The major Thiobacillus ferrooxidans outer membrane protein forms low conductance ion channels in planar lipid bilayers.

A protein isolated and purified from the outer membrane of the acidophilic, chemolithotrophic bacterium, Thiobacillus ferrooxidans with an oligomeric molecular weight of 90,000 Da (p90) was incorporated into phosphatidylethanolamine planar lipid bilayers. The protein formed slightly anionic channels in KCl solutions, with a conductance of 25 pS in 100 mM KCl. The current-voltage relationship was linear between +/- 60 mV, and the conductance was a saturating function of the salt concentration. These channels fluctuated from a single open to closed state at low potentials, but present flickering activity at higher potentials.     

Antibacterial peptide pleurocidin forms ion channels in planar lipid bilayers

Pleurocidin, a 25-residue α helical cationic peptide, isolated from skin mucous secretions of the winter flounder, displays a strong anti-microbial activity and appears to play a role in innate host defence. This peptide would be responsible for pore formation in the membrane of bacteria leading to lysis and therefore death. In this study, we investigated the behaviour of pleurocidin in different planar lipid bilayers to determine its mechanism of membrane permeabilisation. Macroscopic conductance experiments showed that pleurocidin did not display a pore-forming activity in neutral phosphatidylcholine/phosphatidylethanolamine (PC/PE) lipid bilayers. However, in 7:3:1 PC/PE/phosphatidylserine (PS) lipid bilayers, pleurocidin showed reproducible I/V curves at different peptide concentrations. This activity is confirmed by single-channel experiments since well-defined ion channels were obtained if the lipid mixture was containing an anionic lipid (PS). The ion channel characteristics such as—no voltage dependence, only one unitary conductance, linear relation ship current-voltage—, are not in favour of the membrane permeabilisation according to the barrel model but rather by the toroidal pore formation.     

Antibacterial peptide pleurocidin forms ion channels in planar lipid bilayers

Pleurocidin, a 25-residue alpha helical cationic peptide, isolated from skin mucous secretions of the winter flounder, displays a strong anti-microbial activity and appears to play a role in innate host defence. This peptide would be responsible for pore formation in the membrane of bacteria leading to lysis and therefore death. In this study, we investigated the behaviour of pleurocidin in different planar lipid bilayers to determine its mechanism of membrane permeabilisation. Macroscopic conductance experiments showed that pleurocidin did not display a pore-forming activity in neutral phosphatidylcholine/phosphatidylethanolamine (PC/PE) lipid bilayers. However, in 7:3:1 PC/PE/phosphatidylserine (PS) lipid bilayers, pleurocidin showed reproducible I/V curves at different peptide concentrations. This activity is confirmed by single-channel experiments since well-defined ion channels were obtained if the lipid mixture was containing an anionic lipid (PS). The ion channel characteristics such as-no voltage dependence, only one unitary conductance, linear relation ship current-voltage-, are not in favour of the membrane permeabilisation according to the barrel model but rather by the toroidal pore formation.     

The prion peptide forms ion channels in planar lipid bilayers

One of the hypotheses concerning the pathogenic properties of the prion protein considers its influence on cellular ion homeostasis. Using the lipid bilayer technique, the influence of prion-derived peptides on the lipid bilayer conductance was characterized. To evaluate the physiological significance and possible pathological functions of the peptides, their effect on the membrane potential and respiration rate of hippocampal mitochondria was also studied. We used a peptide bearing the human prion protein sequence YSNQNNF (PrP [169-175]), and peptide SSQNNF (PrP [170-175]) bearing a naturally-occurring mutation in position 171 [N(r)S] linked to schizoaffective diseases in humans (Samaia, H.B., Mari, J.J., Vallada, H.P., Moura R.P., Simpson A.J.G., Brentani R.R. A prion-linked psychiatric disorder. Nature 390 (1997) 241). In this report, we show that PrP [170-175] N171S increases the conductance of planar lipid bilayers. Based on the conductance of single channel currents recorded in 500/500 mM KCl (cis/trans), we found a single channel conductance of 8 to 26 pS. The native prion peptide PrP [169-175] does not form ion channels in the lipid bilayer. Neither of the peptides significantly changed the membrane potential or respiration rate of isolated rat hippocampal mitochondria. We propose a possible mechanism for channel formation by aggregation of the prion-derived peptide.     

Refolded outer membrane protein A of Escherichia coli forms ion channels with two conductance states in planar lipid bilayers

Outer membrane protein A (OmpA), a major structural protein of the outer membrane of Escherichia coli, consists of an N-terminal 8-stranded beta-barrel transmembrane domain and a C-terminal periplasmic domain. OmpA has served as an excellent model for studying the mechanism of insertion, folding, and assembly of constitutive integral membrane proteins in vivo and in vitro. The function of OmpA is currently not well understood. Particularly, the question whether or not OmpA forms an ion channel and/or nonspecific pore for uncharged larger solutes, as some other porins do, has been controversial. We have incorporated detergent-purified OmpA into planar lipid bilayers and studied its permeability to ions by single channel conductance measurements. In 1 M KCl, OmpA formed small (50-80 pS) and large (260-320 pS) channels. These two conductance states were interconvertible, presumably corresponding to two different conformations of OmpA in the membrane. The smaller channels are associated with the N-terminal transmembrane domain, whereas both domains are required to form the larger channels. The two channel activities provide a new functional assay for the refolding in vitro of the two respective domains of OmpA. Wild-type and five single tryptophan mutants of urea-denatured OmpA are shown to refold into functional channels in lipid bilayers.     

A high conductance cationic channel from Phaseolus vulgaris roots incorporated into planar lipid bilayers

A previously undescribed plasma membrane cation channel from Phaseolus vulgaris bean roots was studied after its incorporation into planar lipid bilayers. The channel allows the passage of monovalent cations excluding the flux of both anions (Cl-) and divalent cations (Ca2+). The channel presents a high ( approximately 213 pS) conductance in (300 mM Kcis+)/ (150 mMKtrans+) conditions. The probability of opening (Po) is low at all the tested voltages, but it increases significantly at trans-negative potentials. Permeability ratios (Pcation/PK+) under bi-ionic conditions follow the sequence: K+ (1.0)>NH4+ (0.86)>Na+ (0.78). Under the same conditions, the conductance ratios (gamma cation/gamma K+) follow the sequence: NH4+ (1.1) > or = K+ (1.0)>Na+ (0.80). The low probability of opening exhibited by the channel upon its incorporation into a lipid bilayer makes it a candidate to regulation by (and therefore participation in) cellular signalling networks.     

Reconstitution of vacuolar ion channels into planar lipid bilayers.

Vacuolar ion channels were characterized after reconstitution into planar lipid bilayers. (1) Channel activity was observed after incorporation of tonoplast-enriched microsomal membranes, purified tonoplast membranes or of solubilized tonoplast proteins. (2) Channels of varying single-channel conductances were detected after reconstitution. In symmetrical 100 mmol l-1 KCl, conductances between 1 and 110 pS were frequently measured; the largest number of independent reconstitution events was seen for single-channel conductances of 16-25 pS (28 experiments), 30-42 pS (26), 49-56 pS (15) and 64-81 pS (15). Channel current usually increased linearly with voltage. (3) In asymmetrical solutions, cation-, non-selective and, for the first time for the tonoplast, anion-selective channels were detected. Ca(2+)-dependent regulation of channel opening was not observed in our reconstitution system. (4) Permeability was also observed for Cl-, NO3-, SO4(2-) and phosphate. (5) After fractionation of tonoplast proteins by size exclusion chromatography, ion channel activity was recovered in specific fractions. (6) Some of these fractions catalyzed sulfate transport after reconstitution into liposomes. The results suggest that different channels are active at the tonoplast membrane at a larger number than has been concluded from previous work.     

The influence of the trichorzianin C-terminal residues on the ion channel conductance in lipid bilayers

Four natural trichorzianin analogues, channel-forming peptaibols, differing in their C-terminal residues (Gln or Glu, Trpol or Pheol) were tested for their macroscopic and single-channel conductances in planar lipid bilayers. The results indicate that, as regards to the voltage threshold, the most efficient analogue is the charged Trpol-bearing one. In addition, Trpol brings about a drastic lengthening of the open channel life-times. This behaviour is attributed to the dipole moment of the end residues and to the bulkiness and hydrogen bonding ability of Trpol.     

Wave-guide spectroscopy and ion transport in planar lipid bilayers

The refractive indices of the bilayer-electrolyte system allow the membrane to operate as a light-guide. This system is then able to monitor, optically, the flow of ions across the bilayer. The light is coupled into and decoupled from a spherically bulged bilayer by means of optical, single mode fibers. The light wave travels along the curved bilayer for several millimeters. This light transmission depends critically on the angle of incidence between the fiber axis and the tangent to the film. Three transmission peaks were observed when the angle of incidence was varied between 0° and 90°. The transmitted light intensity can be modulated by the application of an electric potential upon the bilayer. The center peak, with maximum light transmission, appears at an angle of incidence which is defined by the launching geometry. A quadratic field dependence (independent of the polarity) is observed, which originates from changes in the shape of the torus transition region. The transmission of the satellite peaks, which appear just before and after the central peak, can also be modulated by an external potential. This modulation signal reflects a linear dependence on the polarity of the external voltage. The phase of the modulation signal changes its sign at each satellite peak. It is shown that this modulation signal originates from the bimolecular area of the lipid film. We present evidence that this transmission modulation occurs as a result of ion transport through the lipid film. This provides the basis for the use of wave-guide spectroscopy to investigate membrane ionic fluxes.     

Proton conductance of influenza virus M2 protein in planar lipid bilayers

Purified M2 protein from the Udorn strain of influenza virus was reconstituted into planar lipid bilayers from liposomes. In 1 mM HCl, the single-channel conductance was measured as 6 pS with open probability of < or =0.03. The current voltage curve is linear over the achievable voltage range. The current amplitude is amantadine sensitive. In HCl solutions, the single-channel current was essentially invariant with changes in [Cl(-)], [Na(+)], and [tetraethylammonium] ([TEA(+)]), but dependent on [H(+)]. The reversal potential, determined with asymmetrical hydrogen chloride solution, is very close to the equilibrium potential of hydrogen. This appears to be the first report of single-channel proton currents with the full-length M2 protein.     

An anion channel of sarcoplasmic reticulum incorporated into planar lipid bilayers: Single-channel behavior and conductance properties

Summary An anion channel of sarcoplasmic reticulum vesicle has been incorporated into planar lipid bilayers by means of a fusion method and its basic properties were investigated. Analysis of fusion processes suggested that one SR vesicle contained approximately one anion channel. The conductance of this channel has several substates and shows a flickering behavior. The occupation probability of each substate was voltage dependent, which induced an inward rectification of macroscopic currents. Further, the anion channel was found to have the following properties. (1) The single-channel conductance is about 200 pS at 100mm Cl^–. (2) The channel does not select among monovalent anions but SO ₄ ^2– hardly permeates through the channel. (3) SO ₄ ^2– added to thecis side (the side to which SR vesicles were added) inhibits Cl^– current competitively in a voltage-dependent manner. (4) An analysis of this voltage dependence suggests that the binding site of SO ₄ ^2– is located at about 36% of the way across the channel from thecis entrance.     

Characterization of the channel properties of tetanus toxin in planar lipid bilayers.

A detailed characterization of the properties of the channel formed by tetanus toxin in planar lipid bilayers is presented. Channel formation proceeds at neutral pH. However, an acidic pH is required to detect the presence of channels in the membrane rapidly and effectively. Acid pH markedly lowers the single-channel conductance, for phosphatidylserine at 0.5 M KCl gamma = 89 pS at pH 7.0 while at pH 4.8, gamma = 30 pS. The toxin channel is cation selective without significant selectivity between potassium and sodium (gamma [K+]/gamma [Na+] greater than or equal to 1.35). In all the lipids studied gamma is larger at positive than at negative voltages. The toxin channel is voltage dependent both at neutral and acidic pH: for phosphatidylserine membranes, the probability of the channel being open is much greater at positive than at negative voltage. In different phospholipids the channel exhibits different voltage dependence. In phosphatidylserine membranes the channel is inactivated at negative voltages, whereas in diphytanoylphosphatidylcholine membranes channels are more active at negative voltages than at positive. The presence of acidic phospholipids in the bilayers increases both the single-channel conductance as well as the probability of the channel being open at positive voltage. A subconductance state is readily identifiable in the single-channel recordings. Accordingly, single-channel conductance histograms are best fitted with a sum of 3 Gaussian distributions corresponding to the closed state, the open subconductance state and the full open state. Channel activity occurs in bursts of openings separated by long closings. Probability density analysis of the open dwell times of the toxin channel indicate the existence of a single open state with a lifetime greater than or equal to 1 ms in all lipids studied. Analysis of intra-bursts closing lifetimes reveals the existence of two components; the slow component is of the order of 1 ms, the fast one is less than or equal to 0.5 ms. The channel activity induced by tetanus toxin in lipid bilayers suggests a mechanism for its neurotoxicity: a voltage dependent, cation selective channel inserted in the postsynaptic membrane would lead to continuous depolarization and, therefore, persistent activation of the postsynaptic cell.     

Regulation of calcium channel activity by lipid domain formation in planar lipid bilayers

The sarcoplasmic reticulum channel (ryanodine receptor) from cardiac myocytes was reconstituted into planar lipid bilayers consisting of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) in varying ratios. The channel activity parameters, i.e., open probability and average open time and its resolved short and long components, were determined as a function of POPE mole fraction (X(PE)) at 22.4 degrees C. Interestingly, all of these parameters exhibited a narrow and pronounced peak at X(PE) approximately 0.80. Differential scanning calorimetric measurements on POPE/POPC liposomes with increasing X(PE) indicated that the lipid bilayer enters a composition-driven transition from the liquid-crystalline state to the gel state at 22.4 degrees C when X(PE) approaches 0.80. Thus, the peaking of the reconstituted channel activity at X(PE) approximately 0.80 in the planar bilayer could result from the appearance of gel/liquid-crystalline domain boundaries at this POPE content. Lipid packing at domain boundaries is known to be looser as compared to the homogenous gel or liquid-crystalline state. We propose that the attractive potential of packing defects at lipid domain boundaries and entropic excluded-volume effects could result in the direct interactions of the transmembrane region of the channel protein with the lipid-packing defects at the lipid/protein interface, which could thus provide a favorable environment for the open state of the protein. The present findings indicate that the activity of the sarcoplasmic reticulum calcium channel could be modulated by lipid domain formation upon slight changes in membrane lipid composition in vivo.     

Formation of ion channels by Colicin B in planar lipid bilayers

Summary The gene for the antibacterial peptide colicin B was cloned and transformed into a host background where it was constitutively overexpressed. The purified gene product was biologically active and formed voltage-dependent, ion-conducting channels in planar phospholipid bilayers composed of asolectin. Colicin B channels exhibited two distinct unitary conductance levels, and a slight preference for Na⁺ over Cl^–. Kinetic analysis of the voltage-driven opening and closing of colicin channels revealed the existence of at least two conducting states and two nonconducting states of the protein. Both the ion selectivity and the kinetics of colicin B channels were highly dependent on pH. Excess colicin protein was readily removed from the system by perfusing the bilayer, but open channels could be washed out only after they were allowed to close. A monospecific polyclonal antiserum generated against electrophoretically purified colicin B eliminated both the biological and in vitro activity of the protein. Membrane-associated channels, whether open or closed, remained functionally unaffected by the presence of the antiserum. Taken together, our results suggest that the voltage-independent binding of colicin B to the membrane is the rate-limiting step for the formation of ion channels, and that this process is accompanied by a major conformational rearrangement of the protein.     

Incorporation in lipid bilayers of a large conductance cationic channel from mitochondrial membranes.

Membranes from subcellular fractions of adrenal medulla were incorporated in phospholipid bilayers formed at the tip of microelectrodes. Current fluctuations recorded in the presence of a transmembrane potential revealed the existence of a voltage-dependent channel of large conductance. This channel is characterized by fast kinetics and four conductance levels separated by jumps of 100, 220 and 220 pS in 150 mM NaCl. It is permeant to Na+,K+, tetraethylammonium, Cl- and acetate and has some cation selectivity. Exposure to trypsin or pronase abolished the voltage-dependence. Upon subcellular fractionation, the activity was found to be associated with mitochondria. A similar activity was observed in mitochondrial fractions from other organs. By its kinetics, its selectivity and its potential-dependence, this channel differs from the voltage-dependent anion channel of outer mitochondrial membranes.     

Lateral diffusion and conductance properties of a fluorescein-labelled alamethicin in planar lipid bilayers

In order to follow alamethicin diffusion within membranes under conditions of pore-formation, a fluorescein isothiocyanate (FITC) analogue was synthesized. To test the influence of the fluorescent probe addition on the pore-forming activity of the new analogue, macroscopic and single-channel experiments into planar lipid bilayers were performed. Although the apparent mean number of monomers per conducting aggregate was equivalent, the voltage-dependence of the new analogue was slightly reduced and hysteresses were broader, in agreement with the much longer duration of the open single-channels. Thus, the conducting aggregates seem to be stabilized by the introduction of the probe, presumably through the interaction of the conjugated cycles with the lipid headgroups, while the added steric hindrance may account for the slightly higher conductances of the open substates. Lateral diffusion of the labelled peptide associated with the bilayer was then investigated by the fluorescence recovery after photobleaching technique. Under applied voltage, associated with high conductance, D, the lateral diffusion coefficient, was reduced by 50% when compared to peptide at rest. These results provide new independent experimental evidence for a voltage-driven insertion of the highly mobile surface-associated peptide into the bilayer as a prominent step in pore formation.     

Asymmetry of the gramicidin channel in bilayers of asymmetric lipid composition: I. Single channel conductance

Summary Gramicidin-doped asymmetric bilayers made by the Montal-Mueller method exhibited an asymmetric current-voltage relationship. The asymmetric conductance was shown to be the product of two components, a rectifying single-channel conductance and an asymmetric voltage dependence of the reaction which leads to the conducting channel. The single-channel conductance was asymmetric in both asymmetric bilayers made of charged lipids and asymmetric bilayers made only of neutral lipids. The single-channel asymmetry decreased with increasing ion concentration. From the comparison of the singlechannel conductance in symmetric and asymmetric bilayers and the dependence of the asymmetry on the solution ion concentrations, it was concluded that (1) the rate of ion entry into the channel is dependent on the lipid composition of the membrane and is asymmetric in asymmetric bilayers; (2) the entry step is rate determining at low ion concentrations; and (3) at higher ion concentrations the rate-determining step is the translocation across the main barrier in the membrane; and this translocation appears insensitive to lipid asymmetry.     

Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.