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K Kunzelmann

Publications and source records attributed to K Kunzelmann.

At least 37 records · Page 2Linked to original sources
PubMed2000

14-3-3 interacts with regulator of G protein signaling proteins and modulates their activity.

Regulator of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) that stimulate the inactivation of heterotrimeric G proteins. We have recently shown that RGS proteins may be regulated on a post-translational level (Benzing, T., Brandes, R., Sellin, L., Schermer, B., Lecker, S., Walz, G., and Kim, E. (1999) Nat. Med. 5, 913-918). However, mechanisms controlling the GAP activity of RGS proteins are poorly understood. Here we show that 14-3-3 proteins associate with RGS7 and RGS3. Binding of 14-3-3 is mediated by a conserved phosphoserine located in the Galpha-interacting portion of the RGS domain; interaction with 14-3-3 inhibits the GAP activity of RGS7, depends upon phosphorylation of a conserved residue within the RGS domain, and results in inhibition of GAP function. Collectively, these data indicate that phosphorylation-dependent binding of 14-3-3 may act as molecular switch that controls the GAP activity keeping a substantial fraction of RGS proteins in a dormant state.

14-3-3 Proteins↗
PubMed2000

Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia.

Many pathogens causing diarrhea do so by modulating ion transport in the gut. Respiratory pathogens are similarly associated with disturbances of fluid balance in the respiratory tract, although it is not known whether they too act by altering epithelial ion transport. Here we show that influenza virus A/PR/8/34 inhibits the amiloride-sensitive Na(+) current across mouse tracheal epithelium with a half-time of about 60 min. We further show that the inhibitory effect of the influenza virus is caused by the binding of viral hemagglutinin to a cell-surface receptor, which then activates phospholipase C and protein kinase C. Given the importance of epithelial Na(+) channels in controlling the amount of fluid in the respiratory tract, we suggest that down-regulation of Na(+) channels induced by influenza virus may play a role in the fluid transport abnormalities that are associated with influenza infections.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Aquaporin 3 cloned from Xenopus laevis is regulated by the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) is essential for epithelial electrolyte transport and has been shown to be a regulator of epithelial Na(+), K(+), and Cl(-) channels. CFTR also enhances osmotic water permeability when activated by cAMP. This was detected initially in Xenopus oocytes and is also present in human airway epithelial cells, however, the mechanisms remain obscure. Here, we show that CFTR activates aquaporin 3 expressed endogenously and exogenously in oocytes of Xenopus laevis. The interaction requires stimulation of wild type CFTR by cAMP and an intact first nucleotide binding domain as demonstrated for other CFTR-protein interactions.

Amino Acid Sequence↗
PubMed2000

Control of epithelial Na+ conductance by the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel expressed in luminal membranes of secretory and reabsorptive epithelia. CFTR plays a predominant role in both cAMP- and Ca2+-activated secretion of electrolytes. Although Ca2+-dependent Cl- channels exist independent of CFTR in the airway epithelium, their physiological significance remains to be determined. However, CFTR seems to be the only relevant Cl- conductance in the colonic epithelium. Apart from its secretory function, CFTR also has a task in regulating the reabsorption of electrolytes by controlling the activity of the epithelial Na+ channel, ENaC. Accordingly, defects in CFTR causing the disease cystic fibrosis (CF) lead to disturbances of both the secretion and absorption of electrolytes. Therefore, it is unclear what is pathophysiologically more important for the development of CF lung disease, the impaired secretion of Cl- or the enhanced reabsorption of Na+ and consecutive hyperabsorption of electrolytes. The mechanisms of how CFTR and ENaC interact are unknown. Previous work has given rise to several interesting working hypothesis, such as direct protein interaction or interaction via cytoskeletal proteins. Recent studies demonstrate the importance of the first nucleotide binding fold of CFTR, not only for the inhibition of ENaC but also for the interaction with other ion channels. Further studies are required to demonstrate whether regulation of other ion channels and membrane transport by CFTR occur by a common mechanism.

Animals↗
PubMed2000

Effect of genistein on native epithelial tissue from normal individuals and CF patients and on ion channels expressed in Xenopus oocytes.

The flavonoid genistein has been shown to activate a Cl(-) conductance in various cell types expressing CFTR. We examined if similar effects can be observed when genistein is applied to native ex vivo tissues from human respiratory tract and rectum. We further compared the effects when genistein was applied to oocytes of Xenopus laevis expressing CFTR. In oocytes, both wtCFTR and DeltaF508-CFTR were activated by genistein while both cyclic AMP (K(v)LQT1) and Ca(2+) (SK4) activated K(+) channels were inhibited at high concentrations of genistein. Biopsies from nasal polyps and rectal mucosa were obtained from normal individuals (non-CF) and CF patients and in the presence of amiloride (10 micromol l(-1); mucosal side) the effects of genistein were assessed using a perfused Ussing chamber. In non-CF airway epithelia, genistein (50 micromol l(-1); mucosal side) increased lumen negative I(sc) but had no additional effects on tissues pre-stimulated with IBMX and forskolin (100 micromol l(-1) and 1 micromol l(-1); both sides). In non-CF rectal biopsies, in the presence of amiloride (10 micromol l(-1); mucosal side) and indomethacin (10 micromol l(-1); basolateral side), genistein increased lumen negative I(sc) and enabled cholinergic (carbachol; CCH, 100 micromol l(-1); basolateral side) stimulation of Cl(-) secretion indicating activation of luminal CFTR Cl(-) channels. However, after stimulation with IBMX/forskolin, genistein induced opposite effects and significantly inhibited CCH activated I(sc). In CF airway and intestinal tissues genistein failed to induce Cl(-) secretion. Thus, genistein is able to activate luminal CFTR Cl(-) conductance in non-CF tissues and mutant CFTR in oocytes. However, additional inhibitory effects on basolateral K(+) conductance and missing effects in native CF tissues do not support the use for pharmacological intervention in CF.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Defective cholinergic Cl(-) secretion and detection of K(+) secretion in rectal biopsies from cystic fibrosis patients.

Rectal biopsies from cystic fibrosis (CF) patients show defective cAMP-activated Cl(-) secretion and an inverse response of the short-circuit current (I(sc)) toward stimulation with carbachol (CCh). Alternative Cl(-) channels are found in airway epithelia and have been attributed to residual Cl(-) secretion in CF colon. The aim of the present study was to investigate ion conductances causing reversed I(sc) upon cholinergic stimulation. Furthermore, the putative role of an alternative Ca(2+)-dependent Cl(-) conductance in human distal colon was examined. Cholinergic ion secretion was assessed in the absence and presence of cAMP-dependent stimulation. Transepithelial voltage and I(sc) were measured in rectal biopsies from non-CF and CF individuals by means of a perfused micro-Ussing chamber. Under baseline conditions, CCh induced a positive I(sc) in CF rectal biopsies but caused a negative I(sc) in non-CF subjects. The CCh-induced negative I(sc) in non-CF biopsies was gradually reversed to a positive response by incubating the biopsies in indomethacin. The positive I(sc) was significantly enhanced in CF and was caused by activation of a luminal K(+) conductance, as shown by the use of the K(+) channel blockers Ba(2+) and tetraethylammonium. Moreover, a cAMP-dependent luminal K(+) conductance was detected in CF individuals. We conclude that the cystic fibrosis transmembrane conductance regulator is the predominant Cl(-) channel in human distal colon. Unlike human airways, no evidence was found for an alternative Cl(-) conductance in native tissues from CF patients. Furthermore, we demonstrated that both Ca(2+)- and cAMP-dependent K(+) secretion are present in human distal colon, which are unmasked in rectal biopsies from CF patients.

Adolescent↗
PubMed2000

Amino acid transport in podocytes.

It has recently been shown that formation of podocyte foot processes is dependent on a constant source of lipids and proteins (Simons M, Saffrich R, Reiser J, and Mundel P. J Am Soc Nephrol 10: 1633-1639, 1999). Here we characterize amino acid transport mechanisms in differentiated cultured podocytes and investigate whether it may be disturbed during podocyte injury. RT-PCR studies detected mRNA for transporters of neutral amino acids (ASCT1, ASCT2, and B(0/+)), cationic AA (CAT1 and CAT3), and anionic AA (EAAT2 and EAAT3). Alanine (Ala), asparagine, cysteine (Cys), glutamine (Gln), glycine (Gly), leucine (Leu), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), glutamic acid (Glu), arginine (Arg), and histidine (His) depolarized podocytes and increased their whole cell conductances. Depletion of extracellular Na(+) completely inhibited the depolarization induced by Ala, Gln, Glu, Gly, Leu, and Pro and decreased the depolarization induced by Arg and His, indicating the presence of Na(+)-dependent amino acid transport. Incubation of podocytes with 100 microg/ml puromycin aminonucleoside for 24 h significantly attenuated the effects induced by the various amino acids by approximately 70%. The data indicate the existence of different amino acid transporter systems in podocytes. Alteration of amino acid transport may participate in podocyte injury and disturbed foot process formation.

Amino Acids↗
PubMed2000

A Bartter's syndrome mutation of ROMK1 exerts dominant negative effects on K(+) conductance.

Mutations in the gene encoding the renal epithelial K(+) channel ROMK1 (Kir 1.1) is one of the causes for Bartter's syndrome, an autosomal recessive disease. It results in defective renal tubular transport in the thick ascending limb of the loop of Henle that leads to hypokalemic metabolic alkalosis and loss of salt. Two novel ROMK1 mutations, L220F/A156V, have been described recently in a compound heterozygote patient demonstrating typical manifestations of Bartter's syndrome. Functional properties of these ROMK1 mutants were studied by coexpressing in Xenopus oocytes and by means of double electrode voltage clamp experiments. When both ROMK1 mutants were coexpressed no K(+) conductance could be detected. The same was found in oocytes expressing A156V-ROMK1 only or coexpressing wild type (wt) ROMK1 together with A156V-ROMK1. In contrast, K(+) conductances were indistinguishable from that of wt-ROMK1 when L220F-ROMK1 was expressed alone. Activation of protein kinase C signaling inhibited the conductance in both L220F-ROMK1 and wt-ROMK1 expressing oocytes. These effects were not seen in A156V-ROMK1 expressing oocytes. Because no further abnormalities in the properties or regulation of L220F-ROMK1 were detected, we conclude that A156V-ROMK1 has a dominant negative effect on L220F-ROMK1 thereby causing Bartter's syndrome type two in this patient.

Bartter Syndrome↗
PubMed2000

Role of K(V)LQT1 in cyclic adenosine monophosphate-mediated Cl(-) secretion in human airway epithelia.

Ion transport defects underlying cystic fibrosis (CF) lung disease are characterized by impaired cyclic adenosine monophosphate (cAMP)-dependent Cl(-) conductance. Activation of Cl(-) secretion in airways depends on simultaneous activation of luminal Cl(-) channels and basolateral K(+) channels. We determined the role of basolateral K(+) conductance in cAMP- dependent Cl(-) secretion in native human airway epithelium obtained from non-CF and CF patients. CF tissues showed typical alterations of short-circuit currents with enhanced amiloride-sensitive Na(+) conductance and defective cAMP-mediated Cl(-) conductance. In non-CF tissues, Cl(-) secretion was significantly inhibited by the chromanol 293B (10 micromol/liter), a specific inhibitor of K(V)LQT1 K(+) channels. Inhibition was increased after cAMP-dependent stimulation. Similar effects were obtained with Ba(2+) (5 mmol/liter). In patch-clamp experiments with a human bronchial epithelial cell line, stimulation with forskolin (10 micromol/liter) simultaneously activated Cl(-) and K(+) conductance. The K(+) conductance was reversibly inhibited by Ba(2+) and 293B. Analysis of reverse-transcribed messenger RNA from non-CF and CF airways showed expression of human K(V)LQT1. We conclude that the K(+) channel K(V)LQT1 is important in maintaining cAMP-dependent Cl(-) secretion in human airways. Activation of K(V)LQT1 in CF airways in parallel with stimulation of residual CF transmembrane conductance regulator Cl(-) channel activity or alternative Cl(-) channels could help to circumvent the secretory defect.

1-Methyl-3-isobutylxanthine↗
PubMed2000

Inhibition of amiloride-sensitive epithelial Na(+) absorption by extracellular nucleotides in human normal and cystic fibrosis airways.

Cystic fibrosis (CF) airway epithelia are characterized by enhanced Na(+) absorption probably due to a lack of downregulation of epithelial Na(+) channels by mutant CF transmembrane conductance regulator. Extracellular nucleotides adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) have been shown to activate alternative Ca(2+)-dependent Cl(-) channels in normal and CF respiratory epithelia. Recent studies suggest additional modulation of Na(+) absorption by extracellular nucleotides. In this study we examined the role of mucosal ATP and UTP in regulating Na(+) transport in native human upper airway tissues from patients with 16 patients with CF and 32 non-CF control subjects. To that end, transepithelial voltage and equivalent short-circuit current (I(SC)) were assessed by means of a perfused micro-Ussing chamber. Mucosal ATP and UTP caused an initial increase in lumen-negative I(SC) that was followed by a sustained decrease of I(sc) in both non-CF and CF tissues. The amiloride-sensitive portion of I(SC) was inhibited significantly in normal and CF tissues in the presence of either ATP or UTP. Both basal Na(+) transport and nucleotide-dependent inhibition of amiloride-sensitive I(SC) were significantly enhanced in CF airways compared with non-CF. Nucleotide-mediated inhibition of Na(+) absorption was attenuated by pretreatment with the Ca(2+)-adenosine triphosphatase inhibitor cyclopiazonic acid but not by inhibition of protein kinase C with bisindolylmaleimide. These data demonstrate sustained inhibition of Na(+) transport in non-CF and CF airways by mucosal ATP and UTP and suggest that this effect is mediated by an increase of intracellular Ca(2+). Because ATP and UTP inhibit Na(+) absorption and stimulate Cl(-) secretion simultaneously, extracellular nucleotides could have a dual therapeutic effect, counteracting the ion transport defect in CF lung disease.

Absorption↗
PubMed2000

Transfer and expression of foreign genes in mammalian cells.

The transfer of foreign genes into eukaryotic cells, in particular mammalian cells, has been essential to our understanding of the functional significance of genes and regulatory sequences as well as the development of gene therapy strategies. To this end, different mammalian expression vector systems have been designed. The choice of a particular expression system depends on the nature and purpose of the study and will involve selecting particular parameters of expression systems such as the type of promoter/enhancer sequences, the type of expression (transient versus stable) and the level of desired expression. In addition, the success of the study depends on efficient gene transfer. The purification of the expression vectors, as well as the transfer method, affects transfection efficiency. Numerous approaches have been developed to facilitate the transfer of genes into cells via physical, chemical or viral strategies. While these systems have all been effective in vitro they need to be optimized for individual cell types and, in particular, for in vivo transfection.

Animals↗
PubMed1999

Feedback inhibition of epithelial Na(+) channels in Xenopus oocytes does not require G(0) or G(i2) proteins.

Regulation of amiloride-sensitive epithelial Na(+) channels (ENaC) is a prerequisite for coordination of electrolyte transport in epithelia. Downregulation of Na(+) conductance occurs when the intracellular Na(+) concentration is increased during reabsorption of electrolytes, known as feedback inhibition. Recent studies have demonstrated the involvement of alphaG(0) and alphaG(i2) proteins in the feedback control of ENaC in mouse salivary duct cells. In this report, we demonstrate that Na(+) feedback inhibition is also present in Xenopus oocytes after expression of rat alpha,beta, gamma-ENaC. Interfering with intracellular alphaG(0) or alphaG(i2) signaling by coexpression of either constitutively active alphaG(0)/alphaG(i2) or dominant negative alphaG(0)/alphaG(i2) and by coinjecting sense or antisense oligonucleotides for alphaG(0) had no impact on Na(+) feedback. Moreover, no evidence for involvement of the intracellular G protein cascade was found in experiments in which a regulator of G protein signaling (RGS3) or beta-adrenergic receptor kinase (betaARK) was coexpressed together with alpha,beta, gamma-ENaC. Although some experiments suggest the presence of an intracellular Na(+) receptor, we may conclude that Na(+) feedback in Xenopus oocytes is different from that described for salivary duct cells in that it does not require G protein signaling.

Animals↗
PubMed1999

Cystic fibrosis transmembrane conductance regulator inhibits epithelial Na+ channels carrying Liddle's syndrome mutations.

Epithelial Na+ channels (ENaC) are inhibited by the cystic fibrosis transmembrane conductance regulator (CFTR) upon activation by protein kinase A. It is, however, still unclear how CFTR regulates the activity of ENaC. In the present study we examined whether CFTR interacts with ENaC by interfering with the Nedd4- and ubiquitin-mediated endocytosis of ENaC. Various C-terminal mutations were introduced into the three alpha-, beta-, and gamma-subunits of the rat epithelial Na+ channel, thereby eliminating PY motifs, which are important binding domains for the ubiquitin ligase Nedd4. When expressed in Xenopus oocytes, most of the ENaC stop (alpha-H647X, beta-P565X, gamma-S608X) or point (alpha-P671A, beta-Y618A, gamma-P(624-626)A) mutations induced enhanced Na+ currents when compared with wild type alpha,beta,gamma-rENaC. However, ENaC currents formed by either of the mutant alpha-, beta-, or gamma-subunits were inhibited during activation of CFTR by forskolin (10 micromol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l). Antibodies to dynamin or ubiquitin enhanced alpha,beta,gamma-rENaC whole cell Na+ conductance but did not interfere with inhibition of ENaC by CFTR. Another mutant, beta-T592M,T593A-ENaC, also showed enhanced Na+ currents, which were down-regulated by CFTR. Moreover, activation of ENaC by extracellular proteases and xCAP1 does not disturb CFTR-dependent inhibition of ENaC. We conclude that regulation of ENaC by CFTR is distal to other regulatory limbs and does not involve Nedd4-dependent ubiquitination.

1-Methyl-3-isobutylxanthine↗
PubMed1999

The first-nucleotide binding domain of the cystic-fibrosis transmembrane conductance regulator is important for inhibition of the epithelial Na+ channel.

The cystic-fibrosis transmembrane conductance regulator (CFTR) functions as a cAMP-regulated Cl- channel and as a regulator of other membrane conductances. cAMP-dependent activation of CFTR inhibits epithelial Na+ channels (ENaC). The specificity of interaction between CFTR and ENaC was examined by coexpression of ENaC and ATP-binding cassette (ABC) proteins other than CFTR. In addition, we identified domains within CFTR that are of particular importance for the inhibition of ENaC. To that end, two-electrode voltage-clamp experiments were performed on Xenopus oocytes coexpressing ENaC together with CFTR, the multidrug resistance protein MDR1, the sulfonyl urea receptor SUR1, or the cadmium permease YCF1. Except for CFTR, none of the other ABC proteins were able to inhibit ENaC. Several truncated versions of CFTR were examined for their inhibitory effects on ENaC. In fact, it is shown that C-terminal truncated CFTR is able to inhibit ENaC on activation by intracellular cAMP. Moreover, the data also show that an intact first-nucleotide binding domain (NBF-1) is important for inhibition of ENaC. We conclude that NBF-1 of CFTR contains a CFTR-specific regulatory site that down-regulates ENaC. It is speculated that this regulatory site also is needed for CFTR-mediated interactions with other membrane proteins and that it is not present in NBF-1 of other ABC proteins.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

The cystic fibrosis transmembrane conductance regulator activates aquaporin 3 in airway epithelial cells.

Enhanced osmotic water permeability has been observed in Xenopus oocytes expressing cystic fibrosis transmembrane conductance regulator (CFTR) protein. Subsequent studies have shown that CFTR activates an endogenous water permeability in oocytes, but that CFTR itself is not the water channel. Here, we show CFTR-dependent activation of endogenous water permeability in normal but not in cystic fibrosis human airway epithelial cells. Cell volume was measured by novel confocal x-z laser scanning microscopy. Glycerol uptake and antisense studies suggest CFTR-dependent regulation of aquaporin 3 (AQP3) water channels in airway epithelial cells. Regulatory interaction was confirmed by coexpression of CFTR and AQP3 cloned from human airways in Xenopus oocytes and of CFTR and rat AQP3 in Chinese hamster ovary cells. These findings indicate that CFTR is a regulator of AQP3 in airway epithelial cells.

Animals↗
PubMed1999

Lack of correlation between CFTR expression, CFTR Cl- currents, amiloride-sensitive Na+ conductance, and cystic fibrosis phenotype.

Cystic fibrosis (CF) is characterized by defective Cl- and enhanced Na+ conductance, both due to malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in airway epithelial cells. In the present study we examined whether expression of CFTR mRNA (CFTR messenger ribonucleic acid) is different in airway epithelia derived from either CF patients or healthy volunteers. Moreover, we tried to correlate differences in epithelial Cl- and Na+ conductance with the level of CFTR mRNA expression and studied whether these properties correlate to the clinical phenotype of CF patients. To that end, CFTR mRNA was determined by means of quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and cyclic adenosine monophosphate (cAMP)-activated Cl- and epithelial Na+ conductances were examined in airway epithelial cells using microelectrode techniques. Complementary in vitro data were obtained from cultured CF and non-CF airway epithelial cell lines. Genotype and Shwachman score were assessed for each patient. We found variable levels of CFTR mRNA expression in airway cells of both CF patients and healthy volunteers. As expected, epithelial Na+ conductance was enhanced and CFTR Cl- conductance was absent in airway cells from CF patients. However, CFTR mRNA expression did not correlate with either electrophysiological properties or Shwachman scores obtained from CF patients. In addition, CFTR mRNA expression did not correlate to Cl- conductance in cultured CF and non-CF airway epithelial cells. These results indicate a lack of correlation between levels of CFTR mRNA and CFTR function, and that only small amounts of CFTR are required for expression of the CFTR Cl- conductance.

Amiloride↗