R)) we recently described in a patient with hypothyroidism, goiter and sensorineural hearing loss. RESULTS: Experiments are summarized showing the functional characterization of wild type SLC26A4 and a mutant (S28R), which we described recently. This mutant protein is transposed towards the cell membrane, however, its transport capability is markedly reduced if compared to wild-type SLC26A4. Furthermore, we show that the SLC26A4 induced chloride uptake in HEK293-Phoenix cells competes with iodide, and, in addition, that the chloride uptake can be blocked by NPPB and niflumic acid, whereas DIDS is ineffective. CONCLUSIONS: The functional characteristics of SLC26A4(S28R) we describe here, are consistent with the clinical phenotype observed in the patient from which the mutant was derived.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2005

ICln159 folds into a pleckstrin homology domain-like structure. Interaction with kinases and the splicing factor LSm4.

ICln is a multifunctional protein involved in regulatory mechanisms as different as membrane ion transport and RNA splicing. The protein is water-soluble, and during regulatory volume decrease after cell swelling, it is able to migrate from the cytosol to the cell membrane. Purified, water-soluble ICln is able to insert into lipid bilayers to form ion channels. Here, we show that ICln159, a truncated ICln mutant, which is also able to form ion channels in lipid bilayers, belongs to the pleckstrin homology (PH) domain superfold family of proteins. The ICln PH domain shows unusual properties as it lacks the electrostatic surface polarization seen in classical PH domains. However, similar to many classical PH domain-containing proteins, ICln interacts with protein kinase C, and in addition, interacts with cAMP-dependent protein kinase and cGMP-dependent protein kinase type II but not cGMP-dependent protein kinase type Ibeta. A major phosphorylation site for all three kinases is Ser-45 within the ICln PH domain. Furthermore, ICln159 interacts with LSm4, a protein involved in splicing and mRNA degradation, suggesting that the ICln159 PH domain may serve as a protein-protein interaction platform.

Amino Acid Sequence↗
PubMed2005

Thiazide-sensitive NaCl-cotransporter in the intestine: possible role of hydrochlorothiazide in the intestinal Ca2+ uptake.

Thiazides, such as hydrochlorothiazide (HCTZ), are used to control blood pressure and to reduce renal calcium excretion. These effects are a result of interactions with the NaCl-cotransporter (NCC). This is demonstrated by the fact that mutations within the NCC protein lead to salt-resistant hypotension and hypocalciuria, paralleled by an increase in bone mineral density. These symptoms are also known as Gitelman syndrome. It has become increasingly evident that the effect of HCTZ on blood pressure and calcium homeostasis cannot be attributed exclusively to kidney functions, where the primary action of HCTZ on NCC is postulated to occur. We demonstrated the presence of the NCC transporter in the rat small intestine (ileum and jejunum) and human HT-29 cells, by using reverse transcription-PCR, Northern blot, Western blot, and immunofluorescence. Furthermore, we show that HCTZ modulates Ca(2+) uptake by intestinal cells, while affecting the electrical parameters of the cellular membrane, thus suggesting a functional interaction between NCC and the epithelial voltage-dependent calcium channel. The experiments presented here support the hypothesis of a direct involvement of the intestinal cells in the interaction between HCTZ and NaCl, as well as calcium homeostasis.

Animals↗
PubMed2005

The expression of wild-type pendrin (SLC26A4) in human embryonic kidney (HEK 293 Phoenix) cells leads to the activation of cationic currents.

OBJECTIVE: The SLC26A4 protein (pendrin) seems to be involved in the exchange of chloride with other anions, therefore being responsible for iodide organification in the thyroid gland and the conditioning of the endolymphatic fluid in the inner ear. Malfunction of SLC26A4 leads to Pendred syndrome, characterized by mild thyroid dysfunction often associated with goiter and/or prelingual deafness. The precise function of the SLC26A4 protein, however, is still elusive. An open question is still whether the SLC26A4-induced ion exchange mechanism is electrogenic or electroneutral. Recently, it has been shown that human pendrin expressed in monkey cells leads to chloride currents. METHODS: We overexpressed the human SLC26A4 isoform in HEK293 Phoenix cells and measured cationic and anionic currents by the patch-clamp technique in whole cell configuration. RESULTS: Here we show that human pendrin expressed in human cells does not lead to the activation of chloride currents, but, in contrast, leads to an increase of cationic currents. CONCLUSION: Our experiments suggest that the SLC26A4-induced chloride transport is electroneutral when expressed in human cellular systems.

Cations↗
PubMed2004

Membrane thickness changes ion-selectivity of channel-proteins.

The plasma membrane is a highly dynamic cell-barrier if the nature and distribution of its constituents are considered. Ion channels are embedded in these double lipid bilayers, which modulate their 3D-structures. The structure modulations by the lipid bilayer can assume such a degree that channel activation depends on them, as was shown for the KcsA potassium channel. Here we show that the cation-over-anion selectivity of reconstituted ICln channels can be varied by the thickness of a bilayer build of phosphatidylcholines. The shorter the acyl-chains and therefore the thinner the bilayers of the membrane are, the more potassium selective the channels are. In contrast, the longer the acyl-chains and therefore the thicker the membranes are, the more chloride selective the channels become.

Animals↗
PubMed2003

A new gene-finding tool: using the Caenorhabditis elegans operons for identifying functional partner proteins in human cells.

How can a large number of different phenotypes be generated by a limited number of genotypes? Promiscuity between different, structurally related and/or unrelated proteins seems to provide a plausible explanation to this pertinent question. Strategies able to predict such functional interrelations between different proteins are important to restrict the number of putative candidate proteins, which can then be subjected to time-consuming functional tests. Here we describe the use of the operon structure of the nematode genome to identify partner proteins in human cells. In this work we focus on ion channels proteins, which build an interface between the cell and the outside world and are responsible for a growing number of diseases in humans. However, the proposed strategy for the partner protein quest is not restricted to this scientific area but can be adopted in virtually every field of human biology where protein-protein interactions are assumed.

Adenosine Triphosphate↗
PubMed2003

Cell swelling stimulates cytosol to membrane transposition of ICln.

ICln is a multifunctional protein that is essential for cell volume regulation. It can be found in the cytosol and is associated with the cell membrane. Besides its role in the splicing process, ICln is critically involved in the generation of ion currents activated during regulatory volume decrease after cell swelling (RVDC). If reconstituted in artificial bilayers, ICln can form ion channels with biophysical properties related to RVDC. We investigated (i) the cytosol versus cell membrane distribution of ICln in rat kidney tubules, NIH 3T3 fibroblasts, Madin-Darby canine kidney (MDCK) cells, and LLC-PK1 epithelial cells, (ii) fluorescence resonance energy transfer (FRET) in living fibroblasts between fluorescently tagged ICln and fluorochromes in the cell membrane, and (iii) possible functional consequences of an enhanced ICln presence at the cell membrane. We demonstrate that ICln distribution in rat kidneys depends on the parenchymal localization and functional state of the tubules and that cell swelling causes ICln redistribution from the cytosol to the cell membrane in NIH 3T3 fibroblasts and LLC-PK1 cells. The addition of purified ICln protein to the extracellular solution or overexpression of farnesylated ICln leads to an increased anion permeability in NIH 3T3 fibroblasts. The swelling-induced redistribution of ICln correlates to altered kinetics of RVDC in NIH 3T3 fibroblasts, LLC-PK1 cells, and MDCK cells. In these cells, RVDC develops more rapidly, and in MDCK cells the rate of swelling-induced depolarization is accelerated if cells are swollen for a second time. This coincides with an enhanced ICln association with the cell membrane.

Animals↗
PubMed2002

ICln channels reconstituted in heart-lipid bilayer are selective to chloride.

ICln is an ion channel cloned from renal epithelial cells. The reconstitution of the protein in 1,2-diphytanoyl- sn-glycero-3-phosphocholine (Diph-PC) bilayer membranes reveals potassium-selective channels, which become more chloride selective in the presence of calcium. Here we show that the ion selectivity of ICln also depends on the lipid environment in which the channels are reconstituted. Diph-PC is a synthetic lipid commonly used for reconstituting ion channels. However, since this lipid is not found in native membranes, we reconstituted the ICln ion channels in a polar heart-lipid extract. Using this lipid mixture the reconstituted ICln ion channels are chloride selective in the presence of calcium and an acidic pH. The relative ion selectivity of ICln under these conditions is similar to the cation versus anion selectivity of native ion channels activated by cell swelling.

Acids↗
PubMed2002

Simvastatin inhibits malignant transformation following expression of the Ha-ras oncogene in NIH 3T3 fibroblasts.

In previous studies we have shown that the expression of the transforming Ha-ras oncogene in NIH 3T3 fibroblasts stimulates cellular calcium entry, which triggers oscillatory calcium induced calcium release from internal stores. The intracellular calcium oscillations lead to cytoskeletal remodeling by actin stress fiber depolymerization and activation of the Na(+)/H(+) exchanger thus mediating cell swelling and intracellular alkalosis, both important mitogenic signals. This is evidenced by abrogation of Ha-ras induced growth factor independent cell proliferation by interference with any of these events, i.e. by inhibition of cellular calcium entry or inhibition of the Na(+)/H(+) exchanger. As shown in this study, simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme for cholesterol biosynthesis, is able to prevent these events following the expression of the transforming Ha-ras oncogene. We show, that simvastatin inhibits farnesylation dependent membrane translocation of a CAAX motive bearing yellow fluorescent protein and suppresses Ha-ras stimulated cellular calcium influx, which can be identified as capacitative calcium entry. In addition simvastatin is able to block regulatory volume decrease channels and to suppress the cytoskeletal remodeling, intracellular alkalinization, increase in cell volume and growth factor independent cell proliferation induced by the oncogene. Thus simvastatin is able to prevent crucial cellular events following expression of the transforming Ha-ras oncogene.

3T3 Cells↗
PubMed2002

Mechanisms sensing and modulating signals arising from cell swelling.

Cell volume alterations are involved in numerous cellular events like epithelial transport, metabolic processes, hormone secretion, cell migration, proliferation and apoptosis. Above all it is a need for every cell to counteract osmotic cell swelling in order to avoid cell damage. The defence against excess cell swelling is accomplished by a reduction of the intracellular osmolarity by release of organic- or inorganic osmolytes from the cell or by synthesis of osmotically less active macromolecules from their specific subunits. De-spite the large amount of experimental data that has accumulated, the intracellular mechanisms underlying the sensing of cell volume perturbations and the activation of volume compensatory processes, commonly summarized as regulatory volume decrease (RVD), are still only partly revealed. Moving into this field opens a complex scenario of molecular rearrangements and interactions involving intracellular messengers such as calcium, phosphoinositides and inositolphosphates as well as phosphoryla-tion/dephosphorylation processes and cytoskeletal reorganization with marked cell type- and tissue specific variations. Even in one and the same cell type significant differences regarding the activated pathways during RVD may be evident. This makes it virtually im-possible to unambigously define common sensing- and sinaling pathways used by differ-ent cells to readjust their celll volume, even if all these pathways converge to the activa-tion of comparatively few sets of effectors serving for osmolyte extrusion, including ion channels and transporters. This review is aimed at providing an insight into the manifold cellular mechanisms and alterations occuring during cell swelling and RVD.

Animals↗
PubMed2001

ICln ion channel splice variants in Caenorhabditis elegans: voltage dependence and interaction with an operon partner protein.

ICln is an ion channel identified by expression cloning using a cDNA library from Madin-Darby canine kidney cells. In all organisms tested so far, only one transcript for the ICln protein could be identified. Here we show that two splice variants of the ICln ion channel can be found in Caenorhabditis elegans. Moreover, we show that these two splice variants of the ICln channel protein, which we termed IClnN1 and IClnN2, can be functionally reconstituted and tested in an artificial lipid bilayer. In these experiments, the IClnN1-induced currents showed no voltage-dependent inactivation, whereas the IClnN2-induced currents fully inactivated at positive potentials. The molecular entity responsible for the voltage-dependent inactivation of IClnN2 is a cluster of positively charged amino acids encoded by exon 2a, which is absent in IClnN1. Our experiments suggest a mechanism of channel inactivation that is similar to the "ball and chain" model proposed for the Shaker potassium channel, i.e. a cluster of positively charged amino acids hinders ion permeation through the channel by a molecular and voltage-dependent interaction at the inner vestibulum of the pore. This hypothesis is supported by the finding that synthetic peptides with the same amino acid sequence as the positive cluster can transform the IClnN1-induced current to the current observed after reconstitution of IClnN2. Furthermore, we show that the nematode ICln gene is embedded in an operon harboring two additional genes, which we termed Nx and Ny. Co-reconstitution of Nx and IClnN2 and functional analysis of the related currents revealed a functional interaction between the two proteins, as evidenced by the fact that the IClnN2-induced current in the presence of Nx was no longer voltage-sensitive. The experiments described indicate that the genome organization in nematodes allows an effective approach for the identification of functional partner proteins of ion channels.

Amino Acid Sequence↗
PubMedDate not supplied

Low-resolution structure refinement in electron microscopy.

A real-space structure refinement method, originally developed for macromolecular X-ray crystallography, has been applied to protein structure analysis by electron microscopy (EM). This method simultaneously optimizes the fit of an atomic model to a density map and the stereo-chemical properties of the model by minimizing an energy function. The performance of this method is characterized at different resolution and signal-to-noise ratio conditions typical for EM electron density maps. A multi-resolution scheme is devised to improve the convergence of the refinement on the global energy minimum. Applications of the method to various model systems are demonstrated here. The first case is the arrangement of FlgE molecules in the helical filament of flagellar hook, in which refinement with segmented rigid bodies improves the density correlation and reduces severe van der Waals contacts among the symmetry-related subunits. The second case is a conformational analysis of the NSF AAA ATPase in which a multi-conformer model is used in the refinement to investigate the arrangement of the two ATPase domains in the molecule. The third case is a docking simulation in which the crystal structure of actin and the NOE data from NMR experiments on the dematin headpiece are combined with a low-resolution EM density map to generate an atomic model of the F-actin-dematin headpiece structure.

Actins↗