Planar Lipid Bilayer Technique Research Articles

Impact of ciguatoxins on human cardiac and neuronal physiology

Ciguatera (CG) is a syndrome induced by Ciguatoxins (CTXs), natural phycotoxins among the most harmful we know. They are produced by benthic dinoflagellates of the Gambierdiscus and Fukuyoa. CG is the result of food poisoning caused by the consumption of contaminated fish meat along the food chain, from herbivorous fish to higher carnivorous fish. It is the most common non-bacterial seafood poisoning in the world with an increasing 50,000 to 100,000 new cases per year although only 2 to 10% of them are actually reported. CG is manifested by multiple undefined symptoms (gastrointestinal, cardiovascular, neurosensory, skin and inflammatory) in the form of polymorphic syndrome. CTXs are known to modify the cellular membrane permeability. Several fundamental studies have highlighted a common action of CTXs by modulating the ion channels (Na+, K+, Ca2+) essential to excitable cells such as cardiomyocytes (CM) and neurons (NR). Our main objective is to understand and characterize the impact of CTXs on human cardiac and neuronal physiology. We hypothesized that CTXs can affect the intracellular calcium handling and therefore the cardiac excitation-contraction coupling (ECC) and neuronal functional and molecular properties. We differentiated human induced pluripotent stem cell-derived ventricular-like cardiomyocytes (hiPSC-CMs) and neurons (hiPSC-NRs), we used 2 types of purified CTXs (CTX1B and CTX3C) and 2 types of hiPSC healthy control (HC). We investigated the effects of CTXs on cytosolic calcium, cells morphology and CM contractile properties. To determine if CTXs modulate the RyR2 channel activity, we performed single-channel measurements using the planar lipid bilayer technique. We observed that CTXs lead to intracellular calcium leaks and increase in cardiac and neuronal basal calcium level. They cause increased activity of the RyR2 channels as reflected by higher opening frequency Fo (Fo of 244 ± 112 events/s for HC vs. 2279 ± 836 events/s for HC + CTX1B, P < 0.01). Exposure of CTXs also cause reduced contractions of the hiPSC-derived cardiac sheets and cardiac hypertrophy as evidenced by an increase in cell area (1997 ± 119 μm2 for HC vs. 2852 ± 475 μm2 for HC + CTX1B, P < 0.01). Our preliminary data suggest that CTXs lead to RyR2 aberrant kinetic properties. CTXs also induce reduced beat rate and hiPSC-CM hypertrophy.

Cell-free electrophysiology of human VDACs incorporated into nanodiscs: An improved method

Voltage-dependent anion-selective channel (VDAC) is one of the main proteins of the outer mitochondrial membrane of all eukaryotes, where it forms aqueous, voltage-sensitive, and ion-selective channels. Its electrophysiological properties have been thoroughly analyzed with the planar lipid bilayer technique. To date, however, available results are based on isolations of VDACs from tissue or from recombinant VDACs produced in bacterial systems. It is well known that the cytosolic overexpression of highly hydrophobic membrane proteins often results in the formation of inclusion bodies containing insoluble aggregates. Purification of properly folded proteins and restoration of their full biological activity requires several procedures that considerably lengthen experimental times. To overcome these restraints, we propose a one-step reaction that combines in vitro cell-free protein expression with nanodisc technology to obtain human VDAC isoforms directly integrated in a native-like lipid bilayer. Reconstitution assays into artificial membranes confirm the reliability of this new methodological approach and provide results comparable to those of VDACs prepared with traditional protein isolation and reconstitution protocols. The use of membrane-mimicking nanodisc systems represents a breakthrough in VDAC electrophysiology and may be adopted to further structural studies.

The PLB measurement for the connector in Phi29 bacteriophage reveals the function of its channel loop

The connector protein, also known as the portal protein, located at the portal vertex in the Phi29 bacteriophage has been found to play a key role in the genome DNA packaging motor. There is a disordered region, composed of 12 sets of 18-residue loops N229–N246, that has been assumed to serve as a “clamp” to retain the DNA within the pressurized capsid when DNA is fully packaged. However, the process remains undefined about how the clamping of DNA occurs and what signal is used to engage the channel loops to clamp the DNA near the end of DNA packaging. In this study, we use the planar lipid bilayer (PLB) membrane technique to study the connector with its loops cleaved. The channel properties are compared with those of the connector with corresponding wild-type loops at different membrane potentials. On the basis of the hypothesis of the Donnan effects in the flashing Brownian ratchet model, we associate the PLB experimental results with the outcomes from the relevant biochemical experiments on the proheads containing the connectors without the loops, which enables us to provide a clear picture about how the DNA clamping occurs. A mathematical relationship between the Donnan potential and the DNA packaging density is established, demonstrating that they are both in essence the same signal that is received and transmitted by the connector to dictate DNA clamping and the termination of DNA packaging. At the end of the study, the PLB technique is proposed as a viral research tool, and its potential use to study the functions of specific domains in a portal protein of the tailed bacteriophages is highlighted.

Exploring the Nature of Cationic Blocker Recognition by the Anthrax Toxin Channel

Obstructing conductive pathways of the channel-forming toxins with targeted blockers is a promising drug design approach. Nearly all tested positively charged ligands have been shown to reversibly block the cation-selective channel-forming protective antigen (PA63) component of the binary anthrax toxin. The cationic ligands with more hydrophobic surfaces, particularly those carrying aromatic moieties, inhibited PA63 more effectively. To understand the physical basis of PA63 selectivity for a particular ligand, detailed information is required on how the blocker structural elements (e.g., positively charged and aromatic groups) influence the molecular kinetics of the blocker/channel binding reactions. In this study, we address this problem using the high-resolution single-channel planar lipid bilayer technique. Several structurally distinct cationic blockers, namely per-6-S-(3-amino) propyl-β-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-α-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-β-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-γ-cyclodextrin, methyltriphenylphosphonium ion, and G0 polyamidoamine dendrimer are tested for their ability to inhibit the heptameric and octameric PA63 variants and PA63F427A mutant. The F427 residues form a hydrophobic constriction region inside the channel, known as the “ϕ-clamp.” We show that the cationic blockers interact with PA63 through a combination of forces. Analysis of the binding reaction kinetics suggests the involvement of cation-π, Coulomb, and salt-concentration-independent π-π or hydrophobic interactions in the cationic cyclodextrin binding. It is possible that these blockers bind to the ϕ-clamp and are also stabilized by the Coulomb interactions of their terminal amino groups with the water-exposed negatively charged channel residues. In PA63F427A, only the suggested Coulomb component of the cyclodextrin interaction remains. Methyltriphenylphosphonium ion and G0 polyamidoamine dendrimer, despite being positively charged, interact primarily with the ϕ-clamp. We also show that seven- and eightfold symmetric cyclodextrins effectively block the heptameric and octameric forms of PA63 interchangeably, adding flexibility to the earlier formulated blocker/target symmetry match requirement.

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

Lipid Composition and Endosomal pH Regulates Anthrax Lethal Toxin Uptake and PA63 Channel Behavior

The tight regulation of cellular pH is critical for the structure and function of cells and organelles. Besides, cells adjust bilayer biophysical parameters by controlling lipid composition and gradient across their plasma and intracellular organelle membranes. Both pH and membrane lipid composition change dramatically along the endocytic pathway, which is the internalization transport process that many bacterial toxins utilize to invade cells. In this study, we investigate the role of the lipid and pH environment in the uptake of the anthrax lethal toxin (composed of protective antigen (PA) and lethal factor (LF)). The anthrax toxin follows a multistep internalization process, which involves PA binding to its cellular receptors, proteolytic cleavage to PA63, oligomerization to form prepores, and LF binding followed by endocytosis. The acidic endosomal environment causes conformational changes in PA63 leading to its insertion into the early endosomal membrane and ion channel formation. The PA63-mediated delivery of the enzymatic LF to cytoplasm was suggested to take place later in the endocytic pathway via the back-fusion of intraluminal vesicles. Given the variations in lipid composition of the early endosome, the intraluminal vesicles, and the late endosomal limiting membrane, it is important to understand how these changes influence the anthrax toxin uptake. Using the planar lipid bilayer technique, we reconstituted single PA63 channels into neutral DOPC, DPhPC and DOPE and negatively-charged DOBMP, DPhPS membranes and varied the solution pH from 7.5 to 4.5 to mimic the changing physical environments of the endosomal pathway. The observed differences in the PA63 single-channel conductance, selectivity, and gating, and in LF binding and internalization were significant. These findings suggest that the anthrax toxin complex represents a robust universal model to study protein/lipid and protein/protein interactions in lipid membranes of different composition.

Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: Characterization and comparison to other RTX-family members

Pediatric septic arthritis in patients under age of four is frequently caused by the oral Gram-negative bacterium Kingella kingae. This organism may be responsible for a severe form of infective endocarditis in otherwise healthy children and adults. A major virulence factor of K. kingae is RtxA, a toxin that belongs to the RTX (Repeats-in-ToXin) group of secreted pore forming toxins. To understand the RtxA effects on host cell membranes, the toxin activity was studied using planar lipid bilayers. K. kingae strain PYKK081 and its isogenic RtxA-deficient strain, KKNB100, were tested for their ability to form pores in artificial membranes of asolectin/n-decane. RtxA, purified from PYKK081, was able to rapidly form pores with an apparent diameter of 1.9nm as measured by the partition of nonelectrolytes in the pores. The RtxA channels are cation-selective and showed strong voltage-dependent gating. In contrast to supernatants of PYKK081, those of KKNB100 did not show any pore forming activity. We concluded that RtxA toxin is the only secreted protein from K. kingae forming large channels in host cell membranes where it induces cation flux leading to programmed cell death. Furthermore, our findings suggested that the planar lipid bilayer technique can effectively be used to test possible inhibitors of RTX toxin activity and to investigate the mechanism of the toxin binding to the membrane.

Pseudo painting/air bubble technique for planar lipid bilayers.

A functional reconstitution of channel proteins in planar lipid bilayers is still very versatile to study structure/function correlates under well-defined conditions at the single protein level. In this study we present an improved planar lipid bilayer technique in which an air bubble is used for stabilizing unstable/leaky bilayers or for removing excess lipids. The bubble can also be used as a tool for reducing the number of channels in the bilayer with the goal of having only one active channel in the membrane. Stable planar lipid bilayers are formed within seconds to minutes. In the case of multiple channel insertion the air bubble can be used to reduce the number of channels within minutes. The simple improvement of the classical folding technique guarantees a very fast creation of stable bilayers even with difficult phospholipids in a conventional vertical bilayer set-up; it requires no modifications of the existing set-up. This technique is very easy to handle and guarantees successful single channel recordings for any kind of planar lipid bilayer experiment.

Wicking: A Rapid Method for Manually Inserting Ion Channels into Planar Lipid Bilayers

The planar lipid bilayer technique has a distinguished history in electrophysiology but is arguably the most technically difficult and time-consuming method in the field. Behind this is a lack of experimental consistency between laboratories, the challenges associated with painting unilamellar bilayers, and the reconstitution of ion channels into them. While there has be a trend towards automation of this technique, there remain many instances where manual bilayer formation and subsequent membrane protein insertion is both required and advantageous. We have developed a comprehensive method, which we have termed “wicking”, that greatly simplifies many experimental aspects of the lipid bilayer system. Wicking allows one to manually insert ion channels into planar lipid bilayers in a matter of seconds, without the use of a magnetic stir bar or the addition of other chemicals to monitor or promote the fusion of proteoliposomes. We used the wicking method in conjunction with a standard membrane capacitance test and a simple method of proteoliposome preparation that generates a heterogeneous mixture of vesicle sizes. To determine the robustness of this technique, we selected two ion channels that have been well characterized in the literature: CLIC1 and α-hemolysin. When reconstituted using the wicking technique, CLIC1 showed biophysical characteristics congruent with published reports from other groups; and α-hemolysin demonstrated Type A and B events when threading single stranded DNA through the pore. We conclude that the wicking method gives the investigator a high degree of control over many aspects of the lipid bilayer system, while greatly reducing the time required for channel reconstitution.

Property of large conductance Ca2+-activated K+ channels from Fasciola hepatica incorporated into planar lipid bilayer

Fasciola hepatica causes biliary epithelial hyperplasia and obstructive jaundice in humans and animals. Using a planar lipid bilayer technique, we further characterized the single channel property of large conductance K+-permeable channels that were previously identified from F. hepatica. The single channel conductance was 254.7±17.9pS under a symmetrical 200/200mM (cis/trans) KCl gradient. Open state probability (Po) varied from channel to channel at a given membrane potential and Ca2+ concentration, but increased with voltage (−60 to +40mV) and cis Ca2+ (1–200μM). Under a near bi-ionic condition of 200mM [K+]cis/200mM [Na+]trans, the permeability ratio of K+ to Na+ was 5.0. Charybdotoxin (1μM) inhibited Po, whereas tetraethylammonium reduced the conductance (KD=67.8mM). Taken together, the results show that the single channel properties of the large conductance K+-permeable channels in F. hepatica are similar to those of large conductance Ca2+-activated K+ (BK) channels in general, but distinct from typical BK channels in the extent of voltage- and Ca2+-dependence, as well as permeability to Na+. This study further reveals a variant BK channel in F. hepatica that could serve as a new drug target to treat fascioliasis.

Exploring protein import pores of cellular organelles at the single molecule level using the planar lipid bilayer technique

Proteins of living cells carry out their specialized functions within various subcellular membranes or aqueous spaces. Approximately half of all the proteins of a typical cell are transported into or across membranes. Targeting and transport to their correct subcellular destinations are essential steps in protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Virtually all proteins of the endosymbiotic organelles, chloroplasts and mitochondria, are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic and biochemical techniques led to rather detailed knowledge on the subunit composition of the various protein transport complexes which carry out the membrane transport of the preproteins. Conclusive concepts on targeting and cytosolic transport of polypeptides emerged, while still few details on the molecular nature and mechanisms of the channel moieties of protein translocation complexes have been achieved. In this paper we will describe the history of how the individual subunits forming the channel pores of the chloroplast, mitochondrial and endoplasmic reticulum protein import machineries were identified and characterized by single channel electrophysiological techniques in planar bilayers. We will also highlight recent developments in the exploration of the molecular properties of protein translocating channels and the regulation of the diverse protein translocation systems using the planar bilayer technique.

A Cytotoxic Peptide from a Marine Sponge, Polytheonamide B; II. Properties for Ion Conduction and Voltage Dependent Gating

A 48-mer peptide from a marine sponge Theonella swinhoei, polytheonamide B (pTB), is highly cytotoxic against eukaryotic cells. Alternating D- and L-chirals throughout the molecule suggest formation of β-helix, which was supported by channel activity having high permeability to Cs+. The 48-mer β-helix is long enough to span the membrane and was shown to form the channel by single peptide. In this study we evaluated the ion conduction and gating properties of pTB channel by use of planar lipid bilayer technique. Single-channel I-V curve in CsCl solution exhibited weak outward rectification. Concentration-dependency of single-channel conductance of pTB channel was examined. For Cs+ permeation, clear saturation in unitary conductance was observed in the concentration range up to 3.0 M, which was contrast to gramicidin A (gA) channel. Gating manner of pTB channel was characteristic. Fast transition between open and closed state was observed, suggesting that the structural changes of single pTB molecule directly link to the gating. In addition to asymmetrical single-channel conductance, distinctive voltage-dependent gating was observed in single-channel and macroscopic current traces of pTB channels. On the basis of the structure of pTB channel, mechanism of ion conduction and voltage-dependent gating will be discussed.

Psalmotoxin-1 Docking to Human Acid-sensing Ion Channel-1

Acid-sensing ion channel-1 (ASIC-1) is a proton-gated ion channel implicated in nociception and neuronal death during ischemia. Recently the first crystal structure of a chicken ASIC was obtained. Expanding upon this work, homology models of the human ASICs were constructed and evaluated. Energy-minimized structures were tested for validity by in silico docking of the models to psalmotoxin-1, which potently inhibits ASIC-1 and not other members of the family. The data are consistent with prior radioligand binding and functional assays while also explaining the selectivity of PcTX-1 for homomeric hASIC-1a. Binding energy calculations suggest that the toxin and channel create a complex that is more stable than the channel alone. The binding is dominated by the coulombic contributions, which account for why the toxin-channel interaction is not observed at low pH. The computational data were experimentally verified with single channel and whole-cell electrophysiological studies. These validated models should allow for the rational design of specific and potent peptidomimetic compounds that may be useful for the treatment of pain or ischemic stroke.

Redox Modification of Ryanodine Receptors Contributes to Sarcoplasmic Reticulum Ca 2+ Leak in Chronic Heart Failure

Abnormal cardiac ryanodine receptor (RyR2) function is recognized as an important factor in the pathogenesis of heart failure (HF). However, the specific molecular causes underlying RyR2 defects in HF remain poorly understood. In the present study, we used a canine model of chronic HF to test the hypothesis that the HF-related alterations in RyR2 function are caused by posttranslational modification by reactive oxygen species generated in the failing heart. Experimental approaches included imaging of cytosolic ([Ca(2+)](c)) and sarcoplasmic reticulum (SR) luminal Ca(2+) ([Ca(2+)]SR) in isolated intact and permeabilized ventricular myocytes and single RyR2 channel recording using the planar lipid bilayer technique. The ratio of reduced to oxidized glutathione, as well as the level of free thiols on RyR2 decreased markedly in failing versus control hearts consistent with increased oxidative stress in HF. RyR2-mediated SR Ca(2+) leak was significantly enhanced in permeabilized myocytes, resulting in reduced [Ca(2+)](SR) in HF compared to control cells. Both SR Ca(2+) leak and [Ca(2+)](SR) were partially normalized by treating HF myocytes with reducing agents. Conversely, oxidizing agents accelerated SR Ca(2+) leak and decreased [Ca(2+)](SR) in cells from normal hearts. Moreover, exposure to antioxidants significantly improved intracellular Ca(2+)-handling parameters in intact HF myocytes. Single RyR2 channel activity was significantly higher in HF versus control because of increased sensitivity to activation by luminal Ca(2+) and was partially normalized by reducing agents through restoring luminal Ca(2+) sensitivity oxidation of control RyR2s enhanced their luminal Ca(2+) sensitivity, thus reproducing the HF phenotype. These findings suggest that redox modification contributes to abnormal function of RyR2s in HF, presenting a potential therapeutic target for treating HF.

The patch-clamp and planar lipid bilayer techniques: powerful and versatile tools to investigate the CFTR Cl − channel

Using the patch-clamp (PC) and planar lipid bilayer (PLB) techniques the molecular behaviour of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channel can be visualised in real-time. The PC technique is a highly powerful and versatile method to investigate CFTR's mechanism of action, interaction with other proteins and physiological role. Using the PLB technique, the structure and function of CFTR can be investigated free from the influence of other proteins. Here we discuss how these techniques are employed to investigate the CFTR Cl − channel with special emphasis on its permeation, conduction and gating properties.

Ion channels of Fasciola hepatica incorporated into planar lipid bilayers.

Ion channels are important target sites of anthelmintics, but little is known about those in Fasciola hepatica. In this work, we applied a planar lipid bilayer technique to characterize the properties of single ion channels in F. hepatica. Under a 200/40 mM KCl gradient, a large conductance channel of 251 pS was observed in 18% of the membranes studied. The channel was selective to K(+) over Cl(-) with a permeability ratio of K(+) to Cl(-) (PK/PCl) of 4.9. Open state probability (Po) of the channel was less than 0.5 and dependent on voltage (-60 to approximately +40 mV) and Ca(2+) (approximately 100 microM). The other two types of single channels observed in 11 and 5% of membranes, respectively, were a K(+)-permeable channel of 80 pS (PK/PCl=4.6) and a Cl(-)-permeable channel of 64 pS (PK/PCl=0.058). Open state probability of both channels showed little voltage dependence. The results indicate that distinct single channels of 60 to approximately 251 pS are present in relative abundance and, in addition, that the planar lipid bilayer technique can be a useful tool for the study of single ion channels in F. hepatica.

Activation of cardiac ryanodine receptors by the calcium channel agonist FPL-64176.

We investigated the possibility that the Ca(2+) channel agonist FPL-64176 (FPL) might also activate the cardiac sarcoplasmic reticulum (SR) Ca(2+) release channel ryanodine receptor (RyR). The effects of FPL were tested on single channel activity of purified and crude vesicular RyR (RyR2) isolated from human and dog hearts using the planar lipid bilayer technique. FPL (100-200 microM) increased single channel open probability (P(o)) when added to the cytoplasmic side of the channel (P(o) = 0.070 +/- 0.021 in control RyR2; 0.378 +/- 0.086 in 150 microM FPL, n = 9, P < 0.01) by prolonging open times and decreasing closed times without changing current magnitude. FPL had no effect on P(o) when added to the trans (luminal) side of the bilayer (P(o) = 0.079 +/- 0.036 in control and 0.103 +/- 0.066 in FPL, n = 4, no significant difference). The bell-shaped [Ca(2+)] dependence of [(3)H]ryanodine binding and of P(o) was altered by FPL, suggesting that the mechanism by which FPL increases channel activity is by an increase in Ca(2+)-induced activation at low [Ca(2+)] (without a change in threshold) and suppression of Ca(2+)-induced inactivation at high [Ca(2+)]. However, the fact that inactivation was restored at elevated [Ca(2+)] suggests a competitive interaction between Ca(2+) and FPL on inactivation. FPL had no effect on RyR skeletal channels (RyR1), where P(o) was 0.039 +/- 0.005 in control versus 0.030 +/- 0.006 in 150 microM FPL (no significant difference). These results suggest that, in addition to its ability to activate the L-type Ca(2+) channels, FPL activates cardiac RyR2 primarily by reducing the Ca(2+) sensitivity of inactivation.

CLIC1 inserts from the aqueous phase into phospholipid membranes, where it functions as an anion channel.

CLIC1 is a member of the CLIC family of proteins, which has been shown to demonstrate chloride channel activity when reconstituted in phospholipid vesicles. CLIC1 exists in cells as an integral membrane protein and as a soluble cytoplasmic protein, implying that CLIC1 might cycle between membrane-inserted and soluble forms. CLIC1 was purified and detergent was removed, yielding an aqueous solution of essentially pure protein. Pure CLIC1 was mixed with vesicles, and chloride permeability was assessed with a chloride efflux assay and with planar lipid bilayer techniques. Soluble CLIC1 confers anion channel activity to preformed membranes that is indistinguishable from the previously reported activity resulting from reconstitution of CLIC1 into membranes by detergent dialysis. The activity is dependent on the amount of CLIC1 added, appears rapidly on mixing of protein and lipid, is inhibited by indanyloxyacetic acid-94, N-ethylmaleimide, and glutathione, is inactivated by heat, and shows sensitivity to pH and to membrane lipid composition. We conclude that CLIC1 in the absence of detergent spontaneously inserts into preformed membranes, where it can function as an anion-selective channel.

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.

CLIC-1 functions as a chloride channel when expressed and purified from bacteria.

CLIC-1 is a member of a family of proteins related to the bovine intracellular chloride channel p64 which has been proposed to function as a chloride channel. We expressed CLIC-1 as a glutathione S-transferase fusion protein in bacteria. The fusion protein was purified by glutathione affinity, and CLIC-1 was released from its fusion partner by digestion with thrombin. After further purification, CLIC-1 was reconstituted into phospholipid vesicles by detergent dialysis. Chloride permeability of reconstituted vesicles was assessed using a valinomycin dependent chloride efflux assay, demonstrating increased vesicular chloride permeability with CLIC-1 compared with control. CLIC-1-dependent chloride permeability was inhibited by indanyloxyacetic acid-94 with an apparent IC(50) of 8.6 micrometer. The single channel properties of CLIC-1 were determined using the planar lipid bilayer technique. We found that CLIC-1 forms a voltage-dependent, Cl-selective channel with a rectifying current-voltage relationship and single channel conductances of 161 +/- 7.9 and 67.5 +/- 6.9 picosiemens in symmetric 300 and 150 mm KCl, respectively. The anion selectivity of this activity is Br approximately Cl > I. The open probability of CLIC-1 channels in planar bilayers was decreased by indanyloxyacetic acid-94 with an apparent IC(50) of 86 micrometer at 50 mV. These data convincingly demonstrate that CLIC-1 is capable of forming a novel, chloride-selective channel in the absence of other subunits or proteins.