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Jan Eggermont

Publications and source records attributed to Jan Eggermont.

10 recordsLinked to original sources
PubMed2005

Stimulation by caveolin-1 of the hypotonicity-induced release of taurine and ATP at basolateral, but not apical, membrane of Caco-2 cells.

Regulatory volume decrease (RVD) is a protective mechanism that allows mammalian cells to restore their volume when exposed to a hypotonic environment. A key component of RVD is the release of K(+), Cl(-), and organic osmolytes, such as taurine, which then drives osmotic water efflux. Previous experiments have indicated that caveolin-1, a coat protein of caveolae microdomains in the plasma membrane, promotes the swelling-induced Cl(-) current (I(Cl,swell)) through volume-regulated anion channels. However, it is not known whether the stimulation by caveolin-1 is restricted to the release of Cl(-) or whether it also affects the swelling-induced release of other components, such as organic osmolytes. To address this problem, we have studied I(Cl,swell) and the hypotonicity-induced release of taurine and ATP in wild-type Caco-2 cells that are caveolin-1 deficient and in stably transfected Caco-2 cells that express caveolin-1. Electrophysiological characterization of wild-type and stably transfected Caco-2 showed that caveolin-1 promoted I(Cl,swell), but not cystic fibrosis transmembrane conductance regulator currents. Furthermore, caveolin-1 expression stimulated the hypotonicity-induced release of taurine and ATP in stably transfected Caco-2 cells grown as a monolayer. Interestingly, the effect of caveolin-1 was polarized because only the release at the basolateral membrane, but not at the apical membrane, was increased. It is therefore concluded that caveolin-1 facilitates the hypotonicity-induced release of Cl(-), taurine, and ATP, and that in polarized epithelial cells, the effect of caveolin-1 is compartmentalized to the basolateral membrane.

Adenosine Triphosphate↗
PubMed2005

Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes.

The epithelial Na+ channel (ENaC) is modulated by various extracellular factors, including Na+, organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni2+ cation on ENaCs, we compared the Na+ permeability and amiloride kinetics of Xenopus ENaCs (xENaCs) and rat ENaCs (rENaCs) heterologously expressed in Xenopus oocytes. We found that the channel cloned from the kidney of the clawed toad Xenopus laevis [wild-type (WT) xENaC] was stimulated by external Ni2+, whereas the divalent cation inhibited the channel cloned from the rat colon (WT rENaC). The kinetics of amiloride binding were determined using noise analysis of blocker-induced fluctuation in current adapted for the transoocyte voltage-clamp method, and Na+ conductance was assessed using the dual electrode voltage-clamp (TEVC) technique. The inhibitory effect of Ni2+ on amiloride binding is not species dependent, because Ni2+ decreased the affinity (mainly reducing the association rate constant) of the blocker in both species in competition with Na+. Importantly, using the TEVC method, we found a prominent difference in channel conductance at hyperpolarizing voltage pulses. In WT xENaCs, the initial ohmic current response was stimulated by Ni2+, whereas the secondary voltage-activated current component remained unaffected. In WT rENaCs, only a voltage-dependent block by Ni2+ was obtained. To further study the origin of the xENaC stimulation by Ni2+, and based on the rationale of the well-known high affinity of Ni2+ for histidine residues, we designed alpha-subunit mutants of xENaCs by substituting histidines that were expressed in oocytes, together with WT beta- and gamma-subunits. Changing His215 to Asp in one putative amiloride-binding domain (WYRFHY) in the extracellular loop between Na+ channel membrane segments M1 and M2 had no influence on the stimulatory effect of Ni2+, and neither did complete deletion of this segment. Next, we mutated His416 flanked by His411 and Cys417, a unique site for possible heavy metal ion chelation, and, with this quality, most proximal (approximately 100 amino acids upstream of the second putative amiloride binding site at the pore entrance), was found localized at M2. Replacing His416 with arginine, aspartate, tyrosine, and alanine clearly affected amiloride binding in all cases, as well as Na+ conductance, as expressed in the xENaC current-voltage relationship, especially with regard to aspartate and tyrosine. However, similarly to those obtained with the WYRFHY stretch, none of these mutations could either abolish the stimulating effect of Ni2+ or reverse it to an inhibitory type.

Amiloride↗
PubMed2005

CBS domains: structure, function, and pathology in human proteins.

The cystathionine-beta-synthase (CBS) domain is an evolutionarily conserved protein domain that is present in the proteome of archaebacteria, prokaryotes, and eukaryotes. CBS domains usually come in tandem repeats and are found in cytosolic and membrane proteins performing different functions (metabolic enzymes, kinases, and channels). Crystallographic studies of bacterial CBS domains have shown that two CBS domains form an intramolecular dimeric structure (CBS pair). Several human hereditary diseases (homocystinuria, retinitis pigmentosa, hypertrophic cardiomyopathy, myotonia congenital, etc.) can be caused by mutations in CBS domains of, respectively, cystathionine-beta-synthase, inosine 5'-monophosphate dehydrogenase, AMP kinase, and chloride channels. Despite their clinical relevance, it remains to be established what the precise function of CBS domains is and how they affect the structural and/or functional properties of an enzyme, kinase, or channel. Depending on the protein in which they occur, CBS domains have been proposed to affect multimerization and sorting of proteins, channel gating, and ligand binding. However, recent experiments revealing that CBS domains can bind adenosine-containing ligands such ATP, AMP, or S-adenosylmethionine have led to the hypothesis that CBS domains function as sensors of intracellular metabolites.

AMP-Activated Protein Kinases↗
PubMed2005

PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes.

The plasma membrane of epithelial cells is subdivided into two physically separated compartments known as the apical and basolateral membranes. To obtain directional transepithelial solute transport, membrane transporters (i.e., ion channels, cotransporters, exchangers, and ion pumps) need to be targeted selectively to either of these membrane domains. In addition, the transport properties of an epithelial cell will be maintained only if these membrane transporters are retained and properly regulated in their specific membrane compartments. Recent reports have indicated that PDZ domain-containing proteins play a dual role in these processes and, in addition, that different apical and basolateral PDZ proteins perform similar tasks in their respective membrane domains. First, although PDZ-based interactions are dispensable for the biosynthetic targeting to the proper membrane domain, the PDZ network ensures that the membrane proteins are efficiently retained at the cell surface. Second, the close spatial positioning of functionally related proteins (e.g., receptors, kinases, channels) into a signal transduction complex (transducisome) allows fast and efficient control of membrane transport processes.

Animals↗
PubMed2004

Long-lasting changes in GABA responsiveness in cultured neurons.

In neuronal cells, GABA evokes an increase in chloride conductance by activating GABA(A) and GABA(C) receptors. In mature neurons, this increase in conductance generally has a hyperpolarizing and inhibitory action. Using gramicidin-based perforated patch recordings, we show that in cultured motor neurons GABA-induced currents are significantly altered following activation of GABA receptors coinciding with changes in membrane potential. Changes in intracellular chloride concentration constituted the mechanism for this modulation. Because of low resting chloride conductance and low activity of chloride transporters, changes in intracellular chloride concentration and hence GABA response were long-lasting (time constant of recovery was about 2.5 min). Cultured dorsal horn and hippocampal neurons exhibited a similar response pattern, suggesting a general property of cultured neuronal cells. These long-lasting changes in GABA responsiveness may have major implications on neuronal excitability.

Animals↗
PubMed2004

Calcium-activated chloride channels: (un)known, (un)loved?

Calcium-activated chloride channels (CaCCs) participate in many different physiologic processes such as transepithelial transport, excitability of neurons and muscle cells, and oocyte fertilization. Within the airways, they contribute to epithelial fluid secretion. This review focuses on three outstanding questions about CaCCs. First, although their biophysical fingerprint (anion selectivity, Ca2+ and voltage dependence, kinetics) is fairly well established, the molecular identity of CaCCs is still unresolved. CLCA, a family of proteins of which four members have so far been identified in humans, has been proposed to mediate calcium-activated chloride currents. However, the biophysical profile and expression pattern of endogenous CaCCs differ from those of the CLCA proteins. Another family of membrane proteins, the bestrophins, has recently been shown in transfected HEK293 cells to confer anion-selective currents that are activated by submicromolar Ca2+ concentrations. Second, pharmacologic tools to manipulate CaCCs are poorly selective. This lack of specificity not only hampers the structural and functional characterization of these channels but also restricts therapeutic options for altering CaCC function. Finally, potential pitfalls with respect to CaCCs as molecular targets for cystic fibrosis therapy are discussed.

Animals↗
PubMed2003

Hypotonicity induces membrane protrusions and actin remodeling via activation of small GTPases Rac and Cdc42 in Rat-1 fibroblasts.

An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types. We demonstrate in this study by means of rhodamine-phalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed in peripheral extensions at the cell border. Moreover, we demonstrate that activation of both Rac and Cdc42, members of the family of small Rho GTPases, forms the link between the hypotonic stimulation and F-actin reorganization. Indeed, inhibition of the small GTPases RhoA, Rac, and Cdc42 (by Clostridium difficile toxin B) prevents the hypotonicity-induced reorganization of the actin cytoskeleton, whereas inhibition of RhoA alone (by C. limosum C3 exoenzyme) does not preclude this rearrangement. Second, a direct activation and translocation toward the actin patches underneath the plasma membrane is observed for endogenous Rac and Cdc42 (but not for RhoA) during cell swelling. Finally, transfection of Rat-1 fibroblasts with constitutively active RhoA, dominant negative Rac, or dominant negative Cdc42 abolishes the swelling-induced actin reorganization. Interestingly, application of cRGD, a competitor peptide for fibronectin-integrin association, induces identical membrane protrusions and changes in the F-actin cytoskeleton that are also inhibited by C. difficile toxin B and dominant negative Rac or Cdc42. Moreover, cRGD also induces a redistribution of endogenous Rac and Cdc42 to the newly formed submembranous F-actin patches. We therefore conclude that hypotonicity and cRGD remodel the F-actin cytoskeleton in Rat-1 fibroblasts in a Rac/Cdc42-dependent way.

Actins↗
PubMed2002

Influence of voltage and extracellular Na(+) on amiloride block and transport kinetics of rat epithelial Na(+) channel expressed in Xenopus oocytes.

We expressed the three subunits of the epithelial amiloride-sensitive Na(+) channel (ENaC) from rat distal colon heterologously in oocytes of Xenopus laevis and analysed blocker-induced fluctuations in current using conventional dual-microelectrode voltage-clamp. To minimize Na(+) accumulation we performed all experiments in low-Na(+) solutions (15 mM). Noise analysis revealed that control or ENaC-injected oocytes did not exhibit spontaneous relaxation noise. However, in ENaC-expressing oocytes, amiloride induced a distinct Lorentzian component in the power density spectra. With three amiloride concentrations and a linear analysis of the respective changes in the corner frequency f(c) (2 pi f(c) plot) we determined the rate constants k(on) and k(off) for the amiloride-ENaC interaction. At a clamp potential (V(m)) of -60 mV k(on) was 80.8 +/- 5.1 microM(-1) s(-1) and k(off) 15.4 +/- 4.2 s(-1). The half-maximal blocker concentration (K(mic,ami)) was 0.19 microM (V(m)=-60 mV). While k(on) was voltage-independent in the range -50 to -100 mV, k(off) and K(mic,ami) decreased significantly with increasing membrane hyperpolarization, resulting in an increased affinity of amiloride for its binding site on ENaC. Increasing extracellular [Na(+)] ([Na(+)](o)) led to saturation of ENaC. Subsequent noise analysis revealed that single-channel current increased non-linearly with [Na(+)](o) and that saturation was not due to a reduction in the number of open channels. The apparent affinity of Na(+) for its binding site on the channel was voltage dependent and increased with hyperpolarization. Noise analysis revealed that k(on) and k(off) for amiloride decreased with increasing [Na(+)](o), while the affinity of the amiloride-binding site did not change. These findings show that the affinity of rat intestinal ENaC for amiloride is voltage dependent and is influenced non-competitively by [Na(+)](o), indicating that Na(+) and amiloride do not compete for the same binding site at the channel.

Amiloride↗
PubMed2002

RhoA exerts a permissive effect on volume-regulated anion channels in vascular endothelial cells.

Cell swelling triggers in most cell types an outwardly rectifying anion current, I(Cl,swell), via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of I(Cl,swell). However, these experiments did not allow us to discriminate between a direct activator role or a permissive effect. We now show that the Rho pathway did not affect VRAC activity if this pathway was activated by transfecting CPAE cells with constitutively active isoforms of Galpha (a Rho activating heterotrimeric G protein subunit), Rho, or Rho kinase. Furthermore, biochemical and morphological analysis failed to demonstrate activation of the Rho pathway during hypotonic cell swelling. Finally, manipulating the Rho pathway with either guanosine 5'-O-(3-thiotriphosphate) or C3 exoenzyme had no effect on VRACs in caveolin-1-expressing Caco-2 cells. We conclude that the Rho pathway exerts a permissive effect on VRACs in CPAE cells, i.e., swelling-induced opening of VRACs requires a functional Rho pathway, but not an activation of the Rho pathway.

Animals↗