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.

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.

SummaryStaphylococcal 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.

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.

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.

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 eis 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 eis and trans entrances were estimated to be ~ 2 nm and ~4 nm respectively.

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.