Comparative analysis of latrotoxin channels of different conductance in planar lipid bilayers. Evidence for cluster organization.

It has been established that channels induced by Latrodectus tredicimguttatus alpha-toxin (LT) in lipid bilayers have a cluster organisation. So far as: (i) the LT-channels had practically identical sizes of its water pores (r = 9.4 +/- 0.6 A) independently on the lipid composition of planar bilayer lipid membrane (BLM) although their conductances might differ from each other more than 10 times (100 mM KCl (pH 7.5)). (ii) affinity of permeable ions to channels had a small variation with distinct group of BLM, although LT-channels conductances varied from 112 +/- 8 pS till 1110 +/- 40 pS for phosphatidylcholine-BLM and from 75 +/- 6 pS till 170 +/- 14 pS for phosphatidylserine-BLM. (iii) Ca/K selectivity was greater in negatively charged membranes but did not also depend on the channel amplitude for the same BLM. Cation-anionic selectivity was identical for all studied channels.

The ionic channels formed by cholera toxin in planar bilayer lipid membranes are entirely attributable to its B-subunit.

The interaction of cholera toxin with planar bilayer lipid membranes (BLM) at low pH results in the formation of ionic channels, the conductance of which can be directly measured in voltage-clamp experiments. It is found that the B-subunit of cholera toxin (CT-B) also is able to induce ionic channels in BLM whereas the A-subunit is not able to do it. The increase of pH inhibited the channel-forming activity of CT-B. The investigation of pH-dependences of both the conductance and the cation-anion selectivity of the CT-B channel allowed us to suggest that the water pore of this channel is confined to the B-subunit of cholera toxin. The effective diameter of the CT-B channels water pores was directly measured in BLM and is equal to 2.1 +/- 0.2 nm. The channels formed by whole toxin and its B-subunit exhibit voltage-dependent activity. We believe these channels are relevant to the mode of action of cholera toxin and especially to the endosomal pathway of the A-subunit into cells.

The mode of action of Vibrio cholerae cytolysin. The influences on both erythrocytes and planar lipid bilayers.

The interaction with erythrocytes of cholera cytolysin (CC) obtained from a non-01 Vibrio cholerae strain results in the osmotic rupture of target cells upon formation by CC of the waterfilled pores in their membranes. The aggregation of several toxin monomers is required for the formation of one CC channel with a radius of 0.9-1.0 nm. The investigations using planar bilayer lipid membranes suggest that the CC-induced pore is an interprotein anion selective channel carrying a fixed positive charge. The role of the charge was supported by the influence of pH on the selectivity, single conductance and voltage gating of the CC channels. The ability of the CC to modify both model and natural membranes has a maximum at pH 6.0-7.0. It was found that CC channels insert into the membrane asymmetrically. The effect of proteolytic treatment of the channel by papain also indicates that the two entrances of the channel protrude from the plane of the membrane into the solution for different distances. It is proposed that the biological effects of the non-01 V. cholera cytolysin are based on its channel-forming activity.

Effects of monoclonal antibodies on alpha-staphylotoxin action against erythrocytes and model phospholipid membranes.

It was found that one of twenty tested monoclonal antibodies (MABs) existed which drastically enhanced ability of Staphylococcus aureus alpha-toxin (ST) to both lysis of human erythrocytes and increase of planar phospholipid bilayer conductance more than 10 and 1,000 times respectively. Other 19 MABs possessed only neutralized effect. The activation could only be observed if the activating MAB (AMAB) interacted with ST in solution but not in membrane. The one molecule of AMAB was able to activate approximately 2-4 molecules of ST. It was assumed that this activation was a result of the AMAB-induced transition of ST from a hydrophilic to an amphiphilic form. The activation could not be observed when the activity of AMAB/ST mixtures was tested on highly sensitive rabbit erythrocytes. All the tested MABs (including AMAB) were able to inhibit the ST-induced lysis of rabbit erythrocytes. The activating effects of AMAB on ST action in BLM and in human erythrocytes as well as their inhibiting influence on the ability of toxin to cause a lysis of rabbit erythrocytes indicate the presence of an ST-specific receptor on the membrane of rabbit erythrocytes.

Lumen geometry of ion channels formed by Vibrio cholerae EL Tor cytolysin elucidated by nonelectrolyte exclusion.

Vibrio cholerae EL Tor cytolysin, a water-soluble protein with a molecular mass of 63 kDa, forms small pores in target cell membranes. In this communication, planar lipid bilayers under voltage clamp conditions were used to investigate the geometric properties of the pores. It was established that all cytolysin channels were inserted into membranes with the same orientation. Sharp asymmetry in the I-V curve of fully open cytolysin channels persisting at high electrolyte concentrations indicated asymmetry in the geometry of the channel lumen. Using the nonelectrolyte exclusion method, evidence was obtained that the cis opening of the channel had a larger diameter (< or = 1.9 nm) than the trans opening (< or = 1.6 nm). The channel lumen appeared constricted, with a diameter of < or = 1.2 nm. Cup-shaped lumen geometry was deduced for both channel openings, which appeared to be connected to each other via a central narrow part. The latter contributed significantly to the total electrical resistance and determined the discontinuous character of channel filling with nonelectrolytes. Comparisons of the properties of pores formed by cytolysins of two V. cholerae biotypes (EL Tor and non-O1) indicated that the two ion channels possessed a similar geometry.

Influence of plant terpenoids on the permeability of mitochondria and lipid bilayers.

Five sesquiterpene alcohol esters of the carotane series, from plants of the genus Ferula, were investigated with regard to their capacity to modify the ion permeability of both planar lipid bilayers and mitochondria. These compounds are subdivided into two structural groups that differ in their effects on membrane permeability. Complex esters of sesquiterpene alcohols with aliphatic acids, which constituted the first group (lapidin and lapiferin), do not possess ionophoric properties. The second group comprised complex esters of sesquiterpene alcohols with aromatic acids (ferutinin, tenuferidin and ferutidin), all of which increase cation permeability of lipid bilayers and mitochondria in a dose-dependent manner. A pronounced selectivity of the terpenoid-modified membranes for divalent cations versus monovalent cations was found. Evidence of a carrier mechanism for terpenoid-induced ion transport is demonstrated. A tentative complex composed of a divalent cation with two molecules of membrane-active terpenoid is proposed.

The hinge portion of the S. aureus alpha-toxin crosses the lipid bilayer and is part of the trans-mouth of the channel.

This paper compares the functional properties of ion channels formed in planar lipid membranes by the wild and mutant Staphylococcus aureus alpha-toxin. It was shown that replacement of the amino acid Gly at position 130 by Cys in the primary structure of the toxin decreases the single-channel conductance with a concomitant decrease in the pH at which the channel becomes unable to discriminate between Cl- and K+ ions. The mutation also induced an increase in the asymmetry in the current-voltage relationship of the channel. The results of our experiments suggest that the trans-mouth of the channel is responsible for all the observed changes in channel properties. It was assumed that this entrance is built by the glycine-rich hinge portion of the toxin and is situated close to the surface of monolayer facing the trans-compartment.

Non stochastic distribution of single channels in planar lipid bilayers.

The selectivity of the planar lipid bilayers modified by two channel-forming proteins (alpha-toxin S. aureus and colicin Ia) was examined. It was established that in all cases the value of zero current potential depended on the amount of open ion channels and increased with the number of channels (from one to about 5-7). These facts point out both the interactions among ion channels and their non stochastic distribution on the membrane surface.

Heparin influence on alpha-staphylotoxin formed channel.

The effects of heparin on ion channels formed by Staphylococcus aureus alpha-toxin (ST channel) in lipid bilayers were studied under voltage clamp conditions. Heparin concentrations as small as 100 pM induced a sharp dose-dependent increase in channel voltage sensitivity. This was only observed when heparin was added to the negative-potential side of lipid bilayers in the presence of divalent cations. Divalent cations differ in their efficiency: Zn2+>Ca2+>Mg2+. The apparent positive gating charge increased 2-3-fold with heparin addition as well as with acidification of the bathing solution. 'Free' carboxyl groups and carboxyl groups in ion pairs of the protein moiety are hypothesized to interact with sulfated groups of heparin through divalent cation bridges. The cis mouth of the channel (that protrudes beyond the membrane plane on the side of ST addition and to which voltage was applied) is less sensitive to heparin than the trans-mouth. It is suggested that charged residues which interact with heparin at the cis mouth of ST channels and which contribute to the effective gating charge at negative voltage may be physically different from those at the trans mouth and at positive voltage.

Cholesterol-dependent hemolytic activity of Passiflora quadrangularis leaves.

Plants used in traditional medicine are rich sources of hemolysins and cytolysins, which are potential bactericidal and anticancer drugs. The present study demonstrates for the first time the presence of a hemolysin in the leaves of Passiflora quadrangularis L. This hemolysin is heat stable, resistant to trypsin treatment, has the capacity to froth, and acts very rapidly. The hemolysin activity is dose-dependent, with a slope greater than 1 in a double-logarithmic plot. Polyethylene glycols of high molecular weight were able to reduce the rate of hemolysis, while liposomes containing cholesterol completely inhibited it. In contrast, liposomes containing phosphatidylcholine were ineffective. The Passiflora hemolysin markedly increased the conductance of planar lipid bilayers containing cholesterol but was ineffective in cholesterol-free bilayers. Successive extraction of the crude hemolysin with n-hexane, chloroform, ethyl acetate, and n-butanol resulted in a 10-fold purification, with the hemolytic activity being recovered in the n-butanol fraction. The data suggest that membrane cholesterol is the primary target for this hemolysin and that several hemolysin molecules form a large transmembrane water pore. The properties of the Passiflora hemolysin, such as its frothing ability, positive color reaction with vanillin, selective extraction with n-butanol, HPLC profile, cholesterol-dependent membrane susceptibility, formation of a stable complex with cholesterol, and rapid erythrocyte lysis kinetics indicate that it is probably a saponin.

Is the mammalian porin channel, VDAC, a perfect cylinder in the high conductance state?

The mammalian porin channel (VDAC, porin-31BM) was reconstituted in planar lipid bilayers under voltage clamp conditions. The radii of both entrances of the channel were examined using a method that consisted in filling the channel with different non-electrolytes through its cis or trans entrances while recording single channel conductances. As a result it was found that the geometry of channels formed by porin-31BM could not be approximated by a perfectly cylindrical pore. In fact there is an asymmetry in the geometry of the channel: the diameters of the cis and trans entrances were estimated to be approximately 2 nm and approximately 4 nm respectively.

The diameter of water pores formed by colicin Ia in planar lipid bilayers.

The effective size of colicin Ia channel was tested by a recently described method (FEMS, Microbiology and Immunology (1992). 105: 93-100) in which the nonelectrolyte molecules with different hydrodynamic diameters (0.52 to 5.0 nm) were used as molecular tools. We have shown that despite low conductance (55-105 pS at 1.5 M KCl, pH 7.0) the ion channels formed by colicin Ia have a fairly large water pore diameter equal to 1.66-1.88 nm. The results are discussed in terms of an energetic barrier for ions passing into the channel lumen.

Diameter of the mammalian porin channel in open and "closed" states: direct measurement at the single channel level in planar lipid bilayer.

Porin isolated from bovine skeletal muscle was reconstitute in planar lipid bilayers under voltage clamp conditions. A set of non-electrolytes were used as molecular probes for determining the pore diameter. The maximal diameter of the open channel was estimated to be 3.02 +/- 0.26 nm. As observed for other porin channels, a large transmembrane potential drove the channel into a "closed" state. The channel transition to the low conductance (closed) state was followed by a decrease in the maximal diameter of the channel to 2.4 +/- 0.08 nm.

Ion transport through channels formed in lipid bilayers by Staphylococcus aureus alpha-toxin.

Staphylotoxin channel appears to be predominantly anion-selective with non-linear and asymmetric current-voltage characteristics (CVC) at neutral pH. Increased salt concentrations induce linearity and asymmetry of CVC and loss of selectivity. At lower pH both the channel conductivity and anion selectivity increase. Higher temperatures raise the channel conductivity in parallel with the changes in electrical conductivity of the salt solution, but do not change selectivity. Experimental dependences are described obtained by approximation of electrical diffusion and considering the interactions of penetrating ions with fixed charges at the entrances and the channel energy profile.

The structure of Staphylococcus aureus alpha-toxin-induced ionic channel.

Polyethylene glycols (PEG) with molecular weight less than or equal to 3000 were shown to effectively protect human erythrocytes from osmotic lysis induced by alpha-staphylotoxin (ST). PEG with MW less than 3000 do not change the conductivity of ion channels induced by ST in bilayer lipid membranes (BLM). Changing the bilayer from a pure phosphatidylcholine (PC) to a negatively charged phosphatidylserine (PS) film results in an asymmetry of the current-voltage characteristics. This is evidenced by the asymmetrical position of the ST-channel pore in bilayer membranes. The results obtained allow to conclude that the ST-channel is an interprotein pore filled with water (with an inner diameter of 2.5-3 nm and a length of approximately 10 nm). It is composed of six molecules of alpha-toxin from Staphylococcus aureus. The ST-channel incorporates into a membrane with only one mouth in contact with the polar lipid heads and the other one protruding 4.5-5 nm from the bilayer plane in water solution.

The interaction of amphotericin B with cell membrane of rat thymocytes.

Amphotericin B (AB) at micromolar concentrations increases cell membrane permeability and induced swelling of rat thymus lymphocytes. Potassium efflux is a precondition for AB to induce swelling of the cells. The rate constants for potassium loss and volume changes were proportional to the 1.24th and the 2nd power of the antibiotic concentration respectively. The reflection coefficients for nonelectrolytes with different hydrodynamic radii were determined, and the equivalent radius of the amphotericin pore in the thymocyte cell membrane was estimated to be 4.1 +/- 0.3 A at polyene concentrations varying between 2.5 mumol/l and 80 mumol/l. It is suggested that channel formation by AB in cell membranes is actually able to modulate immune responses.

Relation between ionic channel conductance and conductivity of media containing different nonelectrolytes. A novel method of pore size determination.

The effects of nonelectrolytes on conductivity and viscosity of KCl solutions as well as on ion channel conductance were studied. Mobility of ions in solutions were found to solely depend on percent concentration (w/w) of the nonelectrolytes added and to be effectively independent on their chemical nature (sugars or polyglycols) and molecular size. Proportional changes in both the ion channel conductance and the conductivity of bulk solution induced by low m. w. nonelectrolytes may be used as a criterion of diffusion mechanism of ion transport through channels. The slope of the dependence of ion channel conductance on conductivity of bulk solution containing different concentrations of nonelectrolytes is a good measure of channel permeability for nonelectrolyte. A new method of pore size determination is introduced. Results of practical application of this simple method to three types of ion channels (formed by alpha-latrotoxin, staphylococcal alpha-toxin and its N-terminal fragment) are shown. The advantages and disadvantages of the method are discussed.

Memory is a property of an ion channels pool: ion channels formed by Staphylococcus aureus alpha-toxin.

The short-time depolarization effects on the integral conductance induced by S. aureus alpha-toxin (ST) in planar lipid bilayer membranes has been studied. Ion channels formed by ST were found to have several potential-induced nonconductance (closed) states. The transitions of ion channels between the states are only through one conductance state. The transition of ST-channels from closed to open state is induced by membrane depolarization. The amplitude current after a series of voltage pulses is a function of pulse number, and is effectively independent of the time interval between the neighbouring pulses. Therefore, a membrane which contains a pool of ion channels "remembers" its previous existence. A simple model can be used to explain this phenomenon.

Channel-sizing experiments in multichannel bilayers.

The possibility of obtaining information about the radius of high and low conductance states of channels in multichannel membranes was tested experimentally. In spite of the interference of non-electrolytes on the numbers of channels that appeared in the membrane, the non-electrolyte-exclusion method was successfully adapted to multichannel bilayers to estimate the radius of the larger opening of the low conductance state of the channel induced by Staphylococcus aureus alpha-toxin. At the pH used, the channel transition to a low conductance state was accompanied by a decrease of the opening radius from 1.3 +/- 0.2 nm to 0.9 +/- 0.1 nm. The determination criteria for maximum size of a channel opening when using the non-electrolyte exclusion method is discussed.

Ion channels in volume regulation of clonal kidney cells.

OBJECTIVES: Clonal kidney cells (Vero cells) are extensively utilized in the manufacture of biological preparations for disease diagnostics and therapeutics and also in preparation of vaccines. In all cells, regulation of volume is an essential function coupled to a variety of physiological processes and is a topic of interest. The objective here was to investigate involvement of ion channels in the process of volume regulation of Vero cells. METHODS: Involvement of ion channels in cell volume regulation was studied using video-microscopy and flow cytometry. Pharmacologically unaltered cells of different sizes, which are presumably at different phases of the cell cycle, were used. RESULTS: Ion transport inhibitors altered all phases of regulatory volume decrease (RVD) of Vero cells, rate of initial cell swelling, V(max) and volume recovery. Effects were dependent on type of inhibitor and on cell size (cell cycle phase). Participation of aquaporins in RVD was suggested. Inhibitors decelerated growth, arresting Vero cells at the G(0) /G(1) phase boundary. Electrophysiological study confirmed presence of volume-activated Cl(-) channels and K(+) channels in plasmatic membranes of the cells. CONCLUSION: Vero cells of all sizes maintained the ability to recover from osmotic swelling. Activity of ion channels was one of the key factors that controlled volume regulation and proliferation of the cells.