Structure-function relationship of the ion channel formed by diphtheria toxin in Vero cell membranes.

Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel, and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin, Deltagm, was initially determined. DT-AB induced a approximately 10-fold lower Deltagm than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Deltagm, whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Deltagm. Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches. No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as Deltagm and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel.

A role for endothelial NO synthase in LTP revealed by adenovirus-mediated inhibition and rescue.

Pharmacological studies support the idea that nitric oxide (NO) serves as a retrograde messenger during long-term potentiation (LTP) in area CA1 of the hippocampus. Mice with a defective form of the gene for neuronal NO synthase (nNOS), however, exhibit normal LTP. The myristoyl protein endothelial NOS (eNOS) is present in the dendrites of CA1 neurons. Recombinant adenovirus vectors containing either a truncated eNOS (a putative dominant negative) or an eNOS fused to a transmembrane protein were used to demonstrate that membrane-targeted eNOS is required for LTP. The membrane localization of eNOS may optimally position the enzyme both to respond to Ca2+ influx and to release NO into the extracellular space during LTP induction.

GPI-anchored diphtheria toxin receptor allows membrane translocation of the toxin without detectable ion channel activity.

We have investigated the role of the transmembrane and cytoplasmic domains of the diphtheria toxin (DT) receptor [heparin-binding epidermal growth factor (HB-EGF) precursor] in the intoxication pathway. Two mutants were constructed in which these domains were replaced by either a 37 amino acid sequence signalling membrane attachment via a glycosylphosphatidylinositol (GPI) anchor (DTR-GPI) or by the transmembrane and cytoplasmic domains of the human EGF receptor (DTR-EGFR). Similar amounts of DTA fragment were translocated through the plasma membrane of NIH 3T3 cells transfected with the wild-type receptor (DTR), DTR-GPI and DTR-EGFR, but translocation was about six times less efficient in the case of DTR-GPI and DTR-EGFR when taking into account the number of receptors expressed. Interestingly, DT-induced 22Na+ influx was weak in DTR-EGFR cells and not detectable in DTR-GPI cells. Whole cell patch-clamp analysis showed the DT at low pH induced depolarization and decreased input resistance in DTR cells (and to a lesser extent also in DTR-EGFR cells) but not in DTR-GPI cells. These results suggest that the transmembrane and cytoplasmic part of the receptor might be involved in channel activity and that translocation of the A fragment is independent of toxin-induced cation channel activity.

Diphtheria toxin endocytosis and membrane translocation are dependent on the intact membrane-anchored receptor (HB-EGF precursor): studies on the cell-associated receptor cleaved by a metalloprotease in phorbol-ester-treated cells.

Preincubation of Vero cells with 1 microM phorbol 12-myristate 13-acetate (PMA) decreased the specific binding of diphtheria toxin by about 50%, whereas the toxic effect, endocytic uptake and membrane translocation were completely blocked. Toxin bound to PMA-treated cells was released upon incubation with heparinase. The effect of PMA was abrogated in the presence of EDTA or N-(DL-[2-(hydroxyaminocarbonyl)methyl]-4-methyl-pentanoyl)-L-3-(2' - naphthyl)-alanyl-L-alanine 2-aminoethyl-amide (TAPI), a specific inhibitor of matrix metalloproteases. The results indicate that PMA induces proteolytic cleavage of the diphtheria-toxin receptor [heparin-binding EGF-like growth factor (HB-EGF)-precursor] outside the membrane anchor, and that about 50% of the growth-factor ecto-domain remains associated with the cells, due to binding to surface proteoglycans containing heparan sulphates. Although the cleaved cell-associated HB-EGF binds diphtheria toxin, it does not serve as a functional receptor, since neither toxin internalization nor translocation occurs. Thus the intact HB-EGF precursor is of crucial importance for its function as the diphtheria-toxin receptor.

Early events of Semliki Forest virus-induced cell-cell fusion.

Insect cells (Aedes albopictus) were infected with Semliki Forest Virus. Cell-cell fusion was then induced by lowering the extracellular pH. The underlying processes were examined by monitoring the intercellular current flow, Ij. Experimentally, this involved the use of cell pairs in conjunction with a dual voltage-clamp method. This approach allowed us to monitor the kinetics of fusion at high temporal resolution. The fusion process began shortly after acidification (delay: 3-138 sec). Initially, Ij increased in a stepwise manner, later on it developed more gradually. Fusion between two cells reached a steady state within 7-70 sec. The steps in Ij are attributable to the formation of cytoplasmic connections between the cells, presumably involving proteinaceous fusion pores. The mean amplitude of Ij steps corresponds to a conductance of 300 pS, consistent with a pore radius of 1 nm. Cytoplasmic connections developed rapidly, i.e., Ij steps occurred within less than 1 msec. The absence of Ij flickering implies that formation of cytoplasmic connections, and hence SFV induced cell-cell fusion, is irreversible.

pH-dependent pore formation in Semliki forest virus-infected Aedes albopictus cells.

The plasma membrane properties of Semliki forest virus-infected Aedes cells were studied using whole-cell patch-clamp recording. Cells exhibited a marked increase in membrane conductance, gm (from 0.48 +/- 0.09 nS to 14.2 +/- 10.8 nS) upon exposure to acidic pH (5.6). The membrane potential depolarized from -40.9 +/- 5.2 mV to -2.43 +/- 7.14 mV under these conditions. In uninfected cells, there was no change in gm after lowering the pH. This implies that viral fusion proteins are involved in changing the membrane properties at low pH. The increased gm in infected cells at low pH was not persistent but declined within minutes. Millimolar concentrations of calcium and zinc prevented or reverted the increase in gm. The results suggest that viral proteins, if brought to their fusogenic conformation by exposure to low pH, form unspecific pores in the residing membrane. These pores might play a role in virus entry.

Arachidonic acid and other unsaturated fatty acids alter membrane potential in PC12 and bovine adrenal chromaffin cells.

The action of arachidonic acid and other fatty acids on membrane potential in PC12 and bovine chromaffin cells was investigated using a membrane potential-sensitive fluorescent dye. Arachidonic acid (1-40 microM) provoked dose-dependent membrane hyperpolarization, thereby reducing hyperpolarization induced by the K(+)-selective ionophore valinomycin. Other cis-unsaturated fatty acids, but not lipoxygenase products or the saturated fatty acid palmitic acid, also affected membrane potential. Tetraethylammonium blocked the arachidonic acid-induced hyperpolarization. These data suggest that cis-unsaturated fatty acids alter membrane potential in PC12 and bovine chromaffin cells by modulating K+ conductances. Valinomycin-generated hyperpolarization had no effect on agonist-induced Ca2+ influx into bovine chromaffin cells, whereas preincubation with arachidonic acid and other cis-unsaturated fatty acids blocked Ca2+ influx and secretion. We propose a model where internally generated fatty acids act as a feedback to desensitize the stimulated cell via inhibition of receptor-dependent Ca2+ influx and induction of membrane hyperpolarization.

Changes in membrane permeability during Semliki Forest virus induced cell fusion.

The infection of Aedes albopictus cells by Semliki Forest virus (SFV) is a non lytic event. Exposure of infected cells to mildly acidic pH (less than 6.2) leads to syncytium formation. This polykaryon formation is accompanied by an influx of protons into the cells (Kempf et al. Biosci. Rep. 7, 761-769, 1987). We have further investigated this permeability change using various fluorescent or radiolabeled compounds. A significant, pH dependent increase of the membrane permeability to low molecular weight compounds (M(r) less than 1000) was observed when infected cells were exposed to a pH less than 6.2. The pH dependence of the permeability change was very similar to the pH dependence of cell-cell fusion. The permeability change was sensitive to divalent cations, protons and anionic antiviral drugs such as trypan blue. The nature of this virus induced, pH dependent permeability change is discussed.