The properties of Bacillus cereus hemolysin II pores depend on environmental conditions

Hemolysin II (HlyII), one of several cytolytic proteins encoded by the opportunistic human pathogen Bacillus cereus, is a member of the family of oligomeric beta-barrel pore-forming toxins. This work has studied the pore-forming properties of HlyII using a number of biochemical and biophysical approaches. According to electron microscopy, HlyII protein interacts with liposomes to form ordered heptamer-like macromolecular assemblies with an inner pore diameter of 1.5-2 nm and an outer diameter of 6-8 nm. This is consistent with inner pore diameter obtained from osmotic protection assay. According to the 3D model obtained, seven HlyII monomers might form a pore, the outer size of which has been estimated to be slightly larger than by the other method, with an inner diameter changing from 1 to 4 nm along the channel length. The hemolysis rate has been found to be temperature-dependent, with an explicit lag at lower temperatures. Temperature jump experiments have indicated the pore structures formed at 37 degrees C and 4 degrees C to be different. The channels formed by HlyII are anion-selective in lipid bilayers and show a rising conductance as the salt concentration increases. The results presented show for the first time that at high salt concentration HlyII pores demonstrate voltage-induced gating observed at low negative potentials. Taken together we have found that the membrane-binding properties of hemolysin II as well as the properties of its pores strongly depend on environmental conditions. The study of the properties together with structural modeling allows a better understanding of channel functioning.     

Flammutoxin, a cytolysin from the edible mushroom Flammulina velutipes, forms two different types of voltage-gated channels in lipid bilayer membranes

Flammutoxin, a 31-kDa cardiotoxic and cytolytic protein from the edible mushroom Flammulina velutipes, has been shown to assemble into a pore-forming annular oligomer with outer and inner diameters of 10 and 5 nm on the target cells [Tomita et al., Biochem. J. 333 (1998) 129-137]. Here we studied electrophysiological properties of flammutoxin channels using planar lipid bilayer technique, and found that flammutoxin formed two types of moderately cation-selective, voltage-gated channels with smaller and larger current amplitudes (1-4.5 pA and 20-30 pA, respectively, at 20 mV) in the lipid bilayers composed of phospholipid and cholesterol. The larger-conductance single channel showed the properties of a wide water-filled pore such as a linear relationship between channel conductance and salt concentration of the bathing solution. The functional diameter of the larger-conductance channel was estimated to be 4-5 nm by measuring the current conductance in the presence of polyethylene glycols of various sizes. In contrast, the smaller-conductance single channels showed a non-linear current to voltage curve and a saturating conductance to increasing salt concentration. These results suggest that the larger-conductance channel of flammutoxin corresponds to the hemolytic pore complex, while the smaller-conductance channel may reflect the intermediate state(s) of the assembling toxin.     

Different modes of membrane permeabilization by two RTX toxins: HlyA from Escherichia coli and CyaA from Bordetella pertussis

This study clarifies the membrane disruption mechanisms of two bacterial RTX toxins: αhemolysin (HlyA) from Escherichia coli and a highly homologous adenylate cyclase toxin (CyaA) from Bordetella pertussis. For this purpose, we employed a fluorescence requenching method using liposomes (extruded through filters of different pore size — 1000 nm, 400 nm or 100 nm) with encapsulated fluorescent dye/quencher pair ANTS/DPX. We showed that both toxins induced a graded leakage of liposome content with different selectivities α for DPX and ANTS. In contrast to HlyA, CyaA exhibited a higher selectivity for cationic quencher DPX, which increased with vesicle diameter. Large unilamellar vesicles (LUV₁₀₀₀) were found to be more suitable for distinguishing between high α values whereas smaller ones (LUV₁₀₀) were more appropriate for discriminating an all-or-none leakage (α = 0) from the graded leakage with low values of α. While disrupting LUV₁₀₀₀, CyaA caused a highly cation-selective leakage (α ~ 15) whereas its mutated form with decreased channel K⁺/Cl⁻ selectivity due to two substitutions in a predicted transmembrane segment (CyaA-E509K + E516K) exhibited much lower selectivity (α ∼ 6). We concluded that the fluorescence requenching method in combination with different size of liposomes is a valuable tool for characterization of pore-forming toxins and their variants.     

Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: Role of voltage and pH

The bifunctional adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis invades target cells via transport through the cytoplasmic membrane. The membrane potential represents thereby an important factor for the uptake in vivo. Previous studies demonstrated that adenylate cyclase (AC) delivery into cells requires a negative membrane potential inside the cells. The results of lipid bilayer experiments with ACT presented here indicated that two different types of pore-like structures are formed by ACT dependent on the orientation of the electrical potential across the membranes. Pore formation at a positive potential at the cis side of the membranes, the side of the addition of the toxin, was fast and its conductance had a defined size, whereas at negative potential the pores were not defined, had a reduced pore-forming activity and a very short lifetime. Fluctuations inserted at positive potentials showed asymmetric current-voltage relationships for positive and negative voltages. Positive potentials at the cis side resulted in an increasing current, whereas at negative potentials the current decreased or remained at a constant level. Calcium ions enhanced the voltage dependence of the ACT pores when they were added to the cis side. The single-pore conductance was strongly affected by the variation of the pH value and increased in 1M KCl with increasing pH from about 4 pS at pH 5 to about 60 pS at pH 9. The ion selectivity remained unaffected by pH. Experiments with ACT mutants revealed, that the adenylate cyclase (AC) and repeat (RT) domains were not involved in voltage and pH sensing.     

Purification and characterization of the pore forming protein of yeast mitochondrial outer membrane

One of the major outer membrane proteins of yeast mitochondria was isolated and purified. It migrated as a single band with an apparent molecular weight of 30 kDa on a SDS-electrophoretogram. When reconstituted in lipid bilayer membranes the protein formed pores with a single channel conductance of 0.45 nS in 0.1 M KCl. The pores had the characteristics of general diffusion pores with an estimated diameter of 1.7 nm. The pore of mitochondrial outer membranes of yeast shared some similarities with the pores formed by mitochondrial and bacterial porins. The pores switched to substates at voltages higher than 20 mV. The possible role of this voltagedependence in the metabolism of mitochondria is discussed.     

Calorimetric scrutiny of lipid binding by sticholysin II toxin mutants

The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid-protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7 × 10⁸ M^− 1). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation. Membrane binding is achieved through two separate mechanisms, that is, recognition of the lipid-water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site. Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation.     

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.     

Nucleopores of the giant amoeba,Pelomyxa carolinensis

Summary As shown in tangential sections of Pelomyxa carolinensis nuclei, there are many pores, each with a surrounding annulus. Each annulus is composed of 8 subannuli or satellites, plus one to three central granules. Each satellite is an electron opaque mass (of much smaller opaque particulates) about 25 nm in diameter. The outer diameter of each annulus is about 115 nm while the inner, or pore diameter, is about 65 nm. The pores occur at distances averaging 185 nm from center to center. Frequently, delicate filaments connect adjacent satellites, and the central granule with the satellites. As seen in cross sections of the nucleus, nucleopores are formed by the fusion of the inner and outer nuclear envelope membranes. The pore appears as a gap, spanned by a delicate diaphragm anchored to the nuclear envelope where its two membranes are fused. Possible functions of the pore-annulus complexes are discussed.Work supported by the U.S. Atomic Energy Commission.The authors acknowledge the assistance of Miss Elaine C. Silver, General Electric Co., Philadelphia, and Dr. Robert Wolfgang of Argonne National Laboratory.     

Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore

Tom40 is the central pore-forming component of the translocase of the outer mitochondrial membrane (TOM complex). Different views exist about the secondary structure and electrophysiological characteristics of Tom40 from Saccharomyces cerevisiae and Neurospora crassa. We have directly compared expressed and renatured Tom40 from both species and find a high content of beta-structure in circular dichroism measurements in agreement with refined secondary structure predictions. The electrophysiological characterization of renatured Tom40 reveals the same characteristics as the purified TOM complex or mitochondrial outer membrane vesicles, with two exceptions. The total conductance of the TOM complex and outer membrane vesicles is twofold higher than the total conductance of renatured Tom40, consistent with the presence of two TOM pores. TOM complex and outer membrane vesicles possess a strongly enhanced sensitivity to a mitochondrial presequence compared to Tom40 alone, in agreement with the presence of several presequence binding sites in the TOM complex, suggesting a role of the non-channel Tom proteins in regulating channel activity.