Holger Repp

Listeriolysin of Listeria monocytogenes forms Ca2+-permeable pores leading to intracellular Ca2+ oscillations.

Listeriolysin (LLO) is a major virulence factor of Listeria monocytogenes, a Gram‐positive bacterium that can cause life‐threatening diseases. Various signalling events and cellular effects, including modulation of gene expression, are triggered by LLO through unknown mechanisms. Here, we demonstrate that LLO applied extracellularly at sublytic concentrations causes long‐lasting oscillations of the intracellular Ca2+ level of human embryonic kidney cells; resulting from a pulsed influx of extracellular Ca2+ through pores that are formed by LLO in the plasma membrane. Calcium influx does not require the activity of endogenous Ca2+ channels. LLO‐formed pores are transient and oscillate between open and closed states. Pore formation and Ca2+ oscillations were also observed after exposure of cells to native Listeria monocytogenes. Our data identify LLO as a tool used by Listeria monocytogenes to manipulate the intracellular Ca2+ level without direct contact of the bacterium with the target cell. As Ca2+ oscillations modulate cellular signalling and gene expression, our findings provide a potential molecular basis for the broad spectrum of Ca2+‐dependent cellular responses induced by LLO during Listeria infection.

Why Escherichia coli α-hemolysin induces calcium oscillations in mammalian cells—the pore is on its own

Escherichia coli α‐hemolysin (HlyA), archetype of a bacterial pore‐forming toxin, has been reported to deregulate physiological Ca2+ channels, thus inducing periodic low‐frequency Ca2+ oscillations that trigger transcriptional processes in mammalian cells. The present study was undertaken to delineate the mechanisms underlying the Ca2+ oscillations. Patch‐clamp experiments were combined with single cell measurements of intracellular Ca2+ and with flow‐cytometric analyses. Application of HlyA at subcytocidal concentrations provoked Ca2+ oscillations in human renal and endothelial cells. However, contrary to the previous report, the phenomenon could not be inhibited by the Ca2+ channel blocker nifedipine and Ca2+ oscillations showed no constant periodicity at all. Ca2+ oscillations were dependent on the pore‐forming activity of HlyA: application of a nonhemolytic but bindable toxin had no effect. Washout experiments revealed that Ca2+ oscillations could not be maintained in the absence of toxin in the medium. Analogously, propidium iodide flux into cells occurred in the presence of HlyA, but cells rapidly became impermeable toward the dye after toxin washout, indicating resealing or removal of the membrane lesions. Finally, patch‐clamp experiments revealed temporal congruence between pore formation and Ca2+ influx. We conclude that the nonperiodic Ca2+ oscillations induced by HlyA are not due to deregulation of physiological Ca2+ channels but derive from pulsed influxes of Ca2+ as a consequence of formation and rapid closure of HlyA pores in mammalian cell membranes.—Koschinski, A., Repp, H., Ünver, B., Dreyer, F., Brockmeier, D., Valeva, A., Bhakdi, S., and Walev, I. Why Escherichia coli α‐hemolysin induces calcium oscillations in mammalian cells—the pore is on its own. FASEB J. 20, E80‐E87 (2006)

Bacillus intermedius ribonuclease as inhibitor of cell proliferation and membrane current.

The antiproliferative action of the guanine-specific ribonuclease secreted by Bacillus intermedius (binase) was studied in different chicken and mouse cell lines. The proliferation rate of chicken embryo fibroblasts, either normal or Rous sarcoma virus-transformed, was significantly reduced by binase treatment. Among mouse fibroblasts, v-ras-transformed NIH3T3 cells were sensitive to binase, whereas the growth of non-transformed, v-src-transformed or v-fms-transformed NIH3T3 cells was not affected. A 48 h treatment with binase inhibited the Ca2+-dependent K+ current of v-ras-transformed NIH3T3 cells but had no effect on this membrane current in non-transformed and in v-src- or v-fms-transformed NIH3T3 cells. Our results suggest that mammalian cells expressing the ras-oncogene are a potential target for the antiproliferative action of binase.

Two types of fast K+ channels in rat myelinated nerve fibres and their sensitivity to dendrotoxin

The effect of dendrotoxin (DTX), a component of the venom of the Eastern green mamba snake, Dendroaspis angusticeps, on K+ currents in rat myelinated nerve fibres was studied in voltage clamp experiments, immunocytochemistry and binding experiments. The analysis of K+ tail currents in 160 mM KCl solution revealed that K+ channels with slow gating kinetics predominate in the intact node of Ranvier. These slow K+ channels were not blocked by DTX. Intact nerve fibres additionally showed fast K+ tail currents of small amplitude which could be blocked by DTX. After enzymatic demyelination with pronase, fast K+ currents of large amplitude appeared. Analysis of the non-monotonous voltage dependence of the fast K+ conductance and the partial pharmacological block by DTX suggest the presence of two subtypes of fast K+ channels in rat nerve fibres similar to the Kf1 and Kf2 channels previously described in the frog [13] and toad node of Ranvier [2]. The DTX concentration required for 50% inhibition (IC50) for the Kf1 component was 8 nM. The IC50 of the blocked Kf2 component was the same as that for Kf1, but the Kf2 component was only partially blocked (about 50%). In contrast to frog nerve, these two fast K+ channel subtypes are located predominantly in the paranodal region. Immunocytochemical staining experiments with DTX using the peroxidase-antiperoxidase technique confirmed the electrophysiological data. In intact nodes, either no staining or only slight staining in some fibres was found. After demyelination, extensive staining of paranodal and internodal regions occurred. Binding of iodine 125DTX to rat nerve membranes revealed a “high affinity” binding site with a KD value in the picomolar range and a binding capacity of 500–700 fmol/mg protein. In addition there was a „low affinity” binding site with a 10-times higher density and a KD of about 15 nM in 160 mM KCl solution. This value is in good agreement with the IC50 of 8 nM for the block of the total fast K+ current obtained in the electrophysiological experiments. Binding of 125I-DTX to rat nerve membranes could be inhibited with decreasing potency by the snake toxin DTX I, the scorpion toxin charybdotoxin and the mast cell degranulating peptide of the honey bee, indicating that these peptide toxins share common binding sites with DTX in rat nerve fibres.

Src-transformation of mouse fibroblasts induces a Ca2+-activated K+ current without changing the T-type Ca2+ current

Membrane currents of src-transformed NIH3T3 mouse fibroblasts were analyzed in comparison with their non-transformed counterparts using the patch-clamp technique. Normal NIH3T3 cells exhibit two types of Ca2+ currents and a membrane current of ohmic behaviour (current amplitude 135 pA at +30 mV) that can partially be blocked by Cd2+. Src-transformed NIH3T3 cells show an additional membrane current that becomes activated after the establishment of the whole-cell configuration with a maximum amplitude of 1040 pA at +30 mV within 30-60 s. This current then inactivates irreversibly within 5-10 min. The additional current is highly K(+)-selective and Ca(2+)-dependent but voltage-independent. It can be blocked by charybdotoxin (IC50 = 20 nM) and by internal tetraethylammonium (TEA; IC50 = 2.9 mM), but it is not sensitive to external TEA (up to 30 mM). Single-channel analysis revealed only one K+ channel type with a conductance of 37 pS at negative potentials and 18 pS at positive potentials (in symmetrical 145 mM K+ solutions), a voltage-independent open-state probability of 0.6 and the same pharmacological properties as the macroscopic KCa current. The properties of the KCa current and the underlying channels of src-transformed NIH3T3 cells are identical to those observed in ras-transformed NIH3T3 cells. In contrast, src- or ras-transformation affects differently the voltage-dependent, transient (T-type) Ca2+ current. While ras-transformation of NIH3T3 cells suppresses their T-type Ca2+ current, this current remains unchanged in src-transformed NIH3T3 cells.

Sulfonylurea-sensitive K+ channels and their probable role for the membrane potential of mouse motor nerve endings.

We studied the effect of the KATP channel blockers tolbutamide and glibenclamide on presynaptic membrane currents in the mouse M. triangularis sterni preparation using the perineural recording technique. Both sulfonylureas blocked part of the fast K+ component within 2 min after application. The block was much more pronounced under glucose-free conditions and was completelyreversible by washing. Addition of glucose to glucose-free bath solution also reduced the K+ component. A further effect of the sulfonylureas was observed under glucose-free conditions. With a delay of 5 to 10 min, the nodal Na+ component began to diminish and disappeared within 30 min. This was associated with a dramatic increase in spontaneous quantal transmitter release suggesting that the block of sulfonylurea-sensitive K+ channels causes depolarization of motor nerve terminals and fibres thus inactivating Na+ channels. Tetraethylammonium (TEA) which blocks ATP-dependent K+ channels in high concentrations caused, under glucose-free conditions, the same delayed effect as the sulfonylureas. This delayed effect was fully reversible by washing with glucose-containing, but not with glucose-free solution. Our findings strongly suggest that KATP channels exist in mammalian motor nerve endings and that under hypoglycemic conditions these channels open and become essential for the maintenance of the membrane potential.

Activation of a Ca2+-dependent K+ current in mouse fibroblasts by lysophosphatidic acid requires a pertussis toxin-sensitive G protein and Ras

Lysophosphatidic acid (LPA) is a bioactive lipid that acts through G protein-coupled plasma membrane receptors and mediates a wide range of cellular responses. Here we report that LPA activates a K+ current in NIH3T3 mouse fibroblasts that leads to membrane hyperpolarization. The activation occurs with an EC50 value of 1.7 nM LPA. The K+ current is Ca2+-dependent, voltage-independent, and completely blocked by the K+ channel blockers charybdotoxin, margatoxin, and iberiotoxin with IC50 values of 1.7, 16, and 62 nM, respectively. The underlying K+ channels possess a single channel conductance of 33 pS in symmetrical K+ solution. Pretreatment of cells with pertussis toxin (PTX), Clostridium sordellii lethal toxin, or a farnesyl protein transferase inhibitor reduced the K+ current amplitude in response to LPA to about 25% of the control value. Incubation of cells with the protein tyrosine kinase inhibitor genistein or microinjection of the neutralizing anti-Ras monoclonal antibody Y13–259 reduced it by more than 50%. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase A activator 8-bromo-cAMP had no effect. These results indicate that the K+ channel activation by LPA is mediated by a signal transduction pathway involving a PTX-sensitive G protein, a protein tyrosine kinase, and Ras. LPA is already known to activate Cl– channels in various cell types, thereby leading to membrane depolarization. In conjunction with our results that demonstrate LPA-induced membrane hyperpolarization by activation of K+ channels, LPA appears to be significantly involved in the regulation of the cellular membrane potential.

Functional transfer of eukaryotic expression plasmids to mammalian cells by Listeria monocytogenes: a mechanistic approach.

Cystic fibrosis (CF) is one of the most common monogenic disorders in the caucasian population. Gene therapy for CF is principally feasible and bacterial transfer systems might provide novel possibilities for therapy. However, transfection efficiencies are low and need to be improved. Thus, more detailed understanding of the DNA transfer mechanism is necessary to systematically eliminate these restrictions.

RNase-induced apoptosis: Fate of calcium-activated potassium channels

The connection between the action of microbial RNases and Ca2+-activated K+ (KCa) channels was investigated in human embryo kidney cells HEKhSK4 artificially expressing the channels. These channels protected HEKhSK4 cells from apoptosis induced by binase and 5K charge reversal mutant of RNase Sa. After the first 24h, potassium current increased without increase in intracellular Ca2+, and mitochondrial potential remained high. After 72 h, the concentration of calcium increased and mitochondria lost their potential. Whole-cell recordings of membrane currents through KCa channels in RNase-treated cells demonstrated a biphasic pattern: initially their activity in cell population increased, peaked at 24h, and then gradually decreased. In each individual cell we observed either an increase of the amplitude of KCa current, or a complete shutdown of the channels. The activity of KCa channels could be restored by removing RNases from the media. Based on this pattern and especially its timing, we hypothesize that toxic RNases downregulate KCa channels at the level of transcription or translation. Our results indicate that new anticancer agents could be created on the basis of microbial RNases targeting KCa channels.

Activation of a Ca2+-dependent K+ current by the oncogenic receptor protein tyrosine kinase v-Fms in mouse fibroblasts

We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor c-Fms, activates a K+ current that is absent in control cells. The activation of the K+ current was Ca2+-dependent, voltage-independent, and was completely blocked by the K+ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC50 values of 3nM, 18 nM and 76nM, respectively. To identify signalling components that mediate the activation of this K+ current, NIH3T3 cells that express different mutants of the wildtype v-Fms receptor were examined. Mutation of the binding site for the Ras-GTPase-activating protein led to a complete abolishment of the K+ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity of v-Fms, led to a 55% reduction of the K+ current. Treatment of wild-type v-Fms cells with Clostridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the K+ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK can modulate K+ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our data suggest that Ras plays a key role for K+ channel activation by the oncogenic RTK v-Fms.