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T Voets

Publications and source records attributed to T Voets.

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

Dissection of three Ca2+-dependent steps leading to secretion in chromaffin cells from mouse adrenal slices.

In neurosecretory cells, intracellular Ca2+ ([Ca2+]i) not only acts as the trigger for secretion but also regulates earlier steps in the secretory pathway. Here, a novel approach was developed to control [Ca2+]i over a broad concentration range, which allowed the quantification of three distinct actions of [Ca2+]i on large dense-core vesicle (LDCV) fusion in chromaffin cells from mouse adrenal slices. Basal [Ca2+]i regulated the transfer of vesicles toward a slowly releasable state, whereas further maturation to the readily releasable state was Ca2+ independent. [Ca2+]i levels above 3 microM triggered exocytosis of all readily and slowly releasable vesicles in two parallel, kinetically distinct fusion reactions. In a molecular context, these results suggest that Ca2+ acts both before and after trans-SNARE complex formation to regulate fusion competence and fusion kinetics of LDCVs.

Adrenal Glands↗
PubMed1999

Reduced intracellular ionic strength as the initial trigger for activation of endothelial volume-regulated anion channels.

Most mammalian cell types, including endothelial cells, respond to cell swelling by activating a Cl- current termed ICl,swell, but it is not known how the physical stimulus of cell swelling is transferred to the channels underlying ICl,swell. We have investigated the precise relation between cell volume and ICl,swell in endothelial cells by performing whole-cell current recordings while continuously monitoring cell thickness (Tc) as a measure for cell volume. The time course of Tc was accurately predicted by a theoretical model that describes volume changes of patch-clamped cells in response to changes in the extracellular osmolality (OSMo). This model also predicts significant changes in intracellular ionic strength (Gammai) when OSMo is altered. Under all experimental conditions ICl,swell closely followed the changes in Gammai, whereas ICl,swell and cell volume were often found to change independently. These results do not support the hypothesis that Gammai regulates the volume set point for activation of ICl,swell. Instead, they are in complete agreement with a model in which a decrease of Gammai rather than an increase in cell volume is the initial trigger for activation of ICl,swell.

Animals↗
PubMed1999

Role of Rho and Rho kinase in the activation of volume-regulated anion channels in bovine endothelial cells.

1. We have studied the modulation of volume-regulated anion channels (VRACs) by the small GTPase Rho and by one of its targets, Rho kinase, in calf pulmonary artery endothelial (CPAE) cells. 2. RT-PCR and immunoblot analysis showed that both RhoA and Rho kinase are expressed in CPAE cells. 3. ICl,swell, the chloride current through VRACs, was activated by challenging CPAE cells with a 25 % hypotonic extracellular solution (HTS) or by intracellular perfusion with a pipette solution containing 100 microM GTPgammaS. 4. Pretreatment of CPAE cells with the Clostridium C2IN-C3 fusion toxin, which inactivates Rho by ADP ribosylation, significantly impaired the activation of ICl,swell in response to the HTS. The current density at +100 mV was 49 +/- 13 pA pF-1 (n = 17) in pretreated cells compared with 172 +/- 17 pA pF-1 (n = 21) in control cells. 5. The volume-independent activation of ICl,swell by intracellular perfusion with GTPgammaS was also impaired in C2IN-C3-pretreated cells (31 +/- 7 pA pF-1, n = 11) compared with non-treated cells (132 +/- 21 pA pF-1, n = 15). 6. Activation of ICl,swell was pertussis toxin (PTX) insensitive. 7. Y-27632, a blocker of Rho kinase, inhibited ICl,swell and delayed its activation. 8. Inhibition of Rho and of Rho kinase by the above-described treatments did not affect the extent of cell swelling in response to HTS. 9. These experiments provide strong evidence that the Rho-Rho kinase pathway is involved in the VRAC activation cascade.

Amides↗
PubMed1999

Inhibition of volume-regulated anion channels by expression of the cystic fibrosis transmembrane conductance regulator.

1. To investigate whether the cystic fibrosis transmembrane conductance regulator (CFTR) interacts with volume regulated anion channels (VRACs), we measured the volume-activated chloride current (ICl,swell) using the whole-cell patch-clamp technique in calf pulmonary artery endothelial (CPAE) cells and in COS cells transiently transfected with wild-type (WT) CFTR and the deletion mutant DeltaF508 CFTR. 2. ICl,swell was significantly reduced in CPAE cells expressing WT CFTR to 66.5 +/- 8.8 % (n = 13; mean +/- s. e.m.) of the control value (n = 11). This reduction was independent of activation of the CFTR channel. 3. Expression of DeltaF508 CFTR resulted in two groups of CPAE cells. In the first group IBMX and forskolin could activate a Cl- current. In these cells ICl,swell was reduced to 52.7 +/- 18.8 % (n = 5) of the control value (n = 21). In the second group IBMX and forskolin could not activate a current. The amplitude of ICl,swell in these cells was not significantly different from the control value (112.4 +/- 13.7 %, n = 11; 21 control cells). 4. Using the same method we showed that expression of WT CFTR in COS cells reduced ICl,swell to 62.1 +/- 11.9 % (n = 14) of the control value (n = 12) without any changes in the kinetics of the current. Non-stationary noise analysis suggested that there is no significant difference in the single channel conductance of VRAC between CFTR expressing and non-expressing COS cells. 5. We conclude that expression of WT CFTR down-regulates ICl, swell in CPAE and COS cells, suggesting an interaction between CFTR and VRAC independent of activation of CFTR.

Animals↗
PubMed1999

Mechanisms underlying phasic and sustained secretion in chromaffin cells from mouse adrenal slices.

Many neurosecretory preparations display two components of depolarization-induced exocytosis: a phasic component synchronized with Ca2+ channel opening, followed by a slower sustained component. We evaluated possible mechanisms underlying this biphasic behavior by stimulating mouse chromaffin cells in situ with both depolarizations and flash photolysis of caged Ca2+. From a direct comparison of the secretory responses to both stimuli, we conclude that phasic and sustained release components originate from a readily releasable pool (RRP) of equally fusion-competent vesicles, suggesting that differences in the vesicles' proximity to Ca2+ channels underlie the biphasic secretory behavior. An intermediate pool in dynamic equilibrium with the RRP ensures rapid recruitment of release-ready vesicles after RRP depletion. Our results are discussed in terms of a refined model for secretion in chromaffin cells.

Adrenal Glands↗
PubMed1999

Block by fluoxetine of volume-regulated anion channels.

1. We have used the whole-cell patch clamp technique to study the effect of fluoxetine, a commonly used antidepressant drug, on the volume-regulated anion channel (VRAC) in calf pulmonary artery endothelial (CPAE) cells. We also examined its effects on other Cl- channels, i.e. the Ca2(+)-activated Cl- current (I(Cl,Ca) and the cystic fibrosis transmembrane conductance regulator (CFTR) to assess the specificity of this compound for VRAC. 2. At pH 7.4 fluoxetine induced a fast and reversible block of the volume-sensitive chloride current (I(Cl,swell)), with a Ki value of 6.0+/-0.5 microM (n = 6-9). The blocking efficiency increased with increasing extracellular pH (Ki= 0.32+/-0.01 microM at pH 8.8, n = 3-9), indicating that the blockade is mediated by the uncharged form of fluoxetine. 3. Fluoxetine inhibited Ca2(+)-activated Cl(-) currents, I(Cl,Ca), activated by loading CPAE cells via the patch pipette with 1000 nM free Ca2+ (Ki= 10.7+/-1.6 microm at pH 7.4, n=3-5). The CFTR channel, transiently transfected in CPAE cells, was also inhibited with a Ki value of 26.9+/-9.4 microM at pH 7.4 (n = 3). 4. This study describes for the first time the effects of fluoxetine on anion channels. Our data reveal a potent block of VRAC at fluoxetine concentrations close to plasma concentrations. The results suggest a hydrophobic interaction with high affinity between uncharged fluoxetine and volume-activated chloride channels. Ca(2+)-activated Cl- currents and CFTR are also blocked by fluoxetine, revealing a novel characteristic of the drug as a chloride channel modulator.

Animals↗
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

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

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

Calcium-activated chloride channels in bovine pulmonary artery endothelial cells.

1. We characterized Ca(2+)-activated Cl- currents in calf pulmonary artery endothelial (CPAE) cells by using a combined patch clamp and fura-2 microfluorescence technique to simultaneously measure ionic currents and the intracellular Ca2+ concentration, [Ca2+]i. 2. Various procedures that increased [Ca2+]i, such as stimulation with ATP or ionomycin, or loading the cells with Ca2+ via the patch pipette, activated a strongly outwardly rectifying current with a reversal potential close to the Cl- equilibrium potential. Changing the extracellular Cl- concentration shifted this reversal potential as predicted for a Cl- current. Buffering Ca2+ rises with BAPTA prevented ATP from activating the current. 3. Ca(2+)-activated Cl- currents could be distinguished from volume-activated Cl- currents, which were sometimes coactivated in the same cell. The latter showed much less outward rectification, their activation was voltage independent, and they could be inhibited by exposing the cells to hypertonic solutions. 4. The permeability ratio for the Ca(2+)-activated conductance of the anions iodide:chloride: gluconate was 1.71 +/- 0.06:1:0.39 +/- 0.03 (n = 12). 5. This Ca(2+)-activated Cl- current, ICl, Ca, inactivated rapidly at negative potentials and activated slowly at positive potentials. Outward tail currents were slowly decaying, while inward tail currents decayed much faster. 6. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic-acid (DIDS) and niflumic acid inhibited Icl,Ca in a voltage-dependent manner, i.e. they exerted a more potent block at positive potentials. The block by N-phenylanthracilic acid (NPA), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and tamoxifen was voltage independent. Niflumic acid and tamoxifen were the most potent blockers. 7. The single-channel conductance was 7.9 +/- 0.7 pS (n = 15) at 300 mM extracellular Cl-. The channel open probability was high at positive potentials, but very small at negative potentials. 8. It is concluded that [Ca2+]i activates small-conductance Cl- channels in endothelial cells, which coexist with the volume-activated Cl- channels described previously.

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