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J Eggermont

Publications and source records attributed to J Eggermont.

At least 37 records · Page 2Linked to original sources
PubMed1998

The exon-intron architecture of human chloride channel genes is not conserved.

The human CLCN6 gene contains a 167 bp exon that is optionally included or excluded in ClC-6 mRNAs. The corresponding region (3.4 kbp) of the human CLCN7 gene has now been cloned and sequenced. A comparison of the human CLCN1, CLCN5, CLCN6 and CLCN7 genes indicates that there is no homologue of the optional CLCN6 exon in the CLCN1, CLCN5 or CLCN7 genes. Thus, the CLCN6 type of alternative splicing and the ensuing structural diversity is not conserved within the CLC gene family.

Alternative Splicing↗
PubMed1998

The GXGXG motif in the pI(Cln) protein is not important for the nucleotide sensitivity of the pI(Cln)-induced Cl- current in Xenopus oocytes.

It has been proposed that the pI(Cln) protein forms a nucleotide-sensitive plasma membrane anion channel with a GXGXG motif being an essential component of the extracellular nucleotide-binding site. To evaluate this hypothesis, we have performed voltage-clamp experiments on Xenopus laevis oocytes injected with RNA encoding a rat mutant pI(Cln) in which the three glycines of the putative nucleotide-binding site have been changed into alanines (G54A; G56A; G58A). The injected oocytes displayed outwardly rectifying anion currents, which were voltage-dependently blocked by extracellular cAMP, but which were not affected by removal of extracellular Ca2+. Furthermore, the mutation did not affect the voltage-dependent inactivation. We therefore conclude that there is no evidence in favour of an extracellular nucleotide-binding site in pI(Cln).

Animals↗
PubMed1998

Evidence for the intracellular location of chloride channel (ClC)-type proteins: co-localization of ClC-6a and ClC-6c with the sarco/endoplasmic-reticulum Ca2+ pump SERCA2b.

Chloride channel protein (ClC)-6a and ClC-6c, a kidney-specific splice variant with a truncated C-terminus, are proteins that belong structurally to the family of voltage-dependent chloride channels. Attempts to characterize functionally ClC-6a or ClC-6c in Xenopus oocytes have so far been negative. Similarly, expression of both ClC-6 isoforms in mammalian cells failed to provide functional information. One possible explanation of these negative results is that ClC-6 is an intracellular chloride channel rather than being located in the plasma membrane. We therefore studied the subcellular location of ClC-6 isoforms by transiently transfecting COS and CHO cells with epitope-tagged versions of ClC-6a and ClC-6c. Confocal imaging of transfected cells revealed for both ClC-6 isoforms an intracellular distribution pattern that clearly differed from the peripheral location of CD2, a plasma-membrane glycoprotein. Furthermore, dual-labelling experiments of COS cells co-transfected with ClC-6a or -6c and the sarco/endoplasmic-reticulum Ca2+ pump (SERCA2b) indicated that the ClC-6 isoforms co-localized with the SERCA2b Ca2+ pump. Thus ClC-6a and ClC-6c are intracellular membrane proteins, most likely residing in the endoplasmic reticulum. In view of their structural similarity to proven chloride channels, ClC-6 isoforms are molecular candidates for intracellular chloride channels.

Animals↗
PubMed1998

Regulation of a swelling-activated chloride current in bovine endothelium by protein tyrosine phosphorylation and G proteins.

1. The role of protein tyrosine phosphorylation and of G proteins in the activation of a swelling-activated Cl- current (ICl,swell) in calf pulmonary artery endothelial (CPAE) cells was studied using the whole-cell patch clamp technique. ICl,swell was activated by reducing the extracellular osmolality by either 12.5% (mild hypotonicity) or 25% (strong hypotonicity). 2. The protein tyrosine kinase (PTK) inhibitors tyrphostin B46, tyrphostin A25 and genistein inhibited ICl,swell with IC50 values of, respectively, 9.2 +/- 0.2, 61.4 +/- 1.7 and 62.9 +/- 1.3 microM. Tyrphostin A1, a tyrphostin analogue with little effect on PTK activity, and daidzein, an inactive genistein analogue, were without effect on ICl,swell. 3. The protein tyrosine phosphatase (PTP) inhibitors Na3VO4 (200 microM) and dephostatin (20 microM) potentiated ICl,swell activated by mild hypotonicity by 47 +/- 9 and 69 +/- 15%, respectively. 4. Intracellular perfusion with GTP gamma S (100 microM) transiently activated a Cl- current with an identical biophysical and pharmacological profile to ICl,swell. This current was inhibited by the tested PTK inhibitors and potentiated by the PTP inhibitors. Hypertonicity-induced cell shrinkage completely inhibited the GTP gamma S-activated Cl- current. 5. Intracellular perfusion with GDP beta S (1 mM) caused a time-dependent inhibition of ICl,swell, which was more pronounced when the current was activated by mild hypotonicity. 6. Our results demonstrate that the activity of endothelial swelling-activated Cl- channels is dependent on tyrosine phosphorylation and suggest that G proteins regulate the sensitivity to cell swelling.

Action Potentials↗
PubMed1998

Activation of volume-regulated chloride currents by reduction of intracellular ionic strength in bovine endothelial cells.

1. We have studied the effects of intracellular ionic strength (gamma 1) on the swelling-activated whole-cell Cl- current (ICl,swell) in cultured calf pulmonary artery endothelial cells (CPAE cells). 2. Reducing gamma 1 from 155 to 95 mM at constant osmolarity and Cl- concentration activates an outwardly rectifying current that is mainly carried by Cl- ions and inactivates at positive potentials. The amplitude of the current is larger at more reduced levels of gamma 1. 3. The permeability ratio for the anions I-, Br-, Cl- and gluconate (PI: PBr: PCl: Pgluc) was 1.35:1.03:1:0.17. 4. Blockers of the swelling-activated Cl- current in CPAE cells also inhibit the current which is activated by a reduction in gamma 1 with an IC50 of 1.1 microM for tamoxifen, 1.3 microM for mibefradil, and 35 microM for quinidine. 5. The protein tyrosine kinase inhibitors tyrphostin B46 (50 microM) and genistein (100 microM), which inhibit ICl,swell in CPAE cells, also inhibited the gamma 1-induced current by 92.9 +/- 2.4% (n = 3) and 41.2 +/- 5.0% (n = 4), respectively. 6. Hypertonic extracellular solutions rapidly and reversibly antagonized the gamma 1-activated current, whereas increasing gamma 1 from 155 to 195 mM precluded activation of ICl,swell by hypotonic shock. 7. It is concluded that a reduction of gamma 1 activates an anion current that is identical to that activated by cell swelling. Changes in intracellular ionic strength may shift the volume set point for activation of ICl,swell.

Action Potentials↗
PubMed1998

Inhibition by inositoltetrakisphosphates of calcium- and volume-activated Cl- currents in macrovascular endothelial cells.

We have used the whole-cell patch-clamp technique to study the effects of inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P4], inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4] and inositol 1,3, 4,5,6-pentacisphosphate [Ins(1,3,4,5,6)P5] on volume-activated Cl- currents (ICl,vol) in cultured endothelial cells from bovine pulmonary artery (CPAE cells). Ins(1,4,5,6)P4 and Ins(3,4,5,6)P4 were applied intracellularly via the patch pipette at concentrations between 10 and 100 muM. Both tetrakisphosphates inhibited the Cl- current ICl,Ca, which was activated by intracellular loading of the cells with 500 nM Ca2+ [for inhibition by Ins(1,4,5,6)P4: 58% at 10 muM, 75% at 100 muM; for Ins(3,4,5,6)P4: 44% at 10 muM, 65% at 100 muM]. Inhibition of ICl,Ca occurred without significant changes in its kinetic properties. The amplitude of ICl,vol activated by a 13.5 or 27% hypotonic solution at +100 mV was strongly reduced in cells loaded with either tetrakisphosphate, i.e. a 73% reduction for Ins(3,4,5,6)P4 and 89% for Ins(1,4,5,6)P4 at 100 muM. Both tetrakisphosphates also inhibited a current probably identical to ICl,vol which was activated by dialysing the cell with 100 muM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]). Ins(1, 3,4,5,6)P5 at a concentration of 30 muM did not significantly reduce ICl, vol. The effects of Ins(3,4,5,6)P4 may represent an inhibitory pathway for the ICl,Ca and ICl,vol in macrovascular endothelium after sustained receptor-mediated activation of phospholipase C.

Animals↗
PubMed1998

Voltage-dependent block of endothelial volume-regulated anion channels by calix[4]arenes.

We have studied the effects of calix[4]arenes on the volume-regulated anion channel (VRAC) currents in cultured calf pulmonary artery endothelial cells. TS- and TS-TM-calix[4]arenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. Maximal block occurred at potentials between 30 and 50 mV. Lowering extracellular pH enhanced the block and shifted the maximum inhibition to more negative potentials. Current inhibition was also accompanied by an increased current noise. From the analysis of the calix[4]arene-induced noise, we obtained a single-channel conductance of 9.3 +/- 2.1 pS (n = 9) at +30 mV. The voltage- and time-dependent block were described using a model in which calix[4]arenes bind to a site at an electrical distance of 0.25 inside the channel with an affinity of 220 microM at 0 mV. Binding occludes VRAC at moderately positive potentials, but calix[4]arenes permeate the channel at more positive potentials. In conclusion, our data suggest an open-channel block of VRAC by calix[4]arenes that also depends on the protonation of the binding site within the pore.

Animals↗
PubMed1997

Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells.

1. We have used the patch-clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole-cell mode, IK(IR) was defined as the Ba(2+)-sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K(+)-selective channel with a conductance of 31 +/- 3 pS. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and functional data suggest that the endothelial IRK is most probably Kir2.1. 3. Intracellular ATP is required to prevent run-down of IRK in whole-cell mode. Single channel activity disappeared in inside-out patches exposed to ATP-free solution and in cell-attached patches on cells exposed to metabolic inhibition (KCN, 2-deoxyglucose). 4. The non-hydrolysable ATP analogues, ATP gamma S and adenylyl imidodiphosphate (AMP-PNP), did not prevent run-down. Run-down did not occur in the presence of okadaic acid, a phosphatase inhibitor, but was enhanced in the presence of protamine, an activator of phosphatase 2A (PP2A). 5. GTP gamma S and AlF4- inhibited IRK, also in the presence of ATP. GTP beta S antagonized the GTP gamma S effect. Pretreatment of the cells with PTX did not affect the GTP gamma S-induced inhibition. Okadaic acid, however, slowed this inhibition. 6. Neither activation of protein kinase A (PKA) nor activation of protein kinase C (PKC) affected IRK. Additionally, neither cytochalasin B nor a high concentration of intracellular Ca2+ affected the time course of IRK run-down. 7. We conclude that run-down of IRK is probably due to dephosphorylation by PP2A. Activation of a PTX-insensitive G protein inhibits this current by a mechanism that is neither mediated via the PKA and PKC pathways nor by intracellular Ca2+, but supposedly by a G protein-dependent activation of a phosphatase.

Adenosine Triphosphate↗
PubMed1997

Alternative splicing of ClC-6 (a member of the CIC chloride-channel family) transcripts generates three truncated isoforms one of which, ClC-6c, is kidney-specific.

ClC-6 is a protein that structurally belongs to the family of ClC-type chloride channels. We now report the identification of three additional ClC-6 isoforms that are truncated because of alternative splicing. We have isolated, from human K562 cells, four types of ClC-6 cDNAs that encode four distinct ClC-6 protein isoforms. ClC-6a (869 amino acids) corresponds to the previously published ClC-6 protein [Brandt and Jentsch (1995) FEBS Lett. 377, 15-20] and it has a canonical ClC structure. However, ClC-6b (320 amino acids), ClC-6c (353 amino acids) and ClC-6d (308 amino acids) are truncated at their C-termini. Hydropathy-plot analysis indicates that the shortened isoforms contain maximally four (ClC-6b and -6d) or seven (ClC-6c) transmembrane domains. Sequence analysis of a human genomic ClC-6 fragment indicates that the cDNA variability arises from alternative splicing at two different positions: the first alternative site consists of an intron flanked by two alternative donor sites and two alternative acceptor sites, the second being due to an exon that is optionally included or excluded. Reverse-transcription-PCR analysis of ClC-6 expression in human cell lines and tissues shows that the majority (83%) of ClC-6 mRNAs consists of ClC-6a or ClC-6c messengers. Furthermore, in a mouse tissue panel, the ClC-6a mRNA has a relatively broad tissue expression pattern, since it could be detected in brain, kidney, testis, skeletal muscle, thymus and pancreas. In contrast, expression of ClC-6c is more restricted, since it was only detected in kidney.

Alternative Splicing↗
PubMed1997

Sequence elements surrounding the acceptor site suppress alternative splicing of the sarco/endoplasmic reticulum Ca2+-ATPase 2 gene transcript.

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) requires activation of an inefficient optional splice process at the 3' end of the primary gene transcript. The sequence elements required for this regulated splice event were studied by modifying a minigene containing the 3' end of the SERCA2 gene. An important requirement appears to be a strong muscle-specific acceptor site, as replacing it by a weak one prevented the induction of muscle-type splicing during myogenic differentiation. The induction of muscle-type splicing did not depend on positive cis-active sequences in the muscle-specific exon. On the other hand, replacement of a broad region around the acceptor site dramatically deregulated the expression pattern, as this modification strongly induced muscle-type splicing in undifferentiated muscle cells and in fibroblasts. This cis-active region is also involved in the suppression of the neuronal type of splicing. Furthermore selective replacement of the acceptor site as well as deletions or replacements in the muscle-specific exon induced muscle-type splicing to various extents in undifferentiated myogenic cells. Therefore sequence elements in the distal part of the optional intron and in the muscle-specific exon contribute to the suppression of muscle-specific SERCA2 splicing.

Alternative Splicing↗
PubMed1997

Expression of human pICln and ClC-6 in Xenopus oocytes induces an identical endogenous chloride conductance.

pICln is a protein that induces an outwardly rectifying, nucleotide-sensitive chloride current (ICln) when expressed in Xenopus oocytes, but its precise function (plasma-membrane anion channel versus cytosolic regulator of a channel) remains controversial. We now report that a chloride current identical to ICln is induced when Xenopus oocytes are injected with human ClC-6 RNA. Indeed, both the pICln and the ClC-6 induced current are outwardly rectifying, they inactivate slowly at positive potentials and have an anion permeability sequence NO3- > I- > Br- > Cl- > gluconate. Cyclamate, NPPB, and extracellular cAMP block the induced currents. The success rate of current expression is significantly increased when the injected Xenopus oocytes are incubated at a higher temperature (24 or 37 degrees C) prior to the analysis. In addition, the ICln current was detected in 6.2% of noninjected control Xenopus oocytes. We therefore conclude that the ICln current in Xenopus oocytes corresponds to an endogenous conductance that can be activated by expression of structurally unrelated proteins. Furthermore, functional, biochemical, and morphological observations did not support the notion that pICln resides in the plasma membrane either permanently or transiently after cell swelling. Thus, it is unlikely that pICln forms the channel that is responsible for the ICln current in Xenopus oocytes.

Animals↗
PubMed1997

Use of a bicistronic GFP-expression vector to characterise ion channels after transfection in mammalian cells.

Transient transfection of ion channels into mammalian cells is a useful method with which to study ion channel properties. However, a general problem in transient transfection procedures is how to select cells that express the transfected cDNA. We have constructed a bicistronic vector, pCINeo/IRES-GFP, which utilises a red-shifted variant of Green Fluorescent Protein as an in vivo cell marker. Incorporation of an ion channel cDNA into the bicistronic unit allows coupled expression of the ion channel and Green Fluorescent Protein. After transient transfection of COS cells with pCINeo/IRES-GFP containing a rat delayed rectifier K+ channel cDNA (RCK1, Kv1.1), all green cells (n = 32) expressed the RCK1 channel as identified by the well known kinetics, K+ selectivity and pharmacology of Kv1.1. In contrast, non-fluorescent cells (n = 24) were negative with respect to RCK1 expression. It is concluded that the bicistronic pCINeo/IRES-GFP vector provides an efficient and non-invasive way of identifying cells which express ion channels after transfection. This novel method should greatly facilitate functional studies of ion channels transfected into mammalian cells.

Animals↗
PubMed1997

Functional effects of expression of hslo Ca2+ activated K+ channels in cultured macrovascular endothelial cells.

The aim of the present study is to elucidate the effects of the expression of large conductance Ca2+ activated K+ channels (BK[Ca]) in an endothelial cell type normally lacking this channel. The human homologue hslo of BK(Ca) was expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which have no endogenous BK(Ca). Membrane potential, ionic currents and Ca2+ signals were investigated in non-transfected and transfected cells using a combined patch clamp and Fura-2 fluorescence technique. In non-transfected control CPAE cells, ATP evoked a Ca2+ activated Cl- current (I[Cl,Ca]). The most prominent current component during ATP stimulation in hslo expressing cells was conducted BK(Ca) which resulted in a pronounced transient hyperpolarization. This hyperpolarization, which was absent in non-transfected cells, was enhanced if I(Cl,Ca) was blocked with niflumic acid. The sustained component of the Ca2+ response during ATP stimulation was significantly larger in hslo transfected cells than in non-transfected cells. This plateau level correlated well with the corresponding effects of ATP on the membrane potential, indicating that the expression of cloned BK(Ca) exerts a positive feedback on Ca2+ signals in endothelial cells by counteracting the negative (depolarizing)effect of stimulation of Ca2+-activated Cl- channels.

Adenosine Triphosphate↗
PubMed1997

Kinetic and pharmacological properties of the calcium-activated chloride-current in macrovascular endothelial cells.

We have studied the kinetic and pharmacological properties of the Ca(2+)-activated Cl- current (ICl,Ca) in cultured cell pulmonary artery endothelial (CPAE) cells by means of combined patch clamp and Fura-2 micro fluorescence measurements. The current was activated by loading the cells via the patch pipettes with Ca(2+)-buffered solutions. Currents activated slowly at positive potentials, and decayed rapidly at negative potentials. The time constant of activation decreased at more positive membrane potentials and more elevated intracellular Ca2+ concentrations ([Ca2+]i). The time constant of deactivation was Ca(2+)-independent and decreased at more negative potentials. Steady-state currents showed strong outward rectification, but the instantaneous current-voltage relationship was almost ohmic. The calmodulin antagonists trifluoperazine (TFP) and calmidazolium inhibit ICl,Ca. Half maximal block for TFP occurred at 5.7 +/- 2.1 microM (n = 16). GTP gamma S did not activate ICl,Ca, but activated a Cl- current similar to the volume-activated Cl- current (ICl,vol). [Ca2+]i for half maximal activation of ICl,Ca was voltage-dependent, and suggests that the apparent binding constant for Ca2+ decreases with depolarization. Its value at 0 mV is 430 nM, and the binding site is 12% within the electrical field from the cytoplasmic side. The Hill-coefficient, nH, of the binding was larger than 1 and increased with depolarization. The maximal Cl- conductance at saturating [Ca2+]i did not depend on the membrane potential. RT-PCR experiments did not provide any evidence that the endothelial Ca(2+)-activated Cl- channel might be identical with a recently cloned Ca(2+)-sensitive Cl- channel (CaCC).

Animals↗
PubMed1997

Downregulation of volume-activated Cl- currents during muscle differentiation.

We have used the whole cell configuration of the patch-clamp technique to investigate volume-activated Cl- currents in BC3H1 and C2C12 cells, two mouse muscle cell lines that can be switched from a proliferating to a differentiated musclelike state. Reducing the extracellular osmolality by 40% evoked large Cl- currents in proliferating BC3H1 and C2C12 cells. These currents were outwardly rectifying and had an anion permeability sequence as follows: I- > Br- > Cl- >> gluconate. They were inhibited by >50% by flufenamic acid (500 microM), niflumic acid (500 microM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 microM) but were relatively insensitive to tamoxifen (100 microM). A reduction in the serum concentration in the culture medium induced growth arrest in both cell lines, and the cells started to differentiate into spindle-shaped nonfusing muscle cells (BC3H1) or myotubes (C2C12). This differentiation was accompanied by a drastic decrease in the magnitude of the volume-activated Cl- currents. The close correlation between volume-activated Cl- currents and cell proliferation suggests that these currents may be involved in cell proliferation.

Animals↗
PubMed1996

The chloride current induced by expression of the protein pICln in Xenopus oocytes differs from the endogenous volume-sensitive chloride current.

1. Phenotypical similarities between ICl,swell, the cell-swelling-induced chloride current and ICln, the nucleotide-sensitive chloride current induced by expression of mammalian pICln in Xenopus oocytes, have led to models which identify pICln either as the volume-sensitive chloride channel or as a cytosolic regulator thereof. 2. To investigate critically the relationship between ICl,swell and pICln two-microelectrode voltage clamp experiments were performed on Xenopus oocytes in which either human pICln was expressed or endogenous ICl,swell was activated. 3. Several criteria that clearly differentiated ICln from ICl,swell were detected. Outward rectification and the discrimination between NO3- and Cl- were more pronounced for ICln. Cyclamate blocked ICln but not ICl,swell. In contrast to ICl,swell, inactivation kinetics of ICln were pH independent and extracellular cAMP blocked only the outward ICln component. Finally, ICln was readily expressed in collagenase-defolliculated oocytes and was not modulated by extracellular hypotonicity, whereas ICl,swell could only be triggered in follicle-enclosed or manually defolliculated oocytes. 4. We therefore conclude that ICln and ICl,swell are two different chloride currents. Consequently, any model which invokes a crucial role for pICln in ICl,swell should be critically reviewed.

Animals↗