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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., (© 2010 Blackwell Publishing Ltd.)

The rate of transbilayer movement (flip-flop) of cholesterol was estimated using planar bilayers with defined initial asymmetry, formed by the opposing monolayers technique. Vibrio cholerae cytolysin (VCC) was utilized as a molecular tool for measuring the cholesterol concentration in the cis leaflet of asymmetric bilayers. To quantify cholesterol flip-flop in planar lipid bilayers, a mathematical model was developed. It considers both the lateral diffusion rate of cholesterol within each monolayer and the flip-flop rate. The difference in initial and steady-state cholesterol contents in bilayer leaflets was used as a start point. Assuming the lateral diffusion coefficient to be of 1 x 10(-8) cm(2) s(-1), the characteristic time of cholesterol flip-flop at 25 +/- 2 degrees C was estimated as <10 s.

Vibrio cholerae cytolysin (VCC) forms oligomeric transmembrane pores in cholesterol-rich membranes. To better understand this process, we used planar bilayer membranes. In symmetric membranes, the rate of the channel formation by VCC has a superlinear dependency on the cholesterol membrane fraction. Thus, more than one cholesterol molecule can facilitate VCC-pore formation. In asymmetric membranes, the rate of pore formation is limited by the leaflet with the lower cholesterol content. Methyl-beta-cyclodextrin, which removes cholesterol from membranes, rapidly inhibits VCC pore formation, even when it is added to the side opposite that of VCC addition. The results suggest that cholesterol in both membrane leaflets aid VCC-pore formation and that either leaflet can function as a kinetic bottleneck with respect to the rate of pore-formation.

While conformational flexibility of proteins is widely recognized as one of their functionally crucial features and enjoys proper attention for this reason, their elastic properties are rarely discussed. In ion channel studies, where the voltage-induced or ligand-induced conformational transitions, gating, are the leading topic of research, the elastic structural deformation by the applied electric field has never been addressed at all. Here we examine elasticity using a model channel of known crystal structure-Staphylococcus aureus alpha-hemolysin. Working with single channels reconstituted into planar lipid bilayers, we first show that their ionic conductance is asymmetric with voltage even at the highest salt concentration used where the static charges in the channel interior are maximally shielded. Second, choosing 18-crown-6 as a molecular probe whose size is close to the size of the narrowest part of the alpha-hemolysin pore, we analyze the blockage of the channel by the crown/K(+) complex. Analysis of the blockage within the framework of the Woodhull model in its generalized form demonstrates that the model is able to correctly describe the crown effect only if the parameters of the model are considered to be voltage-dependent. Specifically, one has to include either a voltage-dependent barrier for crown release to the cis side of the channel or voltage-dependent interactions between the binding site and the crown. We suggest that the voltage sensitivity of both the ionic conductance of the channel seen at the highest salt concentration and its blockage by the crown reflects a field-induced deformation of the pore.

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.

Closing linear poly(ethylene glycol) (PEG) into a circular "crown" dramatically changes its dynamics in the alpha-hemolysin channel. In the electrically neutral crown ether (C2H4O)6, six ethylene oxide monomers are linked into a circle that gives the molecule ion-complexing capacity and increases its rigidity. As with linear PEG, addition of the crown to the membrane-bathing solution decreases the ionic conductance of the channel and generates additional conductance noise. However, in contrast to linear PEG, both the conductance reduction (reporting on crown partitioning into the channel pore) and the noise (reporting on crown dynamics in the pore) now depend on voltage strongly and nonmonotonically. Within the whole frequency range accessible in channel reconstitution experiments, the noise power spectrum is "white", showing that crown exchange between the channel and the bulk solution is fast. Analyzing these data in the framework of a Markovian two-state model, we are able to characterize the process quantitatively. We show that the lifetime of the crown in the channel reaches its maximum (a few microseconds) at about the same voltage (approximately 100 mV, negative from the side of protein addition) where the crown's reduction of the channel conductance is most pronounced. Our interpretation is that, because of its rigidity, the crown feels an effective steric barrier in the narrowest part of the channel pore. This barrier together with crown-ion complexing and resultant interaction with the applied field leads to behavior usually associated with voltage-dependent binding in the channel pore.

To probe the volume changes of the voltage-dependent anion-selective channel (VDAC), the nonelectrolyte exclusion technique was taken because it is one of the few existing methods that may define quite accurately the rough geometry of lumen of ion channels (in membranes) for which there is no structural data.Here, we corroborate the data from our previous study [FEBS Lett. 416 (1997) 187] that the gross structural features of VDAC in its highest conductance state are asymmetric with respect to the plane of the membrane, and state that this asymmetry is not dependent on sign of voltage applied. Hence, the plasticity of VDAC does not play a role in the determination of lumen geometry at this state and the asymmetry is an internal property of the channel. We also show that the apparent diameter of the cis segment of the pore decreases slightly from 2 to 1.8 nm when the channel's conductance decreases from its high to low state. However, the trans funnel segment undergoes a more marked change in polymer accessible volume. Specifically, its larger diameter decreases from approximately 4 to 2.4 nm. Supposing the channel's total length is 4.6 nm, the apparent change in channel volume during this transition is estimated to be about 10 nm(3), i.e. about 40% of the channel's volume in the high conductance state.

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.

Staphylococcal alpha-toxin forms homo-oligomeric channels in lipid bilayers and cell membranes. Here, we report that electrophysiological monitoring of single-channel function using a derivatized cysteine substitution mutant allows accurate determination of the subunit stoichiometry of the oligomer in situ. The electrophysiological phenotype of channels formed in planar lipid bilayers with the cysteine replacement mutant I7C is equal to that of the wild type. When pores were formed with I7C, alterations of several channel properties were observed upon modification with SH reagents. Decreases in conductance then occurred that were seen only as negative voltage was applied. At the level of single channels, these were manifest as stepwise changes in conductance, each step most probably reflecting modification of a single SH group within the oligomer. Because seven steps were observed, the functional channel formed by alpha-toxin in planar lipid membranes is a heptamer.

Asymmetrical (one-sided) application of penetrating water-soluble polymers, polyethylene glycols (PEGs), to a well-defined channel formed by Staphylococcus aureus alpha-toxin is shown to probe channel pore geometry in more detail than their symmetrical (two-sided) application. Polymers added to the cis side of the planar lipid membrane (the side of protein addition) affect channel conductance differently than polymers added to the trans side. Because a satisfactory theory quantitatively describing PEG partitioning into a channel pore does not exist, we apply the simple empirical rules proposed previously (, J. Membr. Biol. 161:83-92) to gauge the size of pore openings as well as the size and position of constrictions along the pore axis. We estimate the radii of the two openings of the channel to be practically identical and equal to 1. 2-1.3 nm. Two apparent constrictions with radii of approximately 0. 9 nm and approximately 0.6-0.7 nm are inferred to be present in the channel lumen, the larger one being closer to the cis side. These structural findings agree well with crystallographic data on the channel structure (, Science. 274:1859-1866) and verify the practicality of polymer probing. The general features of PEG partitioning are examined using available theoretical considerations, assuming there is no attraction between PEG and the channel lumen. It is shown that the sharp dependence of the partition coefficient on polymer molecular weight found under both symmetrical and asymmetrical polymer application can be rationalized within a "hard sphere nonideal solution model." This finding is rather surprising because PEG forms highly flexible coils in water with a Kuhn length of only several Angstroms.

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.

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.

This paper describes a new approach to evaluate the inner structure (including a main constriction and its localization) of the water lumen of an ion channel. The method is based on the determination of channel filling by different nonelectrolyte molecules through each side of an ion channel. The method has two characteristic features that make its use attractive: (i) the possibility to ascertain the existence, localization and size of a narrow part inside an ion channel water lumen and (ii) the chances to determine the maximal size of both entrances of an ion channel and to obtain additional information about the geometry of its water lumen at the same time. Determinations were made on colicin Ia ion channels inserted into planar lipid bilayers. This channel was chosen because there is an apparent contradiction between its low single channel conductance and the large diameter of its water lumen. Our results show that the water lumen of the colicin Ia channel has a funnel-like structure with a small trans-entrance, with a diameter of about 1.0 nm, and a large cis-entrance, with a diameter of approximately 1.8 nm. A constriction with a diameter of approximately 0.7 nm is shown to be located close to the trans-entrance of the channel. The method can also be applied to patch clamp studies of single ion channels.

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.

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.

Staphylococcal alpha-toxin is a single-chain protein with a molecular mass of 33.2 kDa, which can form large water-filled pores both in lipid bilayers and in erythrocyte membranes. Limited proteolysis of the purified toxin with proteinase K led to time-dependent changes of all the functional features of the channels formed by the toxin. Single-channel conductance in planar bilayers was decreased about threefold. The anion selectivity of the channel was replaced with cation selectivity and the asymmetry in the current-voltage relationship of the channel became more pronounced. At the same time the nicked toxin kept its full ability to form ion channels in lipid bilayers, although it lost a considerable part of its hemolytic activity. In planar bilayers and in erythrocyte membranes, the proteolytically nicked toxin actually formed channels with a slightly smaller diameter (approximately 1.2 times) than that formed by the native toxin. This decrease was not marked enough to explain changes in the biological effects of the nicked toxin. The change in channel selectivity induced by the cleavage is considered to be the major determinant of the changes in the biological effects of the nicked toxin.

Replacement of an amino acid residue at position 130 -Gly by Cys- in the primary structure of Staphylococcus aureus alpha-toxin decreases the single-channel conductance induced by the toxin in planar lipid bilayers. Concomitantly, the pH value at which the channel becomes unable to discriminate between Cl- and K+ ions is also decreased. By contrast, the pH dependence of the efficiency of the mutant toxin to form ion channels in lipid bilayers was unchanged (maximum efficiency at pH 5.5-6.0). The asymmetry and nonlinearity of the current-voltage characteristics of the channel were increased by the point mutation but the diameter of the water pore induced by the mutant toxin, evaluated in lipid bilayers and in erythrocyte membranes, was found to be indistinguishable from that formed by wild-type toxin and equal to 2.4-2.6 nm. Alterations at the "trans mouth" were found to be responsible for all observed changes of the channel properties. This mouth is situated close to the surface of the second leaflet of a bilayer lipid membrane. The data obtained allows us to propose that the region around residue 130 in fact determines the main features of the ST-channel and takes part in the formation of the trans entrance of the channel.

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.

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.

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.