Properties of ionic channels formed by the antibiotic syringomycin E in lipid bilayers: dependence on the electrolyte concentration in the bathing solution.

Using the planar lipid bilayer technique, organization of ionic channels formed by the lipodepsipeptide antibiotic syringomycin E applied to one (cis) side of a lipid bilayer was studied. Low concentrations of NaCl (0.01-0.1 M) induced the opening and closing of two types of channels - "small" and "large". The large channels had single channel conductances approximately six times greater than those of the small channels. An increase in the NaCl concentration (0.6-1.0 M) decreased almost completely the chance to reveal the large channels. Although the syringomycin channels exhibited the anion selectivity within the entire range of NaCl concentrations in the bathing solutions (from 0.001 to 1.0 M) whereas the concentration gradients across the bilayers were 2 and 4, the transfer numbers for Cl-decreased with an increase in the mean NaCl concentration (from 0.83 for 0.005 M to 0.70 for 0.5 M). Moreover, at each mean value of NaCl concentration, all conductance levels had the same ion selectivity (identical reversal potential). These results suggest that at low NaCl concentrations the large channels are clusters of small channels which synchronously open and close, while at high electrolyte concentrations the screening of the charged groups responsible for channel interactions prevents the cluster formation. A new theoretical approach for the estimation of the channel radius and the number of elementary charges located at its inner surface (based on the experimental curve of the dependence of transfer number on the NaCl concentration) was developed. Based on this theoretical approach, the channel radius equal to 1 nm and one elementary charge located at its inner surface were obtained.     

Properties of Voltage-gated Ion Channels Formed by Syringomycin E in Planar Lipid Bilayers

Using the planar lipid bilayer technique we demonstrate that the lipodepsipeptide antibiotic, syringomycin E, forms voltage-sensitive ion channels of weak anion selectivity. The formation of channels in bilayers made from dioleoylglycerophosphatidylserine doped with syringomycin E at one side (1–40 μg/ml) was greatly affected by cis-positive voltage. A change of voltage from a positive to a negative value resulted in (i) an abrupt increase in the single channel conductance (the rate of increase was voltage dependent) simultaneous with (ii) a closing of these channels and an exponential decrease in macroscopic conductance over time. The strong voltage dependence of multichannel steady state conductance, the single channel conductance, the rate of opening of channels at positive voltages and closing them at negative voltages, as well as the observed abrupt increase of single channel conductance after voltage sign reversal suggest that the change of the transmembrane field induces a significant rearrangement of syringomycin E channels, including a change in the spacing of charged groups that function as voltage sensors. The conductance induced by syringomycin E increased with the sixth power of syringomycin E concentration suggesting that at least six monomers are required for channel formation. Received: 3 April 1995/Revised: 24 August 1995     

Conductance of phytotoxin channels in the presence of large organic ions

In this study we evaluated the effect of the size of penetrating anions on properties of the channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes. Conductance and the mean lifetime of SRE channels were measured in 0.4 M solutions of sodium chloride, aspartate, and gluconate. A comparison of results of conductometric and electrophysiological measurements has shown the following: (1) the ratio of mobilities of aspartate anions in the channel and in the aqueous solution is five times lower than that of chlorine anions and (2) the conductance of channels in the presence of sodium gluconate is due mainly to cations. The obtained results indicate the binding of penetrating anions to the selective filter of the SRE pore. The radius of the SRE channel selective filter has been established (r ∼ 0.3 nm) and its localization in the cis-mouth of the pore has been found.     

Kinetic parameters of single ion channels and stationary conductivities of phytotoxin modified lipid bilayers

As shown earlier, phytotoxins produced by Pseudomonas syringae pv. syringae form ion channels of "small" and "large" conductance when incorporated into planar lipid membranes. The multilevel conductance is due to cluster organization of the channels (Kaulin et al., 1998; Gurnev et al., 2002). In this study the kinetic parameters of syringomycin E (SRE) and syringostatin A (SSA) channels in negatively charged bilayer lipid membranes were estimated. The average time of open state of the small channels (t(s)(open)) did not depend on transmembrane voltage (in the range of +/- 200 mV). The channel characteristics differed between two phytotoxins: the t(s)(open) for the SRE-channels was much larger than that for SSA-channels. An energetic diagram with two non-conducting states illustrating the formation of the small channel is proposed to explain the voltage independence of the kinetic parameters. The probability for synchronous functioning of small channels with SSA was higher than that with SRE. To analyse the role of the clusters in the biological activities of SRE and SSA, we estimated the cluster contribution to a net transmembrane currents to be 60 and 90%, respectively.     

Kinetics of opening and closure of syringomycin E channels formed in lipid bilayers

A cyclic lipodepsipeptide, syringomycin E (SME), incorporated into planar lipid membranes forms two types of channels ("small" and "large") different in their conductance by approximately a factor of six (Biophys. J. 74:2918-2925 (1998)). We analysed the dynamics of the SME-induced transmembrane current under voltage-clamp conditions to clarify the mechanisms of formation of these channels. The voltage-dependent opening/closure of SME channels in lipid bilayers are interpreted in terms of transitions between three types of clusters including 6-7 SME molecules and some lipid molecules. The initial cluster, the precursor of the other two, was in equilibrium with SME monomer molecules at the membrane surface. The other two types of clusters (State 1 and State 2) were formed from the precursor and also during their interconversions (the consecutive-parallel mechanism of transitions). State 1 was a non-conducting state in equilibrium with small channels, which partially determined the ionic conductance of lipid bilayers modified by SME. State 2 corresponded to large SME channels, major contributors to the conductance of a bilayer. The results of the theoretical analysis based on the chemical kinetics concepts were consistent with experimental observations. Such properties of the SME-induced channels as cluster organisation, voltage dependence and the existence of a non-conducting state are all features shared by many ion channels in biological membranes. This makes it possible to use SME channels as a model to study naturally occurring ion channels.     

Syringomycin E channel: a lipidic pore stabilized by lipopeptide?

Highly reproducible ion channels of the lipopeptide antibiotic syringomycin E demonstrate unprecedented involvement of the host bilayer lipids. We find that in addition to a pronounced influence of lipid species on the open-channel ionic conductance, the membrane lipids play a crucial role in channel gating. The effective gating charge, which characterizes sensitivity of the conformational equilibrium of the syringomycin E channels to the transmembrane voltage, is modified by the lipid charge and lipid dipolar moment. We show that the type of host lipid determines not only the absolute value but also the sign of the gating charge. With negatively charged bilayers, the gating charge sign inverts with increased salt concentration or decreased pH. We also demonstrate that the replacement of lamellar lipid by nonlamellar with the negative spontaneous curvature inhibits channel formation. These observations suggest that the asymmetric channel directly incorporates lipids. The charges and dipoles resulting from the structural inclusion of lipids are important determinants of the overall energetics that underlies channel gating. We conclude that the syringomycin E channel may serve as a biophysical model to link studies of ion channels with those of lipidic pores in membrane fusion.     

Activity of toxins produced by Pseudomonas syringae pv. syringae in model and cell membranes

We studied effects of toxins produced by a bacterium Pseudomonas syringae pv. syringae on the conductance of bilayer lipid membranes (BLM). The used toxins were as follows: syringopeptin 22A (SP22A), syringomycin E (SPE), syringostatin A (SSA), syringotoxin B (STB), and methylated syringomycin E (CH3-SRE). All toxins demonstrated channel-forming activity. The threshold sequence for toxin activity was SP22A > SRE approximately equal to SSA > STB > CH3-SRE, and this sequence was independent of lipid membrane composition, and NaCl concentration (pH 6) in the membrane bathing solution (in the range of 0.1-1.0 M). This sequence correlated with relative bioactivities of toxins. In addition, SRE demonstrated a more potent antifungal activity than CH3-SRE. These findings suggest that ion channel formation may underlie the bioactivities of the above toxins. The properties of single ion channels formed by the toxins in BLMs were found to be similar, which points to the similarity in the channel structures. In negatively charged membranes, bathed with diluted electrolyte solutions (0.1 M NaCl), the channels were seen to open with positive transmembrane potentials (V) (from the side of toxin addition), and close with negative potentials. In uncharged membranes the opposite response to a voltage sign was observed. Increasing the NaCl concentration up to 1 M unified the voltage sensitivity of channels in charged and uncharged membranes: channels opened with negative V, and closed with positive V. With all systems, the voltage current curves of single channels were similarly superlinear in the applied voltage and asymmetric in its sign. It was found that the single channel conductance of STB and SSA was higher than that of other toxin channels. All the toxins formed at least two types of ion channels that were multiple by a factor of either 6 or 4 in their conductance. The results are discussed in terms of the structural features of toxin molecules.     

Glutamate substitution in repeat IV alters divalent and monovalent cation permeation in the heart Ca2+ channel.

In voltage-gated ion channels, residues responsible for ion selectivity were identified in the pore-lining SS1-SS2 segments. Negatively charged glutamate residues (E393, E736, E1145, and E1446) found in each of the four repeats of the alpha 1C subunit were identified as the major determinant of selectivity in Ca2+ channels. Neutralization of glutamate residues by glutamine in repeat I (E393Q), repeat III (E1145Q), and repeat IV (E1446Q) decreased the channel affinity for calcium ions 10-fold from the wild-type channel. In contrast, neutralization of glutamate residues in repeat II failed to significantly alter Ca2+ affinity. Likewise, mutation of neighboring residues in E1149K and D1450N did not affect the channel affinity, further supporting the unique role of glutamate residues E1145 in repeat III and E1446 in repeat IV in determining Ca2+ selectivity. Conservative mutations E1145D and E1446D preserved high-affinity Ca2+ binding, which suggests that the interaction between Ca2+ and the pore ligand sites is predominantly electrostatic and involves charge neutralization. Mutational analysis of E1446 showed additionally that polar residues could achieve higher Ca2+ affinity than small hydrophobic residues could. The role of high-affinity calcium binding sites in channel permeation was investigated at the single-channel level. Neutralization of glutamate residue in repeats I, II, and III did not affect single-channel properties measured with 115 mM BaCl2. However, mutation of the high-affinity binding site E1446 was found to significantly affect the single-channel conductance for Ba2+ and Li+, providing strong evidence that E1446 is located in the narrow region of the channel outer mouth. Side-chain substitutions at 1446 in repeat IV were used to probe the nature of divalent cation-ligand interaction and monovalent cation-ligand interaction in the calcium channel pore. Monovalent permeation was found to be inversely proportional to the volume of the side chain at position 1446, with small neutral residues such as alanine and glycine producing higher Li+ currents than the wild-type channel. This suggests that steric hindrance is a major determinant for monovalent cation conductance. Divalent permeation was more complex. Ba2+ single-channel conductance decreased when small neutral residues such as glycine were replaced by bulkier ones such as glutamine. However, negatively charged amino acids produced single-channel conductance higher than predicted from the size of their side chain. Hence, negatively charged residues at position 1446 in repeat IV are required for divalent cation permeation.     

Effect of temperature on the formation and inactivation of syringomycin E pores in human red blood cells and bimolecular lipid membranes

The effects of temperature on the formation and inactivation of syringomycin E (SRE) pores were investigated with human red blood cells (RBCs) and lipid bilayer membranes (BLMs). SRE enhanced the RBC membrane permeability of 86Rb and monomeric hemoglobin in a temperature dependent manner. The kinetics of 86Rb and hemoglobin effluxes were measured at different temperatures and pore formation was found to be only slightly affected, while inactivation was strongly influenced by temperature. At 37 degrees C, SRE pore inactivation began 15 min after and at 20 degrees C, 40 min after SRE addition. At 6 degrees C, below the phase transition temperature of the major lipid components of the RBC membrane, no inactivation occurred for as long as 90 min. With BLMs, SRE induced a large current that remained stable at 14 degrees C, but at 23 degrees C it decreased over time while the single channel conductance and dwell time did not change. The results show that the temperature dependent inactivation of SRE pores is due to a decrease in the number of open pores.     

Lipid dependence of the channel properties of a colicin E1-lipid toroidal pore

Colicin E1 belongs to a group of bacteriocins whose cytotoxicity toward Escherichia coli is exerted through formation of ion channels that depolarize the cytoplasmic membrane. The lipid dependence of colicin single-channel conductance demonstrated intimate involvement of lipid in the structure of this channel. The colicin formed "small" conductance 60-picosiemens (pS) channels, with properties similar to those previously characterized, in 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (C20) or thinner membranes, whereas it formed a novel "large" conductance 600-pS state in thicker 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22) bilayers. Both channel states were anion-selective and voltage-gated and displayed a requirement for acidic pH. Lipids having negative spontaneous curvature inhibited the formation of both channels but increased the ratio of open 600 pS to 60 pS conductance states. Different diameters of small and large channels, 12 and 16 A, were determined from the dependence of single-channel conductance on the size of nonelectrolyte solute probes. Colicin-induced lipid "flip-flop" and the decrease in anion selectivity of the channel in the presence of negatively charged lipids implied a significant contribution of lipid to the structure of the channel, most readily described as toroidal organization of lipid and protein to form the channel pore.     

Interaction between filamentous actin and lipid bilayer causes the increase of syringomycin E channel-forming activity

The effect of filamentous (F) actin on the channel-forming activity of syringomycin E (SRE) in negatively charged and uncharged bilayer lipid membranes (BLM) was studied. F-actin did not affect the membrane conductance in the absence of SRE. No changes in SRE-induced membrane conductance were observed when the above agents were added to the same side of BLM. However, the opposite side addition of F-actin and SRE provokes a multiple increase in membrane conductance. The similar voltage dependence of membrane conductance, equal values of single channel conductance and the effective gating charge of the channels upon F-actin action suggests that the actin-dependent increase in BLM conductance may result from an increase in the number of opened SRE-channels. BLM conductance kinetics depends on the sequence of SRE and F-actin addition, suggesting that actin-dependent rise of conductance may be induced by BLM structural changes that follow F-actin adsorption. F-actin exerted similar effect on membrane conductance of both negatively charged and uncharged bilayers, as well as on conductance of BLM with high ionic strength bathing solution, suggesting the major role for hydrophobic interactions in F-actin adsorption on lipid bilayer.     

Transport of large organic ions through syringomycin channels in the membranes containing dipole modifiers

The effect of the membrane dipole potential (Phid) on a conductance and a steady-state number of functioning channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes made from phosphocholine and bathed in 0.4 M solution of sodium salts of aspartate, gluconate and chloride was shown. The magnitude of Phid was varied with the introduction to membrane bathing solutions of phloretin, which reduces the Phid, and RH 421, increasing the Phid. It was established that in all studied systems the increase in the membrane dipole potential cause a decrease in the steady-state number of open channels. In the systems containing sodium salts of aspartate (Asp) or gluconate (Glc), changes in the number of functioning channels are in an order of magnitude smaller than in systems containing sodium chloride. At the same time, the conductance (g) of single SRE-channels on the membranes bathed in NaCI solution increases with the increase in Phid, and in the systems containing NaAsp or NaGlc the conductance of single channels does not depend on the Phid. The latter is due to the lack of cation/anion selectivity of the SRE-channels in these systems. The different channel-forming activity of SRE in the experimental systems is defined by the gating charge of the channel and the partition coefficient of the dipole modifiers between the lipid and aqueous phases.     

Ion selectivity of gram-negative bacterial porins.

Twelve different porins from the gram-negative bacteria Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, and Yersinia pestis were reconstituted into lipid bilayer membranes. Most of the porins, except outer membrane protein P, formed large, water-filled, ion-permeable channels with a single-channel conductance between 1.5 and 6 nS in 1 M KCl. The ions used for probing the pore structure had the same relative mobilities while moving through the porin pore as they did while moving in free solution. Thus the single-channel conductances of the individual porins could be used to estimate the effective channel diameters of these porins, yielding values ranging from 1.0 to 2.0 nm. Zero-current potential measurements in the presence of salt gradients across lipid bilayer membranes containing individual porins gave results that were consistent with the conclusions drawn from the single-channel experiments. For all porins except protein P, the channels exhibited a greater cation selectivity for less mobile anions and a greater anion selectivity for less mobile cations, which again indicated that the ions were moving inside the pores in a fashion similar to their movement in the aqueous phase. Three porins, PhoE and NmpC of E. coli and protein P of P. aeruginosa, formed anion-selective pores. PhoE and NmpC were only weakly anion selective, and their selectivity was dependent on the mobility of the ions. In contrast, cations were unable to enter the selectivity filter of the protein P channel. This resulted in a high anion selectivity for all salts tested in this study. The other porins examined, including all of the known constitutive porins of the four gram-negative bacteria studied, were cation selective with a 3- to 40-fold preference for K+ ions over Cl- ions.     

Size selectivity between gap junction channels composed of different connexins

Gap junction channels are traditionally viewed as large, nonspecific pores connecting cells. Recently the diversity in the connexin family has drawn more attention to their permeability characteristics. Several studies have shown that both size and charge contribute to the permeability of gap junctional channels. We have used a graded series of neutral polyethylene glycol probes (PEGs), which eliminate charge contribution completely, to specifically assess the physical exclusion limits of gap junction channels formed by different connexins. Cx 26, 32 and 37 were expressed in paired Xenopus oocytes to form homotypic gap junctional channels. PEG probes were perfused intracellularly into one side of the oocyte pair. A reversible drop in conductance of the gap juctional channels indicated that the probe was small enough to enter the pore and hinder ion flux. Our data suggest that Cx32 channels have a size cut-off between PEG 400 (11.2 A) and PEG 300 (9.6 A) despite their relatively small single channel conductance (approximately 55 pS). Cx26 channels (approximately 130 pS single channel conductance) have a size exclusion limit around PEG 200 (8.0 A), while Cx37 channels show the most restricted size cut-off between PEG 200 (8.0 A) and TriEG (6.8 A), despite having the largest unitary conductance (approximately 300 pS).     

Reconstitution of highly purified saxitoxin-sensitive Na+-channels into planar lipid bilayers.

Highly purified Na+-channels isolated from rat brain have been reconstituted into virtually solvent-free planar lipid bilayer membranes. Two different types of electrically excitable channels were detected in the absence of any neurotoxins. The activity of both channels was blocked by saxitoxin. The first channel type is highly selective for Na+ over K+ (approximately 10:1), it shows a bursting behavior, a conductance of 25 pS in Na+-Ringer and undergoes continuous opening and closing events for periods of minutes within a defined range of negative membranes voltages. The second channel type has a conductance of 150 pS and a lower selectivity for Na+ and K+ (2.2:1); only a few opening and closing events are observed with this channel after one voltage jump. The latter type of channel is also found with highly purified Na+-channel from Electrophorus electricus electroplax. A qualitative analysis of the physicochemical and pharmacological properties of the high conductance channel has been carried out. Channel properties are affected not only by saxitoxin but also by a scorpion (Centruroides suffusus suffusus) toxin and a sea anemone (Anemonia sulcata) toxin both known to be selective for the Na+-channel. The spontaneous transformation of the large conductance channel type into the small one has been considered; the two channel types may represent the expression of activity of different conformational states of the same protein.     

Asymmetry of syringomycin E channel studied by polymer partitioning

To probe the size of the ion channel formed by Pseudomonas syringae lipodepsipeptide syringomycin E, we use the partial blockage of ion current by penetrating poly(ethylene glycol)s. Earlier experiments with symmetric application of these polymers yielded a radius estimate of approximately 1 nm. Now, motivated by the asymmetric non-ohmic current-voltage curves reported for this channel, we explore its structural asymmetry. We gauge this asymmetry by studying the channel conductance after one-sided addition of differently sized poly(ethylene glycol)s. We find that small polymers added to the cis-side of the membrane (the side of lipodepsipeptide addition) reduce channel conductance much less than do the same polymers added to the trans-side. We interpret our results to suggest that the water-filled pore of the channel is conical with cis- and trans-radii differing by a factor of 2-3 and that the smaller cis-radius is in the 0.25-0.35 nm range. In symmetric, two-sided addition, polymers entering the pore from the larger opening dominate blockage.     

Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore

Ionic selectivity in many cation channels is achieved over a short region of the pore known as the selectivity filter, the molecular determinants of which have been identified in Ca(2+), Na(+), and K(+) channels. However, a filter controlling selectivity among different anions has not previously been identified in any Cl(-) channel. In fact, because Cl(-) channels are only weakly selective among small anions, and because their selectivity has proved so resistant to site-directed mutagenesis, the very existence of a discrete anion selectivity filter has been called into question. Here we show that mutation of a putative pore-lining phenylalanine residue, F337, in the sixth membrane-spanning region of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel, dramatically alters the relative permeabilities of different anions in the channel. Specifically, mutations that reduce the size of the amino acid side chain present at this position virtually abolish the relationship between anion permeability and hydration energy, a relationship that characterizes the anion selectivity not only of wild-type CFTR, but of most classes of Cl(-) channels. These results suggest that the pore of CFTR may indeed contain a specialized region, analogous to the selectivity filter of cation channels, at which discrimination between different permeant anions takes place. Because F337 is adjacent to another amino acid residue, T338, which also affects anion selectivity in CFTR, we suggest that selectivity is predominantly determined over a physically discrete region of the pore located near these important residues.     

Determination of ion permeability through the channels made of porins from the outer membrane ofSalmonella typhimurium in lipid bilayer membranes

Summary The three types of porin (matrix-proteins) fromSalmonella typhimurium with molecular weights of 38,000, 39,000 and 40,000 were reconstituted with lipid bilayer membranes either as a trimer or as an oligomer (complex I). The specific conductance of the membranes increased several orders of magnitude after the addition of the porins into the aqueous phase bathing the membranes. A linear relationship between protein concentration in the aqueous phase and membrane conductance was found. In the case of lower protein concentrations (10^–12 m), the conductance increased in a stepwise fashion with a single conductance increment of 2.3 nS in 1m KCl. For a given salt the conductance increment was found to be largely independent of the particular porin (38 K, 39K or 40 K) and on the state of aggregation, although porin oligomers showed an up to 10 times smaller conductance increase in macroscopic conductance measurements. The conductance pathway has an ohmic current voltage characteristic and a poor selectivity for different alkali ions. Further information on the structure of the pores formed by the different porins fromSalmonella was obtained from the selectivity for various ions. From the permeability of the pore for large ions (Tris⁺, glucosamine⁺, Hepes^–_ a minimum pore diameter of 0.8 nm is estimated. This value is in agreement with the size of the pore as calculated from the conductance data for 1m KCl (1.4 nm for a pore length of 7.5 nm). The pore diameter may well account for the sugar permeability which has been found in reconstituted vesicles. The findings reported here are consistent with the assumption that the different porins form large aqueous channels in the lipid bilayer membranes and that the single condutance unit is a trimer. In addition, it is suggested that one trimer contains only one pore rather than a bundle of pores.     

Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c

The Bcl-2 family of proteins, consisting of anti-apoptotic and pro-apoptotic members, regulates cell death by controlling mitochondrial membrane permeability that is crucial for apoptotic signal transduction. We have recently shown that some of these proteins, such as Bcl-x(L), Bax, and Bak, directly modulate the mitochondrial voltage-dependent anion channel (VDAC) and thus regulate apoptogenic cytochrome c release and potential loss. To elucidate the molecular mechanisms of VDAC regulation by Bcl-2 family proteins, an electrophysiological study was carried out. It was found that VDAC and pro-apoptotic Bax created a large pore, with conductance levels 4- and 10-fold greater than those of the VDAC and Bax channels, respectively. Although the VDAC and Bax channels both show ion selectivity and voltage-dependent modulation of their activity, the VDAC-Bax channel had neither of their properties. Anti-apoptotic Bcl-x(L) and its BH4 oligopeptide completely closed the VDAC, in contrast to the Bax. Cytochrome c passed through a single VDAC-Bax channel but not through the VDAC or Bax channel in a planar lipid bilayer. These data provide direct evidence that VDAC forms a novel large pore together with Bax.     

Cluster organization and pore structure of ion channels formed by beticolin 3, a nonpeptidic fungal toxin

Beticolin 3 (B3) belongs to a family of nonpeptidic phytotoxins produced by the fungus Cercospora beticola, which present a broad spectrum of cytotoxic effects. We report here that, at cytotoxic concentration (10 microM), B3 formed voltage-independent, weakly selective ion channels with multiple conductance levels in planar lipid bilayers. In symmetrical standard solutions, conductance values of the first levels were, respectively, 16 +/- 1 pS, 32 +/- 2 pS, and 57 +/- 2 pS (n = 4) and so on, any conductance level being roughly twice the lower one. Whether a cluster organization of elementary channels or different channel structures underlies this particular property was addressed by investigating the ionic selectivity and the pore size corresponding to the first three conductance levels. Both selectivity and pore size were found to be almost independent of the conductance level. This indicated that multiple conductance behavior resulted from a cluster organization of "B3 elementary channels." According to the estimated pore size and analyses of x-ray diffraction of B3 microcrystals, a structural model for "B3 elementary channels" is proposed. The ability to form channels is likely to be involved in the biological activity of beticolins.