Jacqueline C. Tanaka

Actinobacillus actinomycetemcomitans leukotoxin forms large conductance, voltage-gated ion channels when incorporated into planar lipid bilayers.

Actinobacillus actinomycetemcomitans leukotoxin is a member of the bacterial RTX (repeats in toxin) toxin family, produced by a diverse group of Gram-negative pathogens. Members of this group of toxins, although similar in sequence, differ in target cell specificity with Actinobacillus actinomycetemcomitans leukotoxin demonstrating a unique species- and cell-type specificity. Purified A. actinomycetemcomitans leukotoxin added to pre-formed POPE/POPS lipid bilayers showed no spontaneous incorporation (to concentrations of 250 ng/ml). Reproducible channel activity was seen when the bilayer was reformed from lipid monolayers in the presence of toxin (50 ng/ml) in one of the aqueous chambers. Control experiments with heat-inactivated toxin did not display channel activity under the same experimental conditions. The channel behavior showed a complex pattern of multiple conductance levels of 118, 262 and 406 pS in solutions containing 0.140 M NaCl. The first two states showed voltage-dependent channel gating with approximately equal but opposite apparent gating charges of 1.4 electrons. A model accounting for the multiple conducting states and gating properties is presented.

Characterization of Actinobacillus actinomycetemcomitans leukotoxin pore formation in HL60 cells

The mechanism of cell death induced by Actinobacillus actinomycetemcomitans leukotoxin (LTX) has been investigated with flow cytometry and patch electrode recording using cultured HL60 cells. The kinetics of propidium iodide (PI) positive staining of HL60 cells was measured as a function of LTX concentration at 37 degreesC. Results showed a concentration-dependent decrease in the tk times. Cell kill was slow at <1 microg/ml LTX concentrations with fewer than 50% of the cells killed after 1 h; at 1 microg/ml, the tk times ranged from approximately 15 to 30 min. At higher concentrations, the tk times decreased rapidly. The rate of cell kill was appreciably slowed at 20 degreesC. HL60 whole cell currents were recorded with patch electrodes. Immediately following exposure to high concentrations of LTX, large currents were recorded suggesting that the membrane potential of these cells had collapsed due to the large conductance increases. At low toxin concentrations, rapid conductance fluctuations were seen suggestive of a limited number of toxin-mediated events. Cells exposed to low concentrations of LTX exhibited these conductance fluctuations for up to 1 h, whereas toxin-insensitive cells were unaffected by long exposures to high concentrations of toxin. Our results are consistent with LTX-induced pores in susceptible cells which overwhelm the ability of the cell to maintain osmotic homeostasis causing cell death.

Magainin-induced cytotoxicity in eukaryotic cells: kinetics, dose-response and channel characteristics.

Magainin 1 and magainin 2 are broad-spectrum antimicrobial and antifungal peptides initially purified from Xenopus laevis skin glands. The mechanism of cytotoxicity of the naturally occurring magainin 2 and a potent all-D amino acid analogue, MSI-238, was examined for eukaryotic cells using flow cytometric analysis with propidium iodide (PI). Exposure to MSI-238 resulted in cell death within seconds to minutes, depending on the concentration of the peptide. Several cell types were examined including a mouse fibroblast cell line Balb/3T3 and a Rous sarcoma virus Balb/3T3-transformed cell line, SRD/3T3, primary chick embryo fibroblasts and cells derived from a human ovarian carcinoma, OVCA-3. The K0.5 values determined from 5 min exposures ranged from 24 to 80 micrograms/ml for MSI-238 and approximately 600 micrograms/ml for magainin 2. Molecular properties of MSI-238 induced channels were studied in excised membrane patch recordings from Balb/3T3 and SRD/3T3 cells. At low concentrations of 0.1 micrograms/ml, occasional, brief, multiple-level current fluctuations were seen suggesting channels with multiple, rapidly changing conductance levels. At 5 or 10 micrograms/ml of MSI-238, the current fluctuations were larger in magnitude and occurred more frequently producing a general disruption of the membrane similar to the effects of melittin on membranes.

STRUCTURE/FUNCTION ASPECTS OF ACTINOBACILLUS ACTINOMYCETEMCOMITANS LEUKOTOXIN

Actinobacillus actinomycetemcomitans has been implicated as a causative organism in early-onset periodontitis. The mechanisms by which A. actinomycetemcomitans is pathogenic are not known, but the organism produces several potential virulence factors, one of which is a leukotoxin. As a group, bacterial protein toxins are made up of structural domains which control various aspects of toxic activity, such as target cell recognition, membrane insertion, and killing. The purpose of this article is to review the structure of RTX, with special emphasis to its relation to toxin function. In addition, we will propose a model based upon other bacterial proteins whereby the water-soluble A. actinomycetemcomitans leukotoxin is able to achieve insertion into a biological membrane. J Periodontol 1996;67:298-308.

Developmental Appearance of Sodium Channel Subtypes in Rat Skeletal Muscle Cultures

Abstract 22Na influx was measured in the established muscle cell line L‐6 and in primary rat skeletal muscle cultures following activation of sodium channels by veratridine and sea anemone toxin II. Inhibition of the activated channels by tetrodotoxin (TTX) was analyzed with computer‐assisted fits to one‐ or two‐site binding models. In L‐6 cultures, two inhibitable sodium channel populations were resolved at all ages in culture: a TTX‐sensitive (K= 0.6–5.0 × 10−8M) and an insensitive population (Ki= 3.3–4.9 × 10−6M). In primary rat muscle cultures, the sensitivity of the toxin‐stimulated channels to TTX changed with time in culture. In 4‐day‐old cultures, a single sodium channel population was detected using TTX (Ki= 2.4 × 10−7M). A single population was also found in 6‐day‐old cultures (Ki= 5.3 × 10−7M). By day 7 in culture, the inhibition of 22Na influx by TTX could be resolved into two components with high‐ and low‐affinity sites for the toxin (Ki= 1.3 × 10−9M and 9.6 × 10−7M). We conclude that a single, toxin‐activated sodium channel population with low affinity for TTX exists at early stages, whereas a second, high‐affinity population evolves with time in primary rat muscle cultures. The expression of a high‐affinity site apparently does not require ongoing neuronal involvement and may reflect an intrinsic property of the muscle cells.

Conformational studies of Actinobacillus actinomycetemcomitans leukotoxin: partial denaturation enhances toxicity

A 114 kDa, water-soluble, cytotoxin secreted by the Gram-negative bacterium Actinobacillus actinomycetemcomitans (Aa) is similar in sequence to Escherichia coli alpha-hemolysin, but is non-hemolytic, killing leukocytes of select species, including humans. In this work, we investigated aspects of the water-soluble conformation of Aa toxin which relate to its biological, pore-forming activity. The toxin has five native tryptophans and fluorescence spectra were monitored in aqueous solutions in the presence of varying denaturants. Significant changes in the fluorescence spectra, without significant wavelength shifts, were induced by small additions of denaturants and changes in the temperature or pH. The fluorescence changes suggested that small perturbations in the aqueous environment resulted in structural changes in the toxin related not to a large unfolding but to more subtle conformational changes. Analytical ultracentrifugation showed the toxin to be a globular monomer in dilute aqueous solution. Circular dichroism spectroscopy showed about 25% alpha-helical structure which is largely maintained up to a temperature (65 degrees C) known to deactivate toxin activity. Changes in the cytotoxic properties of the toxin were monitored with flow cytometric analysis following preincubation of the toxin under mild conditions similar to those used in the fluorescence studies. These experiments showed that the pretreated toxin exhibited enhanced cell-killing potency on toxin-sensitive cells. The correlation of cytotoxicity with the changes in Trp fluorescence is consistent with the idea that partial unfolding of Aa toxin is an early, obligate step in toxin-induced cell kill.

The effects of ibogaine on dopamine and serotonin transport in rat brain synaptosomes.

Ibogaine has been shown to affect biogenic amine levels in selected brain regions. Because of the involvement of these neurotransmitters in drug addiction, the effects of ibogaine on biogenic amine transport may contribute to the potential anti-addictive properties of ibogaine in vivo. With rat brain synaptosomes as our experimental system, we measured the effects of ibogaine on the uptake and release of dopamine (DA) and serotonin (5-HT). Ibogaine competitively blocked both DA and 5-HT uptake with IC50 values of 20 microM at 75 nM 3H-DA and 2.6 microM at 10 nM 3H-5-HT. Ibogaine had no effect on K+-induced release of 3H-DA from preloaded synaptosomes, but 20 microM and 50 microM ibogaine inhibited roughly 40% and 60%, respectively, of the K(+)-induced release of 3H-5-HT from preloaded synaptosomes. In the absence of a depolarizing stimulus, ibogaine evoked a small release of 3H-DA but not 3H-5-HT. These relatively low-potency effects of ibogaine on DA and 5-HT uptake in synaptosomes are consistent with the low binding affinity of ibogaine that has been previously reported for DA and 5-HT transporters. Our results show that if ibogaine modulates DA and 5-HT levels in the brain by directly blocking their uptake, then a concentration of ibogaine in the micromolar range is required. Furthermore, if the anti-addictive effects of ibogaine require this concentration, then ibogaine likely exerts these effects through a combination of neurotransmitter pathways, because binding affinities and functional potencies of ibogaine in the micromolar range have been reported for a variety of neuronal receptors and transporters.

Divalent effects on cGMP-activated currents in excised patches from amphibian photoreceptors

SummaryThe light-sensitive current in photoreceptors is conducted by a single class of ion channels gated by the binding of multiple molecules of cytoplasmic cGMP. Both Na and Ca ions enter the outer segment through this channel and Ca behaves as a blocking ion, greatly reducing the influx of Na. Because intracellular Ca functions as the cytosolic messenger for light adaptation, and this channel is the major entry point for Ca into the outer segment, we seek a better understanding of the selectivity properties of the channel and how they affect intracellular Ca levels. In these studies, we added divalent cations to the cytoplasmic face of an excised patch at constant, symmetrical [Na]. Our results suggest a novel high-affinity divalent binding site at the internal face of the channel. At constant low levels of cGMP, the addition of 10–100nm cytoplasmic Ca or Mg attenuated the current 5- to 10-fold. There is also a low-affinity site, midway through the transmembrane field; saturation of this site reduces the divalent-free current ∼100-fold. The presence of a high-affinity cytoplasmic site raises the question of whether Ca regulates the photoreceptor current through a direct interaction with the channel perhaps altering the channel selectivity or kinetics.

Skeletal Muscle Sodium Channels

In mammalian muscle, a voltage-sensitive sodium channel controls the transient increase in membrane conductance that produces an action potential in the sarcolemma and T-tubular membranes. The time- and voltage-dependent characteristics of the currents regulated by this channel have been studied extensively over the past few decades, first using the traditional approach of voltage clamp (Adrian et al., 1970) and more recently at the single-channel level using patch-clamp technology (Sigworth and Neher, 1980).

The Voltage-Sensitive Sodium Channel from Mammalian Skeletal Muscle

In mammalian skeletal muscle, action potentials are produced by sequential changes in the conductance of the surface membrane to sodium and potassium ions. These variable conductances are modulated by separate voltage-sensitive ionic channels which span the bilayer and provide gated aqueous pathways for ion movement through the membrane. The characteristics of the currents controlled by the voltage-sensitive sodium channel in skeletal muscle have been the subject of intensive study since the pioneering work of Hodgkin and Huxley in the 1950’s (Hodgkin and Huxley 1952). Early work in muscle relied on the traditional approach of voltage clamp to describe the kinetics of these sodium currents in large populations of channels in a single fiber (Adrian et al. 1970). More recently patch clamp technology has been used to extend this analysis to the behavior of single sodium channels in their native environment (Sigworth and Neher 1980).