A Novel Approach to Study the Geometry of the Water Lumen of Ion Channels: Colicin Ia Channels in Planar Lipid Bilayers

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. Received: 20 February 1997/Revised: 19 August 1997     

Interaction of Nisin with Planar Lipid Bilayers Monitored by Fluorescence Recovery After Photobleaching

Nisin, a prominent member of the lantibiotic family of antimicrobial agents, has wide application as a food preservative despite poor understanding of its mode of action. Fluorescence recovery after photobleaching has been used with planar lipid bilayers as a model membrane system to examine how nisin might interact with the surface of bacterial cells. Nisin associates with planar lipid bilayers in the absence of an applied membrane potential causing an array of effects consistent with adsorption of nisin onto the membrane surface which involves inhibition of the lateral diffusion and fluorescence of the lipid probe N-(7--1,2,3-benzoxadiazol-4-yl) phosphatidylethanolamine (NBD-PE) and a reduction of the capacitance of the bilayer. Nisin adsorption is dependent on phospholipid composition. In the presence of dioleoylphosphatidylcholine (PC): cardiolipin (CL) 4:1, the rate of lateral mobility of phospholipid is reduced to 61% of the control level which decreases to a value of 46% when CL is replaced by 1-palmitoyl-2-oleoylphosphatidylserine (PS). These effects on bilayer parameters are transient, and with time the values return to near original levels. High electrical conductivity is observed on application of a voltage ramp suggesting that insertion into the membrane follows surface association. Results have been interpreted in terms of a model in which nisin initially binds to the surface of the membrane causing a modulation of bilayer properties. Received: 14 August 1995/Revised: 22 February 1996     

Concurrent Neutral Evolution of mRNA Secondary Structures and Encoded Proteins

Messenger RNA sequences often have to preserve functional secondary structure elements in addition to coding for proteins. We present a statistical analysis of retroviral mRNA which supports the hypothesis that the natural genetic code is adapted to such complementary coding. These sequences are still able to explore efficiently the space of possible proteins by point mutations. This is borne out by the observation that, in stem regions of retroviral mRNA foldings, silent mutations on one strand are preferentially accompanied by conservative mutations on the other. Distances between amino acids based on physicochemical properties are used to quantify the conservation of protein function under the constraint of maintained RNA secondary structure. We find that preservation of RNA secondary structure by compensatory mutations is evolutionary compatible with the efficient search for new variants on the protein level. Received: 4 June 1999 / Accepted: 12 October 1999     

Protein-dependent Reduction of the Pyrene Excimer Formation in Membranes

The presence of proteins in lipid bilayers always decreases the excimer formation rate of pyrene and pyrene lipid analogues in a way that is related to the protein-to-lipid ratio. Energy transfer measurements from intrinsic tryptophans to pyrene have shown (Engelke et al., 1994), that in microsomal membranes, the excimer formation rate of pyrene and pyrene fatty acids is heterogeneous within the membrane plane, because a lipid layer of reduced fluidity surrounds the microsomal proteins. This study investigates whether of not liposomes prepared from egg yolk phosphatidylcholine with incorporated gramicidin A give results comparable to those from microsomal membranes. The results indicate that the influence of proteins on the lipid bilayer cannot be described by one unique mechanism: Small proteins such as gramicidin A obviously reduce the excimer formation rate by occupying neighboring positions of the fluorescent probe and thus decrease the pyrene collision frequency homogeneously in the whole membrane plane, while larger proteins are surrounded by a lipid boundary layer of lower fluidity than the bulk lipid. The analysis of the time-resolved tryptophan fluorescence of gramicidin A incorporated liposomes reveals, that the tryptophan quenching by pyrene is stronger for tryptophans located closely below the phospholipid headgroup region because of the pyrene enrichment in this area of the lipid bilayer. Received: 29 December 1996/Revised: 15 May 1996     

Properties of Hexadecaprenyl Monophosphate/Dioleoylphosphatidylcholine Vesicular Lipid Bilayers

In our study we investigated hemispherical phospholipid bilayer membranes and phospholipid vesicles made from hexadecaprenyl monophosphate (C₈₀-P), dioleoylphosphatidylocholine (DOPC) and their mixtures by voltammetric and transmission electron microscopy (TEM) techniques. The current-voltage characteristics, the membrane conductance-temperature relationships and the membrane breakdown voltage have been measured for different mixtures of C₈₀-P/DOPC. The membrane hydrophobic thickness and the activation energy of ion migration across the membrane have been determined. Hexadecaprenyl monophosphate decreased in comparison with DOPC bilayers, the membrane conductance, increased the activation energy and the membrane breakdown voltage for the various value of C₈₀-P/DOPC mole ratio, respectively. The TEM micrographs of C₈₀-P, DOPC and C₈₀-P/DOPC lipid vesicles showed several characteristic structures, which have been described. The data indicate that hexadecaprenyl monophosphate modulates the surface curvature of the membranes by the formation of aggregates in liquid-crystalline phospholipid membranes. We suggest that the dynamics and conformation of hexadecaprenyl monophosphate in membranes depend on the transmembrane electrical potential. The electron micrographs indicate that polyprenyl monophosphates with single isoprenyl chains form lipid vesicular bilayers. The thickness of the bilayer, evaluated from the micrographs, was 11 ± 1 nm. This property creates possibility of forming primitive bilayer lipid membranes by long single-chain polyprenyl phosphates in abiotic conditions. It can be the next step in understanding the origin of protocells. Received: 10 January 2000/Revised: 7 June 2000     

Influence of Cys-130 S. aureus Alpha-toxin on Planar Lipid Bilayer and Erythrocyte Membranes

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. Received: 8 September 1995/Revised: 20 November 1996     

The Effects of Inhibitors Upon Pore Formation by Diphtheria Toxin and Diphtheria Toxin T Domain

The formation of pores by membrane-inserted diphtheria toxin is closely linked to the translocation of its catalytic chain across membranes. In this report a number of aromatic polyanionic molecules were identified that inhibit toxin-induced leakage of molecules from model membrane vesicles. One inhibitor, Cibacron blue, totally blocked pore formation. Aniline blue and Fast Green decreased the size of the molecule released by a given concentration of toxin. Amaranth appeared to reduce the maximal amount of leakage, without greatly affecting the size of the molecule released at a given toxin concentration. Finally, Ponceau S and Cibacron brilliant red appeared to exhibit a mixture of these various types of inhibition. The inhibitors neither prevented the conformational transition of the toxin to form a hydrophobic state at low pH, nor (with the exception of Cibacron Brilliant Red) appeared to strongly inhibit toxin binding to model membranes. Additional experiments showed release of trapped materials from model membranes by isolated T domain of the toxin was similar to that by whole toxin. The effects of inhibitors on T domain induced release was also similar to that they have on whole toxin. Therefore, it is likely that the inhibition of pore formation by whole toxin involves inhibitor interaction with the T domain. The inhibitors identified in this study may be helpful for development of agents that interfere with toxin action in vivo. Received: 10 March 1999/Revised: 22 June 1999     

Tight Junctions and the Experimental Modifications of Lipid Content

Tight junctions (TJs) are cell-to-cell contacts made of strands, which appear as ridges on P faces and complementary furrows on E faces on freeze fracture replicas. Evidences and opinions on whether these strands are composed of either membrane-bound proteins or lipid micelles are somewhat varied. In the present work we alter the lipid composition of Madin-Darby canine kidney monolayers using a novel approach, while studying (i) their transepithelial electrical resistance, a parameter that depends on the degree of sealing of the TJs; (ii) the apical-to-basolateral flux of 4 kD fluorescent dextran (J_DEX), that reflects the permeability of the intercellular spaces; (iii) the ability of TJs to restrict apical-to-basolateral diffusion of membrane lipids; and (iv) the pattern of distribution of endogenous and transfected occludin, the sole membrane protein presently known to form part of the TJs. We show that changing the total composition of phospholipids, sphingolipids, cholesterol and the content of fatty acids, does not alter TER nor the structure of the strands. Interestingly, enrichment with linoleic acid increases the J_DEX by 631%. The fact that this increase is not reflected in a decrease of TER, suggests that junctional strands do not act as simple resistive elements but may contain mobile translocating mechanisms. Received: 7 November 1997/Revised: 20 March 1998     

Characterization of the Sol3 family of nonautonomous transposable elements in tomato and potato

Sol3 transposons are mobile elements defined by long terminal inverted repeats which are found in tomato and potato. Members of the Sol3 family have been isolated from a variety of solanaceous species including Solanum tuberosum (potato), S. demissum, S. chacoense, Lycopersicon esculentum (tomato), and L. hirsutum. While highly conserved elements are found within different species, Sol3 terminal inverted repeats can also flank unrelated sequences. Southern blot analysis indicates that Sol3 elements are less prevalent in the potato (approximately 50 copies) than in the tomato (>100 copies) genome. No Sol3-hybridizing sequences were observed in tobacco. While a number of Sol3 elements ranging in size from 500 bp to 2 kbp were sequenced, no transposase coding domains could be identified within the internal regions of the elements. The data suggest that the Sol3 represent a heterogeneous family of nonautonomous transposable elements associated with an as-yet-unidentified autonomous transposon. Received: 18 September 1996 / Accepted: 11 March 1997     

Permeabilization of Model Lipid Membranes by Bacillus sphaericus Mosquitocidal Binary Toxin and its Individual Components

The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs, BinA formed voltage-dependent channels of ≈100–200 pS with long open times and a high open probability. Larger channels (≥400 pS) were also observed. BinB, which inserted less easily, formed smaller channels (≤100 pS) with shorter mean open times. Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior. Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding component of the toxin, playing a role in promoting channel activity. Received: 29 March 2001/Revised: 20 July 2001     

Phylogeny of the Restriction Endonuclease-Like Superfamily Inferred from Comparison of Protein Structures

To date all attempts to derive a phyletic relationship among restriction endonucleases (ENases) from multiple sequence alignments have been limited by extreme divergence of these enzymes. Based on the approach of Johnson et al. (1990), I report for the first time the evolutionary tree of the ENase-like protein superfamily inferred from quantitative comparison of atomic coordinates of structurally characterized enzymes. The results presented are in harmony with previous comparisons obtained by crystallographic analyses. It is shown that λ-exonuclease initially diverged from the common ancestor and then two ``endonucleolytic' families branched out, separating ``blunt end cutters' from ``5′ four-base overhang cutters.' These data may contribute to a better understanding of ENases and encourage the use of structure-based methods for inference of phylogenetic relationship among extremely divergent proteins. In addition, the comparison of three-dimensional structures of ENase-like domains provides a platform for further clustering analyses of sequence similarities among different branches of this large protein family, rational choice of homology modeling templates, and targets for protein engineering. Received: 14 June 1999 / Accepted: 11 August 1999     

Relationships Between Genomic G+C Content,RNA Secondary Structures,and Optimal Growth Temperature in Prokaryotes

G:C pairs are more stable than A:T pairs because they have an additional hydrogen bond. This has led to many studies on the correlation between the guanine+cytosine (G+C) content of nucleic acids and temperature over the last 20 years. We collected the optimal growth temperatures (T_opt) and the G+C contents of genomic DNA; 23S, 16S, and 5S ribosomal RNAs; and transfer RNAs for 764 prokaryotic species. No correlation was found between genomic G+C content and T_opt, but there were striking correlations between the G+C content of ribosomal and transfer RNA stems and T_opt. Two explanations have been proposed—neutral evolution and selection pressure—for the approximate equalities of G and C (respectively, A and T) contents within each strand of DNA molecules. Our results do not support the notion that selection pressure induces complementary oligonucleotides in close proximity and therefore numerous secondary structures in prokaryotic DNA, as the genomic G+C content does not behave in the same way as that of folded RNA with respect to optimal growth temperature. Received: 25 September 1996 / Accepted: 21 January 1997     

Interconverting Gating Modes of a Nonselective Cation Channel from the Tapeworm Echinococcus granulosus Reconstituted on Planar Lipid Bilayers

A 107-pS (symmetrical 150 mm KCl), nonselective cation channel was reconstituted from a microsomal membrane fraction of the larval stage of the tapeworm Echinococcus granulosus. Most of the time, it displayed a high open probability (>0.95) irrespective of either the applied voltage, Ca²⁺, Ba²⁺, or tetraethylammonium concentration. Nevertheless, in contrast with this ``leaklike' behavior, less frequently this ``all-the-time-open' channel reversibly entered two different kinetic modes. One of them was characterized by lower P _o values and some voltage sensitivity (V _?≅ 129 mV, and an equilibrium constant for channel closing changing e-fold per 63-mV change) the kinetic analysis revealing that it resulted from the appearance of voltage-sensitivity in the mean closed times and a sixfold increase in the equilibrium constant for channel closing at 0 mV. The other mode was characterized by a very fast open-close activity leading to poorly resolved current levels and a P _o around 0.6–0.7 which, occasionally and in a voltage-sensitive manner, entered a long-lived nonconducting state. However, the rare nature of these mode-shifting transitions precluded a more detailed analysis of their kinetics. The conductive properties of the channel were not affected by these switches. Model gating alone does not seem to ensure any physiological role of this channel and, instead, some other channel changes must occur if this phenomenon were to be of regulatory importance in vivo. Thus, mode-shifting might constitute an alternative target for channel activity modulation also in tapeworms. Received: 30 August 1996/Revised: 31 January 1997     

Properties of Chicken Lens MIP Channels Reconstituted into Planar Lipid Bilayers

Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V ₀ = 18.5 mV, n= 4.5 and g _min/g _max= 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration of the structure-function relations of MIP channels. Received: 27 March 1996/Revised: 5 August 1996     

Permeability of Boric Acid Across Lipid Bilayers and Factors Affecting It

Boron enters plant roots as undissociated boric acid (H₃BO₃). Significant differences in B uptake are frequently observed even when plants are grown under identical conditions. It has been theorized that these differences reflect species differences in permeability coefficient of H₃BO₃ across plasma membrane. The permeability coefficient of boric acid however, has not been experimentally determined across any artificial or plant membrane. In the experiments described here the permeability coefficient of boric acid in liposomes made of phosphatidylcholine was 4.9 × 10⁻⁶ cm sec⁻¹, which is in good agreement with the theoretical value. The permeability coefficient varied from 7 × 10⁻⁶ to 9.5 × 10⁻⁹ cm sec⁻¹ with changes in sterols (cholesterol), the type of phospholipid head group, the length of the fatty acyl chain, and the pH of the medium. In this study we also used Arabidopsis thaliana mutants which differ in lipid composition to study the effect of lipid composition on B uptake. The chs1-1 mutant which has lower proportion of sterols shows 30% higher B uptake compared with the wild type, while the act1-1 mutant which has an increased percentage of longer fatty acids, exhibited 35% lower uptake than the wild type. Lipid composition changes in each of the remaining mutants influenced B uptake to various extents. These data suggest that lipid composition of the plasma membrane can affect total B uptake. Received: 15 October 1999/Revised: 11 February 2000     

Molecular Evolution of the Domain Structures of Protein Disulfide Isomerases

Protein disulfide isomerase (PDI) is an enzyme that promotes protein folding by catalyzing disulfide bridge isomerization. PDI and its relatives form a diverse protein family whose members are characterized by thioredoxin-like (TX) domains in the primary structures. The family was classified into four classes by the number and the relative positions of the TX domains. To investigate the evolution of the domain structures, we aligned the amino acid sequences of the TX domains, and the molecular phylogeny was examined by the NJ and ML methods. We found that all of the current members of the PDI family have evolved from an ancestral enzyme, which has two TX domains in the primary structure. The diverse domain structures of the members have been generated through domain duplications and deletions.     

Melatonin Enhances Tamoxifen's Ability to Prevent the Reduction in Microsomal Membrane Fluidity Induced by Lipid Peroxidation

The indoleamine melatonin and the synthetic antiestrogenic drug tamoxifen seem to have similar mechanisms in inhibiting the growth of estrogen receptor positive breast cancer cells. In this study, we compared the ability of these molecules, alone and in combination, in stabilizing microsomal membranes against free radical attack. Hepatic microsomes were obtained from male rats and incubated with or without tamoxifen (50–200 μm), melatonin (1 mm) or both; lipid peroxidation was induced by addition of FeCl₃, NADPH and ADP. After oxidative damage, membrane fluidity, measured by fluorescence polarization techniques, decreased whereas malonaldehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations increased. Incubation of the microsomes with tamoxifen prior to exposure to free radical generating processes inhibited, in a dose-dependent manner, the increase in membrane rigidity and the rise in MDA+4-HDA levels. When melatonin was added, the efficacy of tamoxifen in preventing membrane rigidity was enhanced. Thus, the IC₅₀s for preventing membrane rigidity and for inhibiting lipid peroxidation obtained for tamoxifen in the presence of melatonin were lower than those obtained with tamoxifen alone. Moreover, tamoxifen (50–200 μm) in the presence of melatonin reduced basal membrane fluidity and MDA+4-HDA levels in microsomes. These synergistic effects of tamoxifen and melatonin in stabilizing biological membranes may be important in protecting membranes from free radical damage. Received: 7 July 1997/Revised: 12 November 1997     

Reconstitution in Lipid Bilayers of an ATP-Sensitive K+ Channel from Pig Coronary Smooth Muscle

A K⁺ channel with a main conductance of 29 pS was recorded after the incorporation of coronary artery membrane vesicles into lipid bilayers. This channel was identified as an ATP-sensitive K⁺ channel (K_ATP) because its activity was diminished by the internal application of 50–250 μm ATP-Na₂. Moreover, it was opened when 10–50 μm pinacidil was externally applied. Single-channel records revealed the existence of several (sub)conductance states. At 0 mV and with a 5/250 KCl gradient, the main conductance of the K_ATP channel was 29 pS. The other (sub)conductance states were less frequent and had discrete values of 12, 17 and 22 pS. Pinacidil stabilized the channel open state primarily in the 29 pS conductance level; whereas ATP inhibited all the conductance levels. In general, K_ATP channels were characterized by brief openings followed by long closings (open probability, P _o ≈ 0.02); only occasionally (3 out of 12 experiments) did the K_ATP channels have a high open probability (P _o ≥ 0.7). Channel activity could be increased or rescued by adding 2.5–10 mm UDP-TRIS and 0.5–2 mm MgCl₂ to the internal side of the channel. Received: 7 November 1995/Revised: 10 June 1996